Glycoproteins and glycolipids in disease processes

Glycoproteins and Glycolipids in Disease Processes Earl F. Walborg, Jr., EDITOR The University of Texas System Cancer ...

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Glycoproteins and Glycolipids in

Disease Processes

Earl F. Walborg, Jr., EDITOR The University of Texas System Cancer Center

A symposium sponsored by the ACS Division of Carbohydrate Chemistry at the 175th Meeting of the American Chemical Society, Anaheim, March 14-15, 1978.

ACS SYMPOSIUM SERIES 80

AMERICAN

CHEMICAL

SOCIETY

WASHINGTON, D. C. 1978

In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

Data

Library of Congress

Glycoproteins and glycolipids in disease processes. (ACS symposium series; 80 ISSN 0097-6156) Includes bibliographies and index. 1. Glycoproteins—Congresses. 2. Glycolipids—Congresses. 3. Tumor antigens—Congresses. 4. Carbohydrate metabolism disorders—Genetic aspects—Congresses. I. Walborg, Earl F. II. American Chemical Society. Division of Carbohydrate Chemistry. III. Series : American Chemical Society. ACS symposium series; 80. QP552.G59G6 ISBN 0-8412-0452-7

616.3'99 ASCMC8

78-11920 80 1-480 1978

Copyright © 1978 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of the first page of each article in this volume indicates the copyright owner's consent that reprographic copies of the article may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating new collective works, for resale, or for information storage and retrieval systems. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission, to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. PRINTED IN THE UNITED STATES OF AMERICA


ACS Symposium Series Robert F. Gould, Editor

Advisory Board Kenneth B. Bischoff

Nina I. McClelland

Donald G. Crosby

John B. Pfeiffer

Jeremiah P. Freeman

Joseph V. Rodricks

E. Desmond Goddard

F. Sherwood Rowland

Jack Halpern

Alan C. Sartorelli

Robert A. Hofstader

Raymond B. Seymour

James P. Lodge

Roy L. Whistler

John L. Margrave

Aaron Wold


FOREWORD The ACS SYMPOSIUM SERIES was founded in 1974 to provide

a medium for publishin format of the SERIE parallels that of the continuing I N CHEMISTRY SERIES except that in order to save time the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. As a further means of saving time, the papers are not edited or reviewed except by the symposium chairman, who becomes editor of the book. Papers published in the ACS SYMPOSIUM SERIES are original contributions not published elsewhere in whole or major part and include reports of research as well as reviews since symposia may embrace both types of presentation.


PREFACE T t is fitting that the proceedings of this symposium on "Glycoproteins and Glycolipids in Disease Processes" should be dedicated to the life and scientific contributions of my friend and fellow scientist, Ward W. Pigman. Much of Wards professional effort was devoted to the interchange of scientific information, both by his editorial skills and his support of national and international symposia on glycoconjugate chemistry. This symposium was born in Ward's mind in 1976 during deliberations of the Division of Carbohydrat Society concerning symposia topics for the coming years. Ward felt that sufficient progress had been made concerning the relationship of glycoconjugates to a variety of disease processes to warrant a symposium devoted to this topic. At that early date, the Carbohydrate Chemistry Division made a commitment to sponsor a symposium on some aspect of glycoprotein research and designated Ward as chairman with responsibility to formulate the program. Early in 1977, Ward informed me of his plans for the symposium and asked if I would serve as co-chairman. I gladly agreed and we set about the task of preparing a tentative program and contacting prospective speakers. On September 28, 1977, during the Fourth International Symposium on Glycoconjugates being held at Woods Hole, Ward and I finalized the symposium program. Upon completion of this task Ward expressed to me his personal satisfaction that the years of basic research on glycoconjugates wasfindingincreasing relevance to understanding a variety of disease processes. Ward's sudden death on September 30, 1977, was a shock to the many participants of the Wood's Hole meeting, many of whom were his long-time friends. In a way it was fitting that his last days found him engaged in an international meeting on glycoconjugates, an endeavor he had vigorously promoted and fostered for many years. Many of the participants in the symposium on "Glycoproteins and Glycolipids in Disease Processes" were present at the Wood's Hole meeting and by common consent it was decided that the planned symposium and this monograph should be dedicated to Ward Pigman's life and contributions to glycoconjugate research. The efforts of many persons and agencies were responsible for the success of the symposium and the publication of this monograph. First, I should like to express my appreciation to all of the participants for their contributions to the symposium and to the monograph. The supix In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

port of the Division of Carbohydrate Chemistry, particularly the efforts of Derek H. Ball and Gary D. McGinnis, is gratefully acknowledged. Grants from the National Institute of General Medical Sciences (GM 25226) and the National Cancer Institute (CA 24513), which provided travel support for participants in the symposium, are gratefully acknowl edged. Particular gratitude is extended to Edward Hampp (N.I.G.M.S.) and Mary A. Fink (N.C.I.) for their efforts in making this symposium a reality. On numerous occasions I have been encouraged by Gladys Pigman. Able assistance was obtained from Beverly Sea, Carey Osborn, Reita Maughan, and Pat McKirahan, staff at the Research Division— Science Park, The University of Texas System Cancer Center. Science Park—Research Division

E A R L F. WALBORG JR

The University of Texas July 20, 1978

χ


To Ward W . Pigman



DEDICATION TO WARD W. PIGMAN It is appropriate that this Symposium on "Glycoproteins and Glycolipids in Disease Processes" should be dedicated to the memory of my friend and colleague, Ward W. Pigman, who passed away on September 30, 1977, during his attendance at the 4th International Meeting on Glycoconjugates being held at Woods Hole, Massachusetts. Ward convinced the Carbohydrate Division of the American Chemical Society that a symposium on this subject warranted consideration for the spring meeting of the ACS to b efforts and those of Earl F. Walborg, Jr., this symposium has become a reality. Barely two hours before Ward's death, I had the opportunity to discuss his plans for this symposium, and I realized how deeply he felt about it. In a way, this symposium represents the ultimate goal of the long scientific road he and quite a few others among us have traveled, a road through carbohydrate chemistry that has led us from the use of analytical and physical chemistry in the study of reaction mechanisms to synthetic chemistry andfinallyto the elucidation of the structure of more and more complex carbohydrates, including glycosaminoglycans of connective tissue and glycoproteins from mucous secretions and the surface of the cancer cell. I feel that I have a deep understanding of Ward's research interests because both of us followed quite similar pathways, becoming interested in connective tissue polysaccharides in the late forties. In 1930 at the age of 20, he joined the National Bureau of Standards, where he was active until 1943. During this time, he obtained his B.A. and MA. degrees at George Washington University and his Ph.D. degree at the University of Maryland. At the Bureau he was associated with Horace Isbell, one of the leading American carbohydrate chemists. A series of papers reflecting Isbell's interests in the physical and organic chemistry of carbohydrates resulted from this collaboration. Soon a change in Ward's interests was noticeable, a turn to more biological problems with a study of glycosidases. This turning point was marked by a paper published in collaboration with H. Isbell and B. Helferich and by Ward's obtaining a Lalor Fellowship to spend one year in Leipzig, a stay that was unfortunately interrupted by the start of World War II. He returned to the National Bureau of Standards, and then during the last two years of the war he became a group leader at Corn Products, where most of his efforts were directed toward starch chemistry. Following his brief assoxiii In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

ciation with Corn Products, he moved to the Institute for Paper Chemistry at Appleton, Wisconsin, an institute for basic research supported by the industry, where he also served as a group leader. During the Wisconsin period, Ward's interests included pectins, cellulose, carbohydrate-degrading enzymes, and tannins. In 1949 Ward moved into the academic community, accepting a position at the University of Alabama Dental School where he was Associate Professor until 1960, Professor and then Dean of the Graduate School from 1963 to 1968. He remained associated with this institution as a Visiting Professor until his death. In 1960 Ward assumed the additional burden of a second academic position as Professor and Chairman of the Department of Biological Chemistry at New York Medical College. These moves to academic positions in a dental and medical school correspond to a final shift in series of papers on the reaction between amino acids and carbohydrates, the so-called Browning reaction, initiated at Appleton, papers appeared on dental plaque and saliva, and then the beginning of the work for which Ward is now best known:first,investigations on hyaluronic acid degradation and then an outstanding series of papers on submaxillary mucins. Before this work had been started, the contribution of Ward to carbohydrate chemistry had already been recognized by his selection as the recipient of the Hudson Award by the Carbohydrate Chemistry Division of the American Chemical Society in 1959. Other awards followed, such as medals of the Société de Biochimie de France and of the University of Milan. Enumerated below are a few of Ward's contributions to glycoconjugate chemistry. (a) Elucidation of the mechanism of degradation of hyaluronic acid by ascorbic acid and free radicals. (b) Purification of submaxillary glycoproteins and elucidation of many aspects of their peptide and saccharide structure. (c) Elucidation of the ^-elimination mechanism for cleavage of the glycopeptide bond between 2-acetamido-2-deoxy-D-galactose and serine or threonine, the predominant glycopeptide linkage of the mucin glycoproteins. (d) Suggestion that the peptide moiety of the mucin glycoproteins is composed of repeating sequences of 28 amino acids. Although present evidence suggests a more complex arrangement, this hypothesis will continue to deserve attention.

xiv In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

This symposium and the resulting monograph bear the mark and vision of Ward W. Pigman, and therefore are fitting memorials to his life and scientific contributions to glycoconjugate chemistry. Harvard Medical School

and Massachusetts General Hospital Boston, Massachusetts August 21, 1978

ROGER JEANLOZ

XV In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

Introduction ROGER W. JEANLOZ Laboratory for Carbohydrate Research, Harvard Medical School and Massachusetts General Hospital, Boston, MA 02114

To some chemist member Chemistry of the American Chemical Society, which is organizing this symposium, it may be unexpected that, for the next few days, we w i l l be discussing compounds that encompass lipids and proteins, in addition to carbohydrates, and their relation to disease processes. It is within the life span of many of us that natural product chemists have realized that nature is not compartmentalized into such nice and clean-cut categories as carbohydrates, proteins, and l i p i d s ; in fact, very few carbohydrates exist that are devoid of a peptide or l i p i d component, as well as there are few proteins devoid of a carbohydrate component. The roles of the carbohydrate component, of glycoproteins and glycolipids that are located at the surface of the animal c e l l or present in the connective tissue that links the c e l l s , have received increased recognition in the past decade and w i l l be discussed during the next few days. The subjects of the f i r s t session of this symposium w i l l be the methods of structure identification presently being developed, as well as the biosynthesis and degradation of glycoproteins and glycolipids. Because glycoproteins and glycolipids of biological interest are generally available only in minute amounts, the methods of structure identification, which w i l l be discussed by Dr. Walborg in his general presentation of current concepts of glycoprotein structure, are being developed at the micro- and even nano-gram l e v e l . Dr. Schachter and associates w i l l entertain us with the very active development of glycoprotein biosynthesis presently taking place. For many years, the concept of glycoprotein and glycolipid biosynthesis was dominated by the concept of elongation of the chains by one carbohydrate unit at a time, through sugar nucleotides; this concept, f i r s t developed for such homoglycans as glycogen and starch, was very ably demonstrated for glycoproteins and glycolipids by Roseman's group. Nearly twenty years ago, this unified concept of chain elongation was shown to be invalid for bacterial polysaccharides, and the importance of isoprenoid 3 © 1978 American Chemical Society In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

4

GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES

sugar

phosphate

recognized. work

on

sugar have in

sugar

phosphates

the

intermediates

years in

the

D-glucose

etc.

It

with

the

glycoprotein

is

of

of

more

the

than

polysaccharide group,

established

in

the

the

role

role

component

the

passing

of of

transfer interest

processes:

One

^-glycoproteins

and

involves

rather

where

the

carbohydrate

takes

place

through

transfer at

l i p i d

elongation, are

The which

of

their

these

be

to

units

very

simple

components

two

the we

the

are

faced

processes

animal

and

mechanism, backbone

synthesized

degraded

coexist

now

in

peptide

before

where

sugar

the

chain,

complex

process

from

years

peptide

active

to

few

intermediates

^-glycoproteins,

oligosaccharides

and p a r t i a l l y

a

past

new

sugar

units

nucleotides.

or

are

Time

merely

the

of

blood-group

those

structures

are

found

length

at

the

to

of

eukaryotic

glycoglycerolipids

their

chain

as

b

solely

variation or

large

restricted

composition

process

is

single

residues

their

complex

in

w i l l

diversity of

of

its

inadequat

encompass

pyranosyl and

as

glycolipids

associates The

second

show w h e t h e r

expression

stage

intermediates

the

transferred

w i l l

of

the

a

is

was

for

isoprenoid

The

that

two

plant

of

known

these

to

of

synthesis

already

biosynthesis.

of

core

residues

existence

in

L e l o i r ' s

development

biosynthesis

of

ago,

nucleotides,

witnessed

role

as

Ten

the

up

to

surface

typing,

of

have

for

50

of

the

a-D-galacto-

chain

of

carbohydrate

units

the

organisms,

glycosphingolipids.

example

within

(evidence

having

result

(for

only

and

has

cells

been

of

reported),

during

the

specific

genetic

defects,

a l l

reasons

for

been

glycolipids), chains

oncogenic their

active

role

investiga

tion. Finally, the

most

microgram

specific chains

the

by

ago,

Dr.

of

we

by

were

Maley

Eylar of

D.

not

Dr.

degradation

methods the

identification of

this

his

been

are

we

and to

of

It

able

the the

more

progresses

glycoproteins these

to

my

the

I

laboratory structure such

that

present

addition of established and

structures

of

α -acid an

glycolipids disease

endo-

found

extract

that

biochemical

chemical i n

glyco

x

of

the

25

exo-

enzyme, in

be

when,

with

lack

such

recently

with

carbohydrate

remember

time

made

off

at

the

and which w i l l

the

was

elucidations

without

s p l i t

linkage

deploring

group,

is

the

in

on

were

his

structure possible

associates.

working

pneumoniae

great

correlation of

subject

was

manipulating.

allowed

chemical

have

that

r e a l i z i n g at

Kobata

has the

not

and

human p l a s m a ,

glycosidase later

recent

carbohydrate-peptide

Dr.

glycosidases protein

the would

endo-glycosidases

near

discussed years

of

level

methods

that

structural

and

made

possible

processes,

Symposium.

RECEIVED August 2, 1978.


the

2 Biosynthesis and Catabolism of Glycoproteins HARRY SCHACHTER, SAROJA NARASIMHAN, and JAMES R. WILSON Research Institute, Hospital for Sick Children, Toronto, Canada

Glycoproteins are biosynthesis is, not unexpectedly Although much has been learned about this process in the past ten years, there are many aspects which we do not understand. In the limited space permitted for this article, it will be possible only to outline the major steps in the biosynthesis of some of the gly coprotein types; more detailed reviews are available (Schachter and Rodén, 1973; Montreuil, 1975; Schachter, 1974a,b, 1977, 1978; Waechter and Lennarz, 1976). I. Initiation of oligosaccharides of the asparagine-N-acetyl-Dglucosamine linkage type. The largest group of animal glycoproteins are those which contain only or predominantly oligosaccharides that are linked to the polypeptide back-bone by an N-glycosidic linkage between N-acetyl-D-glucosamine (GlcNAc) and asparagine (Asn). Many secreted glycoproteins (α1-acid glycoprotein and other plasma globulins, immunoglobulins, various gonadotrophins, ovalbumin, various enzymes, etc.) and membrane-bound glycoproteins (red cell membrane glycophorin, rhodopsin, the envelope glycoproteins of certain viruses such as vesicular stomatitis virus and Sindbis virus) contain Asn-GlcNAc linkage type oligosaccharides. It has become clear that the synthesis of the sugar-amino acid linkage pre-determines the general nature of the oligosaccharide that is subsequently assembled, i.e., that the synthesis of this linkage is an important control point. It is therefore important to understand the initiation process. In recent years it has become apparent that many (but not all) Asn-GlcNAc oligosaccharides share a common "core" structure (Montreuil, 1975) : Man-α1,3 Man-al,6

Man-31,4-GlcNAc-31,4-GlcNAc-Asn

0-8412-0452-7/78/47-080-021$06.50/0 © 1978 American Chemical Society In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

22


Further,

the

Hemming,

Heath,

1976,

and

strong

work

is

evidence

in

Soon

after

suggested cells. that

It

tissues

of

of

mammalian

core

is

scheme

Jeanloz,

and

has

Lennarz,

provided

pre-assembled prior

for

role

of

the

to

as

a

incorporation

initiation

p o l y p r e n o l - l i n k e d sugars

polysaccharide

a

process

similar however,

the

1);

be

pioneering

evidence and

nucleotides

(Fig.

might

assembly,

reaction

the

to

work

for

the

of

had

in

Leloir s

group

1

transfer

i n

mammalian

N-acetylglucosamine

from a

phosphate,

phosphate

workers

higher

dolichol

dolichol

been

various

occuring

their

phosphoryla-

sugars

formed

are,

Man-3-monophos-

GlcNAc-a-pyro-phosphate-dolichol, Glc-3

and

intermediates tissues

thyroid,

hen

ha

(rat

and p i g

oviduct,

l i v e r ,

human

mouse

myeloma

lymphocytes,

calf

tumor,

pancreas,

. Another

covery that

of

rat

glucose

liver

to

thought

to

water,

in

but

the

be

protein.

This

of

study

in

this of

the

product

product and

an

which

was

in

to

dolichol

the

was

of

radioactive

was

at

f i r s t

subsequently

shown

w h i c h was

et

aJU ,

pyrophosphate

ac to

insoluble

chloroform-methanol

(Behrens

dis

noted

endogenous

chloroform-methanol-water

solvent

was It

transfer

glucose

oligosaccharide acid

group

1

effect

acid-insoluble

soluble

The

length

of

the

tissue

and w i t h

reports

from

contain

about

mannose

and

mouse

20

residues

η

assembly

esting

to

formation

the

led

be

in

(2:1,

(10:10:3,

1971)

v/v)

the

oligosaccharides

units

residues. for

oligosaccharide of

GlcNAc

and

attached

conditions

laboratory

microsomes,

;

way in

5-7

used

indicated

to

for

the of

With

tissues

other

appeared

the to

residues

of

varies The

oligosaccharide

consisting example),

dolichol

synthesis.

to

N-acetylglucosamine, (hen

oviduct

predominant

lack

glucose

mannose,

and

as

and

dolichol contain

follows:

-Man-31,4-GlcNAc-31,4-GlcNAc-pyrophosphate-dolichol of

note of

oligosaccharide

glycose

glucose

myeloma

(Man-α-)

the

Leloir's

pyrophosphate

from

could

L e l o i r s

oligosaccharide.

tissues.

with

The

an

from

monophosphate

trichloroacetic was

intense

two

microsomes

pyrophosphate

discovery

many

contribution pyrophosphate

dolichol

form

dolichol

v/v)

major

dolichol

from

ceptor

to

reviews)

intermediate

general

mannose

l i p i d

(Leloir,

Waechter

1.

the

sugar

phate-dolichol

a

The

see

recent

common

glucose,

polyprenol

etc)

this

strong

respectively,

bovine

for

provided

of

laboratories others,

bacterial

was,

respective ted

in

that

first

1978,

that

Figure

established

and

oligosaccharide

polypeptide.

shown

several

Schachter,

lipid-linked into

of

Lennarz

this the

structure

inversions

3-linked

mannose

is of

shown

in

anomeric

from

Fig.

1.

It

is

configuration

GDP-a-Man and

inter

in

α-linked

the mannose

Man-3-monophosphate-dolichol. M.J.

carried

Spiro

out

containing

et

careful dolichol

a l .

(1976a,b)

and

investigations pyrophosphate

on

R.G. the

Spiro

et

formation

oligosaccharides

al of by

(1976)

have

glucoseseveral


2.

Glycoprotein

SCHACHTER ET AL.

tissues.

Working

with

these

workers

phate

oligosaccharide

Biosynthesis

thyroid

demonstrated with

slices

and

rather

than a

23

Catabolism with

the

formation

of

the

tentative

structure:

microsomes,

dolichol

pyrophos

(Man-α-) -(Glc) _ ~(Man-α-) -Man-GlcNAc-GlcNAcβ

1

2

4

PY r o p h o Evidence and

was a l s o

thymus

pyrophosphate preparations these

large

molecules lack

slices calf

dolichol

which

Parodi

for the of

pyrophosphate only

However, et

It

of

dolichol

pyrophosphat

glucose

believed

to

is

so

that

dolichol The

final

asparagine work and the to

i n i t i a t i o n process

and Lennarz, labelled

that

membrane the

that

that

is

of the

unglycosylated presence

of

by

retory

The

is

It

always

of

attached

pathway

transfer

However

to

the presence

pyrophosphate

of

of

of

further

forms

of

pathway. for

from

that

any amino sequence

the

dolichol

three

which is

sec

under

oligosaccharide

acid

does

A. effects

not

i n the tripeptide

almost this

time

i n

sec

and ribonuclease

polypeptide

some

a

glycoprotein

the transferase

for

rates

evidence

oligosaccharide

into

synthesis

ovalbumin,

the dolichol

denatured

an asparagine X can be

that

i n secretory

α-lactalbumin

recognized to

by

obtained

specificity

(Thr)-where

dolichol

has

tunicamycin,

normal

oligosaccharide

has been

i n h i b i t e d by

almost

appears

synthesis

however,

continued

oligosaccharide

substrate

Asn-X-Ser 1974).

the

suggested

since

i t

to

proteins

i n the

work,

endo

bound

secretory

only

and

The

1977);

at

been

asparagine

i n i t i a l l y

of

cells

intermediate

a l l tightly

recent is

l i p i d

an

catalyzed

reactions.

known

synthesis

have

the d o l i c h o l

showing

More

myeloma

has not yet

between

involved

glycosylated

proteins-ovalbumin,

incorporation stood.

is

(1977)

i t

of

to

Earlier

and Lennarz,

tunicamycin,

glycoprotein,

synthesis

the

from

findings

be

transfer

preparations

were as

ovalbumin

ovalbumin

and Lennarz

pyrophosphate

of

(Struck

retory

of

these

might

the

(Fig. 1).

i n these

identified

blocks

Pless

involvement

linkage

glycoproteins.

which

glucose-

configuration.

is

rat l i v e r ,

however,

experiments

study;

glycosylation

antibiotic

with

microsome

synthesized

pathway

N-acetylglucosamine

and

et a l . ,

oligosaccharide

chain

oligosaccharide

n o t be

under

dolichol

membrane-bound

shown an

tissues

the

1976) that

acceptors;

i n these

and could

smaller

(Herscovics

large

anomeric

pyrophosphate

an N - g l y c o s i d i c

acceptors

of

i n the polypeptide

N-acetylglucosamine

of

dolichol

protein

but

glucose-3-monophosphate-

from

i n the

of

microsome

produce

(Behrens

pancreas

step

transfer

genous

calf

synthesizing

glucose

established

established

of

of

hen oviduct endogenous

rat liver

inversion

residue

(Waechter

from

single

from

that

do n o t

and N-acetylglucosamine

a

oligosaccharide

kidney dolichol

b

transfer

involves

interesting

oligosaccharides

and from

capable

containing cules

is

mannose

microsomes

a l . , 1972) were

by o v i d u c t ,

glucose-containing

t h y r o i d and hen oviduct

contain

a l . , 1977a,b)

formation

similar

oligosaccharides. from

glucose.

1971; et

obtained

tissue

sphate-do1icho1

sequence(Marshall,

not

necessarily


24


result a

i n

glycosylation

protein

may,

cosylated (Thr)The

forms

site

work

but

that

A l l

may

be

completely

lactalbumin sequence

the

three and

ineffective

fully

protein A)

must

the

required tripeptide

and

the

Although serve

smaller This

into

observation transfer that

GlcNAc

contain

structures clear by

such

and

oligosaccha-

have

not

as yet

which

lack

and

since

1975)

o fa l l

glucose

stomatitis stages in

1978).

involve The

t othis

the

the

The

viral

class

Work

and

Sindbis

assembly

leads

e ta l . , envelope

plasmacytoma processing

Asn-GlcNAc

oligosaccharides

oligosaccharide i n

2.

F i g . may

f o l -

mannose

evidence has

i n d i -

envelope

subsequent 1977;

glyco-

rough

processing

o f mannose Robbins,

glycoproteins processing since

c e l l

(Tabas

line

GlcNAc,

Man a n d

endoplasmic

are

reprehowever,

biosynthesis o f was

e tal_.,

initiation

(and

1977;

i s , also

shown

1978) .

and

I t i spostulated

undergo

containing the

be

enveloped

virus)

t o removal

glycoproteins;

in

containing

must

the

o f the

type

type

residues, i t

reported with

o fAsn-GlcNAc oligosaccharides

summarized

occurs

Asn-

a n d o f some

recently

o fglycoprotein,

a mouse

are

process

o f the

o f mannose

residues;

(Hunt

processing large

large

N-

protein-bound oligosaccharide i s

mannose

oligosaccharide

initiation

have

virus

in

viruses,

rich

by

the

contain

3 mannose

residues

processing".

o fmembrane-bound IgG

oligosaccharides. primarily

oligosaccharide

the

residues

confined

oligo-

the

Asn-GlcNAc linkage

only

11 r e s i d u e s

glucose)

secretory

i n

than

N-acetyllactosamine

and

laboratories

a tearly

e ta l . ,

effective

acceptors

which

o f the the

contain

probably sentative

of

type.

o f glucose

and

more

involves

incorporation o fa large

o f these

moieties

glucose.

glycoproteins

This

accept

incorporation

pyrophosphate

envelope

Tabas

the

protein

pathway

these

a

not

were

proteins

oligosaccharide

9 times

oligosaccharides

(vesicular

large

i n

oligosaccharides

glucose

Several

that

proteins quite

vivo

"oligosaccharide

viruses

to

denatured

o fp r o t e i n - b o u n d o l i g o s a c c h a r i d e s

removal

residues.

cated

dolichol

(Montreuil,

that

residues

lowed

a

tripeptide

sequence

oligosaccharides

small

donors

about

mannose

linkage Since

not

are

pyrophosphate

Processing

not

glycosylation t o

tha

i n

acetylglucosamine, II.

relatively

t o endogenous

glucose-free

suggests

dolichol

of

site.

protein

oligosaccharides

saccharide

for

this

did

such

acceptors

(ovalbumin, a -

glycosylation

pyrophosphate

carry

asoligosaccharide

important phate

1-2

for

for

ungly-

-Asn-X-Ser

exogenous

several

and

a tanother

required

without

sequence

requirements

dolichol

and which

oligosaccharide

is

a tone

proteins

1977);

Lennarz,

resolved.

glucose

do

with

the

and

glycosylated

unfolded

however,

ride

can

be

acceptor

with been

(1977)

proteins

asacceptors;

Pless

both

glycosylated

contained

several

exact

i n

unglycosylated

Lennarz

effective

RNase

whereas

be

exist

or

and

1974;

(Marshall,

example,

o fPless

indicates occur.

for

by Glc

reticulum

subsequent that

a l l

transfer (Fig. (see


of

2). next

2.

Glycoprotein

SCHACHTER ET AL.

Biosynthesis

25

and Catabolism

UOP-GicNAc Dd-P *UMP G l c N Acc*-PP- - P - D o l U D P ^ G l c N A c J-

(GOP^Mon) (GOP^M •

n 1

n

γυΟΡ

(Dol-P)4

(

n

GlcNAc GlcNAc*P-P-Dol IcNAc^P-P-Dol GDP-Man *GDP

ί I " ^Man^P-Dol)

£

Mon-GlcNAc^GlcNAc^P-P-Dol—^(Man) *Man*GlcNAc*GlcNAc*P-P-Dol n

"

l U 0 1

P

Acceptor

)

Nooi-p-p ( Man\,

Man - GlcN Ac - GlcN Ac * A S N

Figure 1. Initiation of Asn-GlcNAc-type gosaccharides are preassembled tions: Dol,

oligosaccharides.

Oli

Peptide ^

(Glc) -(Man) -(GlcNAc) -P-P-Dolichol 2

n

(Glc) -(Man) -(GlcNAc) -Asn2

2

n

2

Oligosaccharide processing

V M-) CHUOH

CH9OH

I

HC-NH

I

HC - OH

I

(CH9)..

I

2

CH-

1

*

3

I

CH9OH

I

HC-NH

9

I

I

9

HC-NH

I

I

9

1

H C - OH

H C - OH

HC

HC

II

II

I

I

CH

(ÇH2)12 CH^ Sphinganine

CH9OH

I

4-Sphingenine

CH

(ÇH2)W

HC— N H 9

I

HC -

I

OH

HC-OH

I

(CH9)n

I CH

3

CH^ 4-Eicosasphingenine

4-D-Hydroxysphinganine The fatty acids occurring in the ceramide moiety are a variable mixture of saturated and monounsaturated homologs from C ^ to C 2 , and the α-hydroxy derivatives of these fatty acids. Lignoceric (24:uf and nervonic (24:1, Δ ) acids are generally major components, along with substantial amounts of palmitic (16:0), stearic (18:0), arachidic (20:0), behenic (22:0) and tricosanoic (23:0) acids. In some cases the α - h y d r o x y fatty acids are the dominant species. Comparisons of the fatty acid composition of a wide variety of glycosphingolipids have been reported

01).

In the glycosphingolipids, ceramide is attached to a mono- or oligosaccharide by a glycosidic linkage with the primary hydroxyl group of the long-chain base. Although it has only been proved conclusively in a few instances by nmr spectroscopy or chemical synthesis, it is assumed that the glycosidic linkage between ceramide and the carbohydrate moiety


3.

SWEELEY ET AL.

Glycolipid

Structure

and

Metabolism

49

has the 3 configuration, and that the sugar residues exist in the pyranose ring form. An example is shown in Figure 1, galactosyl-(gl->-4)-glucosyl31+D-ceramide (lactosylceramide). A class of phosphoglycosphingolipids occurs in higher plants and fungi; these structures consist of carbohydrate groups with an inositol residue linked to ceramide through a phosphodiester bridge. A substance of this type from corn was proposed to be GlcNal^4GlcUAal^6inositol(2^1aMan)-l-0-phosphorylceramide (169). A related compound from tobacco leaves (170) has been shown to be GlcNAcal+4GlcUAal^2myo-inositol-l-Q-phosphorylceramide (171). In the glycoglycerolipids studied thus far, the carbohydrate residues are attached to C-3 of sn-glycerol by either a- or 3-glycosidic linkages. Three important types of glycoglycerolipids are 3-3-galactosyl-l,2-diacylsn-glycerol (12), 3'-sulfo-3-3-galactosyl-l-alkyl-2-acyl-sn-glycerol (seminolipid) (13,14), and a sulfotriglycosyldiglyceride of human gastric secretions, 3-ot-(6'-sulfoglucosyl-(a l+6)-glucosyl-(al+6)-glucosyl)-l-alkyl2-acyl-sn-glycerol (15). Structural variatio glycosphingolipids is so diverse that a special nomenclature has become necessary in referring to these substances. A semisystematic nomencla ture has been recommended by the IUPAC-IUB Commission on Biochemi cal Nomenclature (^6) and has been reviewed in some detail (6,17,18). Compounds containing a single monosaccharide unit were historically called cerebrosides. Now they are identified by the sugar they contain; examples are galactosylceramide (GalCer) and glucosylceramide (GlcCer). Similarly, the diglycosylceramide containing a lactose unit is called lactosylceramide (LacCer). More complex glycosphingolipids are given names that identify a particular portion of the carbohydrate moiety, indicating the family to which it belongs and the number of glycose units in the chain. Prefixes (given below) for the various families imply a specific structure, including the sequential arrangement of the glycose units as well as the positions and anomeric configuration of the glycosidic linkages. Thus, the glycosphingolipid of the globo type that contains all four sugar residues is referred to as globotetraglycosylceramide and is abbreviated GbOse^Cer. Similarly, the glycosphingolipid of the ganglio type with three sugar units is called gangliotriglycosylceramide (GgOse^Cer); this substance is G a l NAc3l->4Gal31^4GlcCer. Substituents that are not part of the root oligosaccharide are given at the beginning of the name, using a Roman numeral to indicate the monosaccharide unit (counting from ceramide) on which the substituent is located and a superscript Arabic numeral to indicate the position of the glycosidic linkage. According to this system, Forssman hapten (Gal Ν Ac al-* 3GalNAc31+3Galal+4Gal3 l+4GlcCer) should he called IV -a-N-acetylgalactosaminyl-globotetraglycosylceramide (I\raGalNAc-GbOse^Cer). Methods of Analysis of Glycolipids Determination of the complete chemical structure of a glycolipid usually requires a combination of several techniques. Some of the methods can be used with nanomolar amounts of sample, whereas others


50


Figure 1. Lactosylceramide (LacCer), galactopyranosyl-(βl -» 4)-glucopyranosyl—(1 -» r)-ceramide


3.

SWEELEY ET AL.

Glycolipid

Prefix

Abbreviation

lacto lactoneo muco gala globo globoiso ganglio

Lc Lcn Mc Ga Gb Gbi Gg

Structure

and Metabolism

51

Structure GaKg l+3)GlcNAc(e l+3)Gal(B l-*4)Glc Gal(Bl+4)GlcNAc(ei+3)Gaiei+4)Glc Gal( β 1 +3)Gal( β 1 +4)Gal( β 1+ 4)Glc GalNAc( l+3)Gal( l+4)Gal(al+4)Gal* GalNAc(ei+3)Gal(al-*4)Gal(31+4)Glc GalNAc( Bl+3)Gal(al+3)Gal(6 l-*4)Glc Gal( β ΐ +3)GalNAc( ei+4)Gal(6 l+4)Glc

^Substances more complex than Galal->-4GalCer in this family have not so far been isolated from biological material. require about one micromole; carbon-13 nmr spectroscopy is applicable at present only if a large amount (ca. 150 mg) of sample is available. Most of the structural studies chromatographic separation of the glycolipid (column and thin-layer chromatography), followed by several commonly used analytical proce dures: fatty acid, long-chain base and carbohydrate composition by gasliquid chromatography after acid-catalyzed methanolysis; stepwise degra dation of the carbohydrate moiety with specific glycosidases; and analysis of the partially methylated alditol acetates derived from permethylated glycolipid. Frequent use of mass spectrometry has been made in recent studies to validate the structures of the partially methylated alditol acetates and to determine some structural features of the intact permethylated glycolipid. Some of the newest techniques are reviewed briefly in the following section. Excellent separations of glycolipids according to fatty acid differ ences have been obtained by high pressure liquid chromatography of perbenzoylated or O-acetyl-N-p-nitrobenzoyl derivatives (19-21), requir ing only nanomolar amounts of samples for detection at 254 nm with a suitable flow-through detector. Mixtures of natural glycolipids from GlcCer to GbOse. Cer can be separated by chromatography on Sorbax Sil (22). Though the technique appears to be ideally suited for the separation of substances with different molecular weights, sufficient studies have not been made to determine whether isomeric glycolipids (different sugar arrangement, different positions and/or configuration of glycosidic link ages, different epimeric components) can be separated effectively. An alternative chromatographic approach involves the careful analysis by thin-layer chromatography of the effluent material from a column. This technique appears to be most effective when use is made of an ion exchange material such as DEAE-cellulose or DEAE-Sephadex and when a very large number of samples from the column are spotted adjacent to each other on the thin-layer plates. The resulting map looks like a two-dimensional chromatogram with excellent resolution of components that would overlap if analyzed together on the plate. This procedure has been exploited in the study of blood group glycolipids from human erythrocytes (23) and for analyses of the ganglioside composition of brain (24,25). Additional fractionation of gangliosides has been obtained


52


on a preparative scale by DEAE-Sephadex column chromatography and subsequent separation on a new spherical silica, called Iatrobeads (26-29). Analysis of carbohydrate composition by gas-liquid chromatography is common practice. Trimethylsilyl derivatives of methyl glycosides (obtained by acid-catalyzed methanolysis) or alditol acetates (obtained by acetolysis and hydrolysis) can be used with suitable internal standards to obtain molar ratios of the sugar components with about 100 nanomoles of sample ( 3 0 - 3 2 ) · These conditions cannot be used to distinguish between N acetyl and N-glycolyl derivatives of neuraminic acid in the intact glycolipids. Mild acid-catalyzed methanolysis (0.05 Ν methanolic HC1 at 8 0 ° for 1 hr) has been used instead, giving high yields of the β - m e t h y l ketosides of N-acetyl and N-glycolylneuraminic acid, the trimethylsilyl derivatives of which separate readily by gas chromatography (33). The sequential arrangement of the sugar residues in complex glycolipids can be deduced enzymatically or by mild acid hydrolysis or methanolysis. The enzymatic procedure involves the stepwise hydrolysis of the glycolipid with specifi evidence for the nature of the terminal glycose residue at each step as well as the anomeric configuration of the glycosidic linkage (34). It is customary to first remove sialic residues from gangliosides by 1 M formic acid hydrolysis for 1 hr at 1 0 0 ° (35), a procedure which also liberates fucose (36). Negative results in the enzymatic analysis need to be interpreted with some care since they might be caused by improper detergent in the reaction mixture or a glycosidase specificity that gives poor K m or maximal velocity values with a particular structure (34). It is important to realize that the procedure requires a set of highly pure glycosidases. The other method involves partial methanolysis with 0.3 M HC1 in chloroform/methanol at 6 0 ° for 40 min; it has been used effectively for analyses of a heptaglycosylceramide (37) and various blood group Α - a c t i v e glycolipids from gastric mucosa (36). Two methods make use of permethylation for the analysis of glycolipids and can be used together to obtain information about the sequential arrangement of carbohydrate residues and the positions of glycosidic linkages. Permethylation is generally carried out in dimethylsulfoxide with a mixture of sodium dimethylsulfinylcarbanion and methyl iodide (38), a mild procedure (room temperature) which gives high yields of completely methylated product. Precautions have to be taken to exclude plasticizers from the starting materials, particularly dimethylsulfoxide. Mass spectra of the permethylated intact glycolipid reveal several kinds of structural detail, including the composition of the ceramide moiety, the nature of the terminal glycose unit, the total number of glycose units and, to some extent, the sequence of sugars (3945). Spectra of the product obtained by reduction of the N-methyl amide linkage with lithium aluminum hydride are somewhat superior as ions at high mass are stabilized and the volatility of the derivative increases to some extent (39-45). Useful information results from the cleavage reactions (at glycosidic linkages) that result from random charge localization on ring oxygen atoms. Unfortunately these ions are obscured to some extent and often represent weak contributions to the total mass spectrum, due to the presence of the ceramide moiety.


3.

SWEELEY ET AL.

Glycolipid

Structure

and

Metabolism

53

An alternative approach involves conversion of the glycolipid to an oligosaccharide by ozonolysis (46) or treatment with osmium tetroxide (47), both of which attack at the double bond of the sphingosine moiety, yielding a product that is readily hydrolyzed to the free oligosaccharide. The permethylated oligosaccharide has excellent mass spectral character istics, as illustrated in the case of lacto-N-tetraose and difucohexaose I in Figures 2 and 3. The molecular ion was not observed in either case upon electron impact ionization but may be present in chemical ionization mass spectra. The ion at highest mass in the case of the lacto-N-tetraose derivative is at m/e 858; it results from the loss of a C-6 substituent (· CHÔMe) from whichever ring bears the charge (on oxygen) in the molecular ion. Three ions (below) of approximately equal intensity, at m/e 219, 464 and 668 in Figure 2, are predicted for the sequence hexoseN-acetylhexosamine-hexose-R by arithmetic calculations; they result from the cleavage of different glycosidic linkages, with charge retention on the ring oxygen and loss of the remainder of the chain as a free radical.

Ν ME AC Further loss of methanol from Jând a disaccharide from 3 both give m/e 187 (4), whereas loss of the terminal hexose residue from 2^ gives an intense ion at m/e 228 (5). The presence of m/e 464 (a hexosaminecontaining disaccharide moiety) and the absence of m/e 260 (a terminal N acetylhexosamine unit) are strong evidence for the hexose-hexosamine



71

100

ΙΟΙ

150

668

159

650 100

M

I 'Ί 1

\

150

300

1

1

I

\

1

I

1

I

1

I

J I 'Ί I I ' I

850

1

858

/

MeOÇHs Ν

'Se/' /423

8CO

I 11

/66Θ

/

\ MtOÇHg

668\

350

r

2

T

1

Γ

τ

450

432

Χ3

1

950

I M 'Ί ' I ' 1

1000

1

500

464

M Ί I I I I I I M ' I

MW 903

Τ ' I

900

r

9

400

LACTO-N-TETRAOSE

464 \

I M M ι I ' I

2I9\

250

228

1

HO lb

Figure 2. Mass spectrum of permethyhted hcto-N-tetraose, obtained by electron impact ionization at 70 eV with a Varian MAT CH-5 double-focusing mass spectrometer (direct probe)

600

200

187

V-i • f • |-»|t f Ι Γ Ι | + | Λ * ι ' Λ Γ f i

50

100 η

ο

η

S

S!

S 3

ο ο ο ο

>

3

8

3.

SWEELEY ET AL.

55

Glycolipid Structure and Metabolism

tUC

S •S Se

1 ο ο ο

1 Ο

.s

Ë a.

3 as shown by these i n v e s t i g a t o r s . In contrast to the branched s t r u c t u r e of (Man)3(GlcNAc)2Asn, a s s o c i a t e d with the core of the complex o l i g o s a c c h a r i d e s , that i s o l a t e d from the α - m a n n o s i d a s e d i g e s t i o n of (Man)5(GlcNAc)2Asn, was determined by methylation (Table IV) and periodate o x i d a t i o n to be l i n e a r and to possess the p e r i p h e r a l s t r u c t u r e shown i n F i g u r e 10. Both (Man)4(GlcNAc)2Asn and l i n e a r (Man)3(GlcNAc)2Asn appear to be hydrolyzed as r a p i d l y by endo-H as i t s most a c t i v e s u b s t r a t e , which to date i s (Man)5(GlcNAc)2Asn, and c o n t r a s t s sharply w i t h the rather slow h y d r o l y s i s by t h i s enzyme of branched (Man)3(GlcNAc)2Asn (Figures 2 and 3). By u t i l i z i n g a modified Smith degradation on (Man)5(GlcNAc)2Asn (21, 22), (Man)2(GlcNAc)2Asn could be i s o l a t e d and v e r i f i e d by compositional a n a l y s i s , methyla t i o n (Table I V ) , and chromatography (Figure 9B). T h i s compound was hydrolyzed at only h a l f the r a t e of branched (Man)3(GlcNAc)2~ Asn, while (Man)1(GlcNAc)2Asn i s probably not hydrolyzed at a l l by endo-H. Thus, the statement i n our a b s t r a c t that (Man)2(GlcNAc)2Asn i s as good a substrate as (Man)5(GlcNAc)2Asn i s i n e r r o r and should be corrected by r e p l a c i n g the former compound with l i n e a r (Man)3(GlcNAc)2Asn. (Man)1(GlcNAc)2Asn, however, could be very e f f e c t i v e l y hydrolyzed to Man31->4GlcNAc + GlcNAc-Asn by endo-L, the enzyme r e f e r r e d to e a r l i e r as enabling us to i s o l a t e and to c h a r a c t e r i z e t h i s d i s a c c h a r i d e (1). That t h i s enzyme i n not c h i t o b i a s e i s i n d i c a t e d by i t s i n a b i l i t y to hydrolyze (GlcNAc) 2 Asn. Endo-L has now been p u r i f i e d about 300 f o l d and possesses proper t i e s d i s t i n c t l y d i f f e r e n t from endo-H, not the l e a s t of which i s t h e i r substrate s p e c i f i c i t i e s . Other d i f f e r e n c e s are a s s o c i a t e d with the higher molecular weight of endo-L and i t s sharper pH optimum (23). A comparison of these enzymes and t h e i r capacity to


96


175

35

52.5

70

0.1 M Ν α - C I T R A T E (pH 2.60), ml Figure 9.

Chromatographic separation of glycosyl asparagine derivatives on the amino acid analyzer using AG-50 X-4 resin.

The various stands used (prepared from ovalbumin) are indicated by the dashed line and repre sent (Man) (GlcNAc) Asn, 1-2-6; (Man) (GlcNAc) Asn, 1-2-5; (Man) (GlcNAc) Asn 1-2-1; (GlcNAc) Asn, 1-2; GlcN Ac-Asn, 1-1. Peaks a and b resulted from a limited a-mannosidase digestion of 1-2-5 as shown in Figure 10; peak c in Β was prepared by the periodate oxidation of 1-2-5; peak d in C was obtained by treating 1-2-2 with periodate. The elution times (t), measured at peaks of various glycosyl-Asn derivatives, were plotted as shown in the insert vs. the number of mannose residues in the compounds. As indicated, compounds a, b , and c fall on the line at 4, 3, and 2 mannose residues respectively, which is consistent with their mannosyl content (Table IV). The proposed mannosyl linkages shown in Figure 10 were obtained from the methylation data in Table IV. More specific details on the methodology used are in prepa ration. 6

2

5

2

t

2

2


y

In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978. 1.00

(Man) 2 (GlcNAc) 2 A s n b

endo-H

1.0

0.84

2.2

1.4

1.1

1.3 1.0

1.0

Methylated mannitol products (2,3,4,6) (2,3,4) (2,4,6) (2,4)

s u b s t r a t e s for

'Periodate o x i d a t i o n product from (Man)r (GlcNAc)οAsn.

Prepared from IgM (9) ; R = p e p t i d e .

1.96

3.00

2.00

1.55

(Man) 3 (GlcNAc) 2 - R a 1.80

2.84

1.82

1.00

4.06

1.85

(Man) 3 (GlcNAc) 2 A s n

Man

Analysis GlcNAc

1.00

Asn

of compounds tested as

(Man) 4 (GlcNAc) 2 A s n

Isolated compound

Composition

TABLE IV

98


hydrolyze s p e c i f i c dansylated substrates prepared from ovalbumin (Figure 11) r e v e a l s that endo-L i s not very e f f e c t i v e against sub s t r a t e s c o n t a i n i n g more than two r e s i d u e s of mannose. In a s i m i l a r v e i n , endo-H i s not e f f e c t i v e against compounds c o n t a i n i n g two mannosyl residues or l e s s . We cannot be c e r t a i n at present whether the small amount of a c t i v i t y seen with (Man)3(GlcNAc)2Asn as s u b s t r a t e i s n a t i v e to endo-L and i s not due to traces of endo-H. There i s , however, a greater degree of confidence that homogenous endo-H decreases i n a c t i v i t y by 10,000 f o l d i n going from l i n e a r (Man)3(GlcNAc)2Asn to (Man)2(GlcNAc)2Asn. These recent s p e c i f i c i t y s t u d i e s suggest the need for c a u t i o n i n e s t i m a t i n g o l i g o s a c c h a r i d e s i z e based on the s e n s i t i v i t y or r e s i s t a n c e of glycopeptides to endo-H. Following our c h a r a c t e r i z a t i o n of the p r o p e r t i e s of endo-H (2, 6, 15) and that of endo-D by Koide and Muramatsu (14) a number of other u s e f u l endoglycosidases have been i d e n t i f i e d . Thus, an endo-3-N-acetyl-galactosaminidas f i l t r a t e s of Diplococcu from E s c h e r i c h i a f r e u n d i i (25), two endo-3-N-acetylglucosaminidases from C l o s t r i d i u m perfringens (26), one with s l i g h t l y d i f f e r ent s p e c i f i c i t y than endo-H, and more r e c e n t l y an amidase from almond emulsin (27). The amidase, although not p u r i f i e d to a s i g n i f i c a n t extent, possesses the i n t r i g u i n g property of r e l e a s i n g o l i g o s a c c h a r i d e s from a stem bromelein glycopeptide by h y d r o l y z i n g the glucosaminyl asparagine bond. If t h i s enzyme i s more gener a l l y a p p l i c a b l e , i t should be extremely u s e f u l i n the study of glycopeptides and p o s s i b l y g l y o c p r o t e i n s , but not enough i n known about i t s s p e c i f i c i t y at the present time. As far as the existence of an animal endoglycosidase with p r o p e r t i e s s i m i l a r to those of endo-H or endo-D, i t s presence can be i n f e r r e d from the s t r u c t u r e of o l i g o s a c c h a r i d e s excreted i n the u r i n e and present i n the t i s s u e s of animals with c e r t a i n lysosomal storage d i s e a s e s . Thus, mannosyl o l i g o s a c c h a r i d e s with a s i n g l e N-acetylglucosamine on the reducing end were found i n the u r i n e and t i s s u e of b l a c k angus c a t t l e (28) and humans (29) a f f l i c t e d with α - m a n n o s i d o s i s . O l i g o s a c c h a r i d e s c o n s i s t i n g of 7 to 10 sugar r e s i d u e s with the complex t e t r a s a c c h a r i d e core of (Man^GlcNAc have a l s o been found i n p a t i e n t s with GM]^ or GM2 g a n g l i o s i d o s i s (30, 31, 32), and f i b r o b l a s t s from p a t i e n t s with α - f u c o s i d o s i s (32) apparently accumulate the d i s a c c h a r i d e Fucal-*6GlcNAc, a d i s a c c h a r i d e found at the reducing end of many "complex" type oligosaccharides. Whether these products accumulate due to the above s p e c i f i c exoglycosidase d e f i c i e n c i e s combined with cleavages a f f e c t e d by a s i n g l e endoglycosidase i s not known, but we have described an enzyme i n animal t i s s u e s p a r t i a l l y capable of e x p l a i n i n g these f i n d i n g s (33). The enzyme was p u r i f i e d over 1,500 f o l d from hen-oviduct and as shown i n F i g u r e 12 hydrolyzed [ l ^ C ] - N - a c e t y l a t e d g l y c o s y l - A s n d e r i v a t i v e s derived from ovalbumin i n a manner s i m i l a r to that described for endo-H. As an added bonus, t h i s enzyme p r e p a r a t i o n


MALEY ET AL.

Endo-β-acetylglucosaminidases

Manal - ^ 6 Μ β η β 1 ^ » 4 G 1 c N A c 0 1

•>

4G1 c N A c - A s n

10.

b) H

+

Manal , ^ 6 Manal

^

Manal ^

ManÊl Manal

Manal

4G1 cNAcS1

4G1 c N A c - A s n

^

r

6Manal

a-Mannosidase

Man 31
-Gal

+ Neu Ν A c

GANGLIOSIDOSIS

GANGLIOSIDOSIS

Gal

+ Gal Ν Ac

IN

+

IN

catabolism in GM-l-gangliosidosis, Gauchers Disease

Cer

Cer-GIc

Cer-Glc-Gal

NeuNAc

J 1 i— I

Cer-Glc-Gal

-BLOCKED

— I

-NeuNAc

I

Cer-Glc-Gal-GalNAc

•

2)NeuNAc

1

C e r ( 1 « — 1 . β ) G l c ( 4 « - 1 . β) G a l ( 4 « - 1 , ^ g ) G a l Ν A c ( 3 * - 1 . β)

S «s:

Ci

Ο > w Η

142


Fuc F u c - G a l - GlcNAc - M a n

1

M a n - G l c N A c —GlcNAc - A s n Fuc-Gal - GlcNAc-Man A

^ (Endo-0-N-glucosaminidase)

^

Fuc-Gal-GlcNAc-Man

Fuc \ Man —GlcNAc

I GlcNAc-Asn

+

Fuc- Gal-GlcN Ac-Man Β Gal — G l c N A c —Man ΓM a n — G l c N A c

+

Fuc

Gal — G l c N A c — M a n ' C

\

G l c N A c - A s n + Fuc N-aspartyl-

^

glucosaminidase

( β-Galactosidase)

GlcNAc

+ Asn

GlcNAc — Man

\

G l c N A c —Man

/

Man —GlcNAc

+ Gal

( β-hexosaminidase)

i

Man, Man

>

Man

(Q£

1

-GlcNAc

Man

GlcNAc

mannosidase)

I an —GlcNAc M

(β

+

+ Man

mannosidase) +

GlcNAc

Figure 2. Probable catabolic pathway for the degradation of oligosaccharide chains derived from brain glycoproteins. Reactions B, C, D, and Ε are blocked in fucosidosis, GMl-gangliosidosis, GM2-gangliosidosis, and mannosidosis, respec tively.


7.

BRUNNGRABER

Lysosomal

Enzyme

Deficiency

Diseases

143

Gaucher*s Disease ( d e f i c i e n c y of glucocerebrosidase) Whether p a r t i a l degradation products derived from g l y c o p r o t e i n s accumulate i n the b r a i n of the neuropathic forms of Gaucher s disease i s not known. Kanfer et a l (34) t r e a t e d de f a t t e d l i v e r t i s s u e with p r o t e o l y t i c enzymes. Glycopeptides d e r i v e d from g l y c o p r o t e i n s were markedly elevated i n the Gaucher l i v e r ; a 20 f o l d e l e v a t i o n i n hexose, hexosamine, fucose and NeuNAc was recorded. The accumulated m a t e r i a l contained Gal and GlcNAc as the major sugars; Glc and Man were present i n t r a c e s . The disease i s caused by d e f i c i t i n glucocerebrosidase, a 3-glucosidase. Although glucose-containing g l y c o p r o t e i n s un doubtedly e x i s t (1), there i s no evidence f o r the accumulation of G l c - c o n t a i n i n g glycopeptides or o l i g o s a c c h a r i d e s . Gaucher spleen appears to produce an excess of a g l y c o p r o t e i n a c t i v a t i n g f a c t o r , capable of a c t i v a t i n g the glucocerebrosidase from normal spieen (35-37)· While the a c t i v a t o r from Gaucher spleen was a g l y c o p r o t e i n , that fro composition of the two of the g l y c o p r o t e i n a c t i v a t o r remains obscure. Metachromatic Leukodystrophy (MLD) and M u l t i p l e S u l f a t a s e D e f i c i e n c y (MSP). 1

The s i a l o g l y c o p e p t i d e p r e p a r a t i o n from b r a i n contains mole cules which c a r r y s u l f a t e - e s t e r groups. Consequently, i t ap peared l i k e l y that p a r t i a l degradation products derived from g l y c o p r o t e i n s which bear s u l f a t e d o l i g o s a c c h a r i d e chains might accumulate i n MLD, a disease caused by the absence of a f u n c t i o n ing s u l f a t a s e A. However, s u l f a t i d e s are the n a t u r a l substrate f o r t h i s enzyme. The hexosamine l e v e l s i n the d e l i p i d a t e d t i s s u e residue from cases of MLD, i n which i t was e s t a b l i s h e d that s u l f a t a s e A i s d e f i c i e n t , f e l l w i t h i n the normal range (39,40). The s i t u a t i o n d i f f e r s i n MSD, a v a r i a n t of MLD i n which the t i s s u e s of the p a t i e n t s l a c k s u l f a t a s e s A, B, and C. The non l i p i d hexosamine i n t i s s u e s from such p a t i e n t s show an e l e v a t i o n of protein-bound hexosamine (41), and i t has been shown that t h i s i s due, at l e a s t i n p a r t , by the e l e v a t i o n of glycosaminoglycans· I t i s not known whether glycopeptides or o l i g o s a c c h a r i d e s de r i v e d from g l y c o p r o t e i n s accumulate i n t h i s d i s e a s e . I t has been suggested (42) that O-sulfated glycosaminoglycans are the n a t u r a l substrate f o r s u l f a t a s e B, s i n c e t h i s enzyme i s d e f i c i e n t i n two diseases (MSD and Maroteaux-Lamy disease) i n which O-sulfated glycosaminoglycans accumulate (mainly dermatan s u l f a t e ) . However, c o - c u l t i v a t i o n of MSD f i b r o b l a s t s with S a n f i l i p p o A or Hunter c e l l s d i d not c o r r e c t f o r the degradation defect i n glycosaminoglycan-SO/j-S metabolism. S a n f i l i p p o and Hunter f i b r o b l a s t s l a c k heparan s u l f a t a s e and dermatan s u l f a t a s e , but a r y l s u l f a t a s e Β i n these c e l l s i s normal or elevated (42). These f i n d i n g s l e d to the suggestion that s u l f a t a s e Β may be i n v o l v e d i n degrading s u l f a t e d glycopeptides (43). 35


144


S u l f a t a s e Β appears to correspond to N-acetylgalactosamine 4 - s u l f a t a s e (44). The s u l f a t a s e r e s p o n s i b l e f o r the accumula t i o n of protein-bound hexosamine s t i l l remains to be i d e n t i f i e d . Nor i s i t yet e s t a b l i s h e d whether s u l f a t e d glycopeptides d e r i v e d from g l y c o p r o t e i n s accumulate i n MSD. The unknown s u l f a t a s e may be a component of s u l f a t a s e A or B, both of which e x i s t i n m u l t i p l e forms, or a s u l f a t a s e which does not respond to the s y n t h e t i c substrates commonly i n use to detect s u l f a t a s e a c t i " vity. The mucopolysaccharidoses. The defects i n H u r l e r , Hunter, and S a n f i l i p p o A and Β syndromes, those forms of the mucopolysaccharidoses i n v o l v i n g severe n e u r o l o g i c a l d e f i c i t s , are caused by nonfunctioning α - i d u r o n i d a s e , s u l f o i d u r o n a t e s u l f a t a s e , heparan-N-S-sulfatase, and Ofr-N-acetylglucosaminidase, r e s p e c t i v e l y . These linkages a r e , as f a r as i s known, absent i n g l y c o p r o t e i n s . Consequently, no a b e r r a t i o n i n g l y c o p r o t e i such a defect been r e p o r t e d b r a i n of a Hunter p a t i e n t showed no remarkable changes ( 4 5 ) · Diseases with a primary l e s i o n i n g l y c o p r o t e i n c a t a b o l i s m . Mannose-rich o l i g o s a c c h a r i d e s accumulate i n the b r a i n of mannosidosis p a t i e n t s (46-48): Man-Man-Gl cNAc? Man-Man-Man-Gl cNAc Man-Man

^Man-GlcNAc

Man' The disease occurs a l s o i n Angus c a l v e s , and the defatted b r a i n t i s s u e residue of the a f f l i c t e d animals showed a 4-6 f o l d e l e v a t i o n i n Man content (49). D e f i c i e n c y of α - m a n n o s i d a s e causes a b l o c k i n the catabolism of the h e t e r o p o l y s a c c h a r i d e c h a i n s : mannose-containing glycopeptides or o l i g o s a c c h a r i d e s are d e r i v e d from the s i a l o g l y c o p e p t i d e trimannoside c o r e , as w e l l as from the mannoglycopeptides· In p a t i e n t s with a d e f i c i e n c y of ot-fucosidase (fucosidosis), f u c o s e - c o n t a i n i n g substances were shown to accumulate i n the d e l i p i d a t e d t i s s u e , i n c l u d i n g that of the b r a i n (50). Aqueous e x t r a c t s of sonicated b r a i n t i s s u e y i e l d e d an o l i g o s a c c h a r i d e (27) : Fuc-Gal-GlcNAc-Man V

Man-GlcNAc Fuc-Gal-GfccNAc-Man^


7.

BRUNNGRABER

Lysosomal

Enzyme

Deficiency

Diseases

145

A disaccharide, fucosylated N-acetylglucosamine, was also de tected i n the brains of fucosidosis patients (Fig. 2). Aspartylglucosaminuria i s caused by a d e f i c i t i n the enzyme l-aspartamido-3-N-acetylglucosamine amidohydrolase. Aspartylglucosaminylamine and aspartyloligosaccharides accumulate i n organs, including the brain (51,52). The mucolipidoses are caused by a deficiency of α-neuramini dase (53) resulting i n the excessive excretion of s i a l y l o l i g o saccharides. Most of the oligosaccharides isolated had a core consisting of three mannose residues to which two GlcNAc-GalNeuNAc chains, or incomplete portions of these chains, were attached (54). Although not a l l of the linkages i n the oligo saccharides of brain tissue have been definitively established, i t appears l i k e l y that their structure w i l l prove to be identi cal to that of one of the sialoglycopeptides isolated from urine (55): NeuNAc ( α , 2+6) Gal ( β , 1+4)GlcNA

/

Man(β,1+4)GlcNAc

NeuNAc ( α , 2+6 ) Gal ( 3,1+4 ) GlcNAc ( β , 1+2 )Man (α, 1+6 )' A sequence of the steps involved i n the catabolism of the heteropolysaccharides of brain glycoproteins has been postulated (Fig. 2) based on the structures of the oligosaccharides which accumulate i n the various lysosomal enzyme diseases discussed above. Niemann-Pick's Disease The primary lesion i n the i n f a n t i l e , type A, form of Nie mann Pick's disease i s a deficiency of the enzyme, sphingomye linase. While sphingomyelin i s the primary storage product, many cases which have been examined have demonstrated an increase i n gangliosides and cholesterol i n brain tissue. Tingey (16) and Norman et a l (56) have reported an elevation of proteinbound hexosamine as well. Brunngraber et a l (57) noted a two fold elevation of glycoproteins-carbohydrate which was recovered as sialoglycopeptides upon proteolytic digestion of the d e l i p i dated tissue residue from gray matter. Mannoglycopeptide levels remained within the normal range. The composition of the s i a l o glycopeptides was altered, since the number of -GlcNAc-GalNeuNAc chains attached to the internal trimannoside core was reduced. I t i s possible, but not demonstrated, that the accumu lated glycopeptides i n Niemann-Pick disease may correspond to the sialylated forms of the oligosaccharides which accumulate i n the GMl-gangliosidoses. I t was suggested that sphingomyelin catabolism may be an early step i n the degradation of plasma mem branes, and a lesion i n the catabolism of this phospholipid may have the secondary effect of impeding the degradation of other plasma membrane components.


146


Neuronal-Ceroid L i p o f u s c i n o s e s (NCF). A form of NCF may i n v o l v e g l y c o p r o t e i n storage. Adelman et a l (58) found a n e a r l y two f o l d i n c r e a s e i n the NeuNAc con tent of s i a l o g l y c o p e p t i d e p r e p a r a t i o n s obtained from t i s s u e sec t i o n s of the c e r e b r a l c o r t e x of a case with c l i n i c a l f e a t u r e s resembling those of Batten's d i s e a s e . C e l l r e c o g n i t i o n of lysosomal enzymes. Studies u t i l i z i n g c u l t u r e d f i b r o b l a s t s d e r i v e d from p a t i e n t s w i t h lysosomal enzyme d e f i c i e n c y diseases have shown that these c e l l s s e c r e t e lysosomal g l y c o s i d a s e s i n t o the medium, and are capable of r e - a s s i m i l a t i n g these enzymes. Hickman and Neuman (59,60) have proposed t h a t lysosomal enzymes are t r a n s p o r t e d from t h e i r s i t e of s y n t h e s i s and g l y c o s y l a t i o n i n the G o l g i apparatus to the lysosome by t h i s process of e x o c y t o s i s and subsequent p i n o c y t o t i c r e - i n c o r p o r a t i o n . Such a mechanism may be a p a r t of a membrane r e c y c l i n g mechanism s i n c e i n t e r n a l i z a t i o n of the enzyme i s patch of plasma membrane to fuse with the lysosome (61). F i b r o b l a s t s from p a t i e n t s a f f l i c t e d with one of the l y s o s o mal d e f i c i e n c y diseases are capable of a s s i m i l a t i n g the d e f i c i e n t enzyme which had been added to the c u l t u r e medium, thereby c o r r e c t i n g the metabolic d e f e c t . Uptake of lysosomal g l y c o s i dases i s dependent on the r e c o g n i t i o n of the h e t e r o p o l y s a c c h a r i d e c h a i n of the g l y c o p r o t e i n enzyme by a r e c e p t o r on the s u r f a c e of the f i b r o b l a s t . Thus, the t e r m i n a l Gal r e s i d u e appeared to be important f o r the r e c o g n i t i o n and uptake o f a-N-acetylglucosaminidase (62) by f i b r o b l a s t s of S a n f i l i p p o Β p a t i e n t s . Mannose residues appeared to be important f o r uptake of 3-galactosidase by f i b r o b l a s t s from p a t i e n t s with G M l - g a n g l i o s i d o s i s (63). Cultured f i b r o b l a s t s appear to recognize phosphohexosyl groups i n 3-glucuronidase, β-hexosaminidase, and 3-galactosidase (64,65) and a-L-iduronidase (66). The r e c o g n i t i o n of phospho hexosyl groups may be a general c h a r a c t e r i s t i c of p i n o c y t o s i s of lyosomal g l y c o s i d a s e s . These f i n d i n g s may be r e l a t e d to the e a r l i e r r e p o r t s on the i s o l a t i o n of a phosphoglycoprotein from b r a i n (67-69). Phosphorylated mannoglycopeptides were i s o l a t e d from the phosphoglycoprotein, and the phosphate r e s i d u e which i s attached to one of the h y d r o x y l groups of mannose, was removed by the a c t i o n of a l k a l i n e phosphatase (69). The r e l a t i o n of t h i s phosphoglycoprotein to the lysosomal g l y c o s i d a s e s remains to be e s t a b l i s h e d . I f the c a p a c i t y of the f i b r o b l a s t to s e c r e t e and r e - a s s i m i l a t e lysosomal enzymes i s a s p e c i f i c example of a more general phenomena, phosphoglycoproteins and/or g l y c o s i d a s e s may p l a y an important r o l e i n the turnover of the plasma membrane.


7.

BRUNNGRABER

1. 2. 3. 4. 5. 6. 7· 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

Lysosomal

Enzyme

Deficiency

Diseases

147

Literature Cited Brunngraber, E. G.; Hof, H.; Susz, J.; Brown, B. D.; Aro, Α.; and Chang, I. Biochim Biophys Acta, 304; 781, 1973. Krusius, T. FEBS Lett, 66: 86, 1976. Hof, Η. I.; Susz, J. P.; Javaid, J. I.; and Brunngraber, E. G. Neurobiology, 5.: 347, 1975. Krusius, T.; Finne, J.; and Rauvala, H. FEBS Lett, 71: 117, 1976. Brunngraber, E. G. Adv Exptl Med and Biol, 25: 255, 1972. Krusius, T.; and Finne, J. Eur J Biochem, 78: 369, 1977. Finne, J.; Krusius, T.; and Rauvala, H. Biochem Biophys Res Communs, 74: 405, 1977. Javaid, J. I.; Hof, H. I.; and Brunngraber, E. G. Biochim Biophys Acta, 404: 74, 1975. Aronson, Ν. Ν., Jr.; and DeDuve C J Biol Chem 243: 4564, 1968. Opheim, D. J.; and Touster, 0. J Bio Chem, 252: 739, 1977. Tarentino, A. L.; and Maley, F. J Biol Chem, 251: 6537, 1976. Tarentino, A. L.; and Maley, F. Anal Biochem, 77: 185, 1976. Trimble, R. B.; and Maley, F. Biochem Biophys Res Communs, 78: 935, 1977. Kohno, M.; and Yamashina, I. Biochim Bipphys Acta, 258: 600, 1972. Fidler, I. J.; Montgomery, P.C.;and Cesarini, J. P. Cancer Res, 33: 1376, 1973. Tingey, A. J Neurochem, _3 230, 1959. Stefanko, St.; Guminska, M.; and Pietrzykowa, B. Schweiz Arch Neurol Neurochir Psychiat, 90: 295, 1962. Edgar, G. W. F. Proc of the Fifth Internatl Congr Neuro pathology, Zurich, 1965. Amsterdam, Excerpta Med Found, 1966, p. 328. Thieffry, S.; Bertrand, I.; Bargeton, E.; and Edgar, G.W.F. Rev Neurol, 102: 130, 1960. Suzuki, Y.; Jacob, J.C.;Suzuki,K.;Kutty, Κ.N.;and Suzuki, K. Neurology, 21: 313, 1971. Norman, R. M.; Tingey, A. H.; Newman, C. H. H.; and Ward, S. P. Arch Dis Child, 39: 634, 1964. Brunngraber, E.G.;Haberland,C.;and Brown, B. D. Brain Res, 38: 151, 1972. Brunngraber, E. G.; Brown, B. D.; and Aro, A. J Neurochem, 22: 125, 1974. Brunngraber, E. G.; Davis, L. G.; Javaid, J. I.; and Berra, B. Adv Exptl Med and Biol,68:31, 1976. Ng Ying Kin, Ν. M. K.; and Wolfe, L. S. Biochem Biophys Res Communs, 59: 837, 1974. Wolfe, L. S.; and Ng Ying Kin, Ν. M. K. Adv Exptl Med and Biol, 68: 15, 1976. Tsay, G.C.;and Dawson, G. J Neurochem, 27: 733, 1976. American Chemicaf Society Library 1155 16th St. N. W. W a shiGlycolipids ngton, D.inC.Disease 200Processes; 36 In Glycoproteins and Walborg, E.; :

ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

148


28. Bearpark, T.; Bouquelet, S.; Fournet, B.; Montreuil, J; Spik, G.; Stirling, J.; Strecker, G. FEBS Lett, 84: 379, 1977. 29. Wolfe, L. S.; Senior, R. G.; and Ng Ying Kin, Ν. M. K. J Biol Chem, 249: 1828, 1974. 30. Ng Ying Kin, Ν. M. K.; and Wolfe, L. S. Biochem Biophys Res Communs, 66: 123, 1975. 31. Brunngraber, E. G.; Berra, B.; and Zambotti, V. FEBS Lett, 34: 350, 1973. 32. Berra, B.; Di Palma, S.; and Brunngraber, E. G. Clin Chim Acta, 57: 301, 1974. 33. Patel, V.; Goebel, H. E.; Watanabe, I.; and Zeman, W. Acta Neuropathol, 30: 155, 1974. 34. Kanfer, J. N.; Stein, M.; and Spielvogel, C. Adv Exptl Med and Biol, 19: 225, 1972. 35. Ho, M. W.; and O'Brien J S Proc Natl Acad Sci US 68: 2810, 1971. 36. Ho, M. W.; O'Brien Biochem J, 131; 173, 1973. 37. Ho, M. W. Biochem J, 136: 721, 1973. 38. Peters, S. P.; Coyle, P.; Coffee, C. J.; Glew, R. H.; Kuhlenschmidt, M. S.; Rosenfeld, L.; and Lee, Y. C. J Biol Chem, 252: 563, 1977. 39. Haberland, C.; Brunngraber, E.G.;Witting, L. Α.; and Daniels, A. Acta Neuropathol, 26: 93, 1973. 40. Federico, Α.: Guazzi, G. C.; and Di Benedetta, C. Eur Neurol, 15: 163, 1977. 41. Eto, Y.; Meier, C.; and Herschkowitz, N. N. J Neurochem, 27;1071,1976. 42. Austin, J. Arch Neurol, 28: 258, 1973. 43. Eto, Y.; Wiesmann, U.; and Hierschkowitz, Ν. N. Arch Neurol, 30: 153, 1974. 44. Dorfman, Α.; Arbogast, B.; and Matalon, R. Adv Exptl Med and Biol, 68: 261, 1976. 45. Roukema, P. Α.; Overkerk, C. E. and Van Den Berg, G. Clin Chim Acta, 44: 277, 1973. 46. Norden, Ν. E.; Lundblad, Α.; Svensson, S.; Ockerman, P. Α.; and Autio, S. J Biol Chem, 248: 6210, 1973. 47. Norden, Ν. E.; Lundblad, Α.; Svensson, S.; and Autio, S. Biochemistry, 13: 871, 1974. 48. Tsay, G. C.; Dawson,G.;and Matalon, R. J Pediat, 84: 865, 1974. 49. Norden, Ν. E.; Lundblad, Α.; Ockerman, P. A. and Jolly, R. D. FEBS Lett, 35: 209, 1973. 50. Van Eoff, F.; and Eers, E. G. Eur J Biochem, 7: 34, 1968. 51. Palo, J.; and Savolainen, E. J Chromatog, 65: 447, 1972. 52. Palo, J. and Savolainen, E. Ann Clin Res, 5: 156, 1973. 53. Cantz, M.; Gehler, J.; and Spranger, J. W. Biochem Bipphys Res Communs, 74: 732, 1977.


7. BRUNNGRABER

149 Lysosomal Enzyme Deficiency Diseases

54. Michalski, J. C.; Strecker, G.; and Fournet, B. FEBS Lett, 79: 101, 1977. 55. Strecker, G.; Peers, M. C.; Michalski, J. C.; Hondi-Assah, T.; Fournet, B.; Spik, G.; Montreuil, J.; Farriaux, J. P.; Maroteaux, P.; and Durand, P. Eur J Biochem, 75: 391, 1977. 56. Norman, R. M.; Tingey, A. H.; and Fowler, M. C. Proc of the Fifth Internatl Congr Neuropathol, Zurich, 1965, Amster dam, Excerpta Med Found, 1966, p. 143. 57. Brunngraber, E. G.; Berra, B.; and Zambotti, V. Clin Chim Acta, 48: 173, 1973. 58. Adelman, L. S.; Young, E.; and Bass, Ν. H. Neurology, 24: 1045, 1974. 59. Hickman, S.; and Neufeld, E. F. Biochem Biophys Res Communs, 49: 992, 1972. 60. Neufeld, E. F.; Sando, G. N.; Sarvin, A. J.; and Rome, L. H. J Supramol Struct 6: 95 1977 61. Brunngraber, E. G Springfield, Ill. , 62. Von Figura, Κ.; and Kresse, H. Prot Biol Fluids, 22: 275, 1975. 63. Hieber, V.; Distler, J.; Myerowitz, R.; Schmickel, R. D.; and Jourdian, G. W. Biochem Biophys Res Communs, 73: 710, 1966. 64. Kaplan, Α.; Fischer, D.; Achord, D.; and Sly, W. J Clin Invest, 60: 1088, 1977. 65. Kaplan, Α.; Achord, D. T.; and Sly, W. S. Proc Natl Acad Sci US, 74: 2026, 1977. 66. Sando, G. N.; and Neufeld, E. F. Cell, 12: 619, 1977. 67. Davis, L. G.; Javaid, J. I.; and Brunngraber, E. G. FEBS Lett, 65: 30, 1976. 68. Davis, L. G.; Costello, A. J. R.; Javaid, J. I.; and Brunngraber, E. G. FEBS Lett, 65: 35, 1976. 69. Davis, L. G.; Brettschneider, I.; and Brunngraber, E. G. Fed Proc, 36: 750, 1977. RECEIVED

March 15, 1978.


8 Incorporation of Exogenous Enzymes into Lysosomes A Theoretical and Practical Means for Correcting Lysosomal Blockage P. G. PENTCHEV, J.A. BARRANGER, A. E. GAL, F. S. FURBISH, and R. O. BRADY Developmental and Metabolic Neurology Branch, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, MD 20014

The lysosomal storag types for delineating th inherited metabolic disorders. Progress has led to an identification of most primary enzymatic lesions in these disorders and to a better understanding of the cellular manifestations resulting from these enzymopathies. Specific enzymatic tests are now available for the unequivocal diagnosis of most of these storage diseases. Reliable heterozygote detection and prenatal diagnosis has enabled genetic counseling to effectively reduce the incidence of many of these crippling diseases (1). Investigators have also addressed considerable effort to the intriguing possibility of enzyme replacement as an effective therapeutic measure for the treatment of the lysosomal storage disorders. Although more than a decade of effort has been channelled in this direction, an unequivocal answer has yet to appear. An evaluation of the status of this research program can perhaps be best initiated by examining the cellular and biochemical bases for enzyme replacement. Remarkable insight into this phenomena was given in 1964, at a time when only one lysosomal storage disorder was recognized: "Both in our pathogic speculations and in our therapeutic attempts it may be well to keep in mind that any substance which is taken up intracellularly by an endocytic process is likely to end up within lysosomes. This obviously opens up many possibilities for interaction including replacement therapy."(2). The implication of the dynamic nature of lysosomal interactions is that one can, in theory, equate the ability of endogenous and exogenous enzymes to stimulate intralysosomal digestion (Figure I). It is now well documented that enzymes that are initially found in the extracellular environment can be incorporated into the lysosomal architecture of cells (Table I). It has 150 This chapter not subject to U.S. copyright Published 1978 American Chemical Society In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

PENTCHEV ET AL.

Lysosomal

PRIMARY L Y S O S O M E

Blockage

AUT0PHAG0S0ME

ENZ

RESIDUAL B O D Y

SECONDARY LYSOSOME

-Λ /

PHAGOSOME

ENDOCYTOSIS

PRIMARY L Y S O S O M E

AUTOPHAGOSOME

r

/

/ •

SECONDARY L Y S O S O M E ,

PHAGOSOME

ÊNZ

RESIDUAL B O D Y

Λ

ENDOCYTOSIS

Figure 1. Representation of lysosomal interactions. (Top) Nor mal lysosomal digestion. (Bottom) Lysosomal digestion stimuhited by exogenous enzyme.


152


also been shown that these "migrant" enzymes are functionally active in reversing previously blocked metabolic pathways and/or reversing abnormal morphology of lysosomes (Table II). In 1966, i t was predicted that Gaucher^ disease might prove to be the ideal model for enzyme replacement therapy (]_5). The accumulation of glycolipid occurs predominantly within the cells of reticuloendethelial system. Consequently intravenously admin istered enzyme preparations were expected to have ready access to major pathological depots of stored l i p i d . Our enzyme infusion studies with Gaucher patients began in 1973 with the successful isolation of human placental glucocerebrosidase (1_6). A brief summary of the results of these i n i t i a l t r i a l s is presented in Table III. Encouraged by these preliminary findings and having developed a more efficient and practical means of purifying large quantities of the enzyme (1_8), we began a series of infusions in patients using considerably more glucocerebrosidase than i n i t i a l l y employed. The results o ing. No significant changes in either the circulating or l i v e r levels of glycolipid were observed following the intravenous ad ministration of native glucocerebrosidase (Brady, R. et al_., un published observations). Concomittant c l i n i c a l observations such as regression of hepatomegaly also prove negative. On the other hand, Belchetz and collaborators (1_9) and Beutler's group (20) have reported a decrease in l i v e r size when glucocerebrosidase was administered over relatively long periods of time (13 and 3-1/2 months respectively). Furthermore, Belchetz, et al_., injected glucocerebrosidase that had been entrapped in liposomes while Beutler, et al^., used resealed erythrocyte ghosts to deliver the placental enzyme. A possible reason for the refractiveness of biochemical and c l i n i c a l responses to exogenous enzyme in our t r i a l s with the new glucocerebrosidase preparations may be an unfavorable cellular disposition of the enzyme. It has been shown that approximately 30% of the infused enzyme is taken up by the l i v e r in rats and monkeys (5). More recent studies have shown that approximately 90% of this uptake represents incorpora tion into hepatocytes ( 2 Π ) . The predominate uptake of enzyme by hepatocytes is an inefficient route for inducing l i p i d clearance since the accumulation of glycolipid occurs mainly within reticuloendothetial cells (22J. Modification and/or encapsulation of the enzymes will now have to be seriously considered in an attempt to specifically target the administered enzyme to these c e l l s . The eventual effectiveness of enzyme supplementation in the treatment of Gaucher's disease will also depend on several other parameters which unfortunately have not been experimentally evalu ated due to the lack of a suitable animal model (Table IV). To begin with, the inherent extent of enzymatically induced clearance will depend on the degree of accessibility of exogenous enzymes to stored l i p i d . Complete clearance of glucocerebroside can be effected when specimens of Gaucher l i v e r tissue are incubated with glucocerebrosidase in vitro (Figure II). However, the in situ


8.

PENTCHEV ET AL. Table I.

Lysosomal

Blockage

Demonstrated Lysosomal Uptake of Exogenously Administered Enzymes

Enzyme

Reference

Tissue

3-Glucuronidase

Cultured

a-Glucosidase

Rat Liver

(4)

Glucocerebrosidase

Rhesus Monkey Liver

(5)

Hexosaminidase

Rat Liver

(6)

Table II.

Fibroblasts

(3)

Experimental Models for Correction of Lysosomal Accumulation with Exogenous Enzymes

Effect

Reference No.

Invertase and 3-glucosidase

Reversal of Sucrose and cellobiose accumulation in macrophases

7

Deficient lysosomal hydrolases:

Metabolic correction in patients' fibroblasts:

Mucopolysacchari dases

Mucopolysacchari doses

8

Arylsulfatase-A

Metachromatic leukodystrophy

9

Acid lipase

Wolman's Disease

10

ofr-Galactosidase

Fabry's Disease

11

3-N-Acetylhexosaminidase

Sandhoff Disease

Enzyme Addition

B. Invertase

Dextranase

12

Animals

Prevention of hepatic sucrose accumulation in rat

13

Reversal of hepatic dextran accumulation in rat

14


154


Table III.

Initial Results of Enzyme Replacement Trials in Gaucher's Disease (17)

1.

Intravenous infusions of highly purified human placental glucocerebrosidase were well tolerated by patients with no untoward immunological or pyrogenic response.

2.

Exogenous enzyme tion.

3.

A clearance of the elevated glycolipid levels was observed in the blood stream.

4.

Following enzyme infusion, lower levels of glycolipid were found in l i v e r biopsy samples.

Table IV.

Assessment of Lysosomal Responses in Evaluating the Effectiveness of Enzyme Replacement for the Treatment of Storage Disorders

1.

The extent of enzymatically induced clearance of stored material.

2.

The rate at which stored material

3.

The degree of lysosomal overloading in relationship to c l i n i c a l and pathological manifestations.

4.

The c l i n i c a l consequence of reversing intralysosomal accumulation.

accumulates.


PENTCHEV ET AL.

Lysosomal

Blockage

Figure 2. Clearance of glucocerebroside from Gaucher liver tissue by exogenous glucocerebrosidase in vitro. Fresh liver needle biopsy samples from a Gaucher patient were incubated in phosphate-buffered saline at pH = 6.0 for 12 hr at 37°C without and with 1000 units of highly purified glucocerebrosidase as indicated. Glucocerebroside in these preparations and in nonincubated samples was subsequently quantitated as previously described (23). Gl I = glucocerebroside; Cer-ceramide, PE = phosphatidyl ethanolamine.



Adult

Adult

Adult

F

F

M

M

F

F

F

F

F

3

4

5

6

7

8

9

10

Π

F&M

Adult

M

2

itrol

Adult

F

1

Adult

Adult

Adult

Adult

Juvenile

Adult

Sex

Patient

Type

Table V.

20-35

21

23

49

39

30

20

30

15

54

15

44

Age

100

29

25

23

23

23

0.03 - 0.07

22.90

17.90

17.80

16.50

16.40

15.40

11.20

-27

4.30

1.63

0.70

0.25

(mg/gram wet t i s s u e )

Liver Glucocerebroside

29

30

21

16

(% of Control)

Residual Glucocerebrosidase A c t i v i t y i n Leukocytes

Glycolipid Accumulation in Livers of Gaucher Patients

8.

PENTCHEV ET AL.

Lysosomal

Blockage

157

accessability of l i p i d may be more restrictive and perhaps a limiting factor. Preliminary studies with mouse l i v e r have shown that residual bodies have a lower tendency to fuse with lysosomes containing exogenous material (24). Secondly, the efficiency and course of enzyme replacement regimens w i l l depend on the degree to which an induced clearance can be sustained. Obviously, a rapid rate of accumulation relative to clearance w i l l limit the u t i l i t y of this form of treatment. It may be useful to attempt to stabilize the exogenous enzyme in order to prolong its intralysosomal digestive effect. The h a l f - l i f e of human glucocerebrosidase in the l i v e r of rats and Rhesus monkeys is approximately 8 hours (5). Generalized hypothermia (33°C) extends this halfl i f e to over thirty hours (5). Lipids have also been shown to stabilize the enzyme in vitro (Furbish, F. and Brady, R., unpublished observations). Stabilization and protection of the enzyme may also be achieved by cross-linking or binding to matrices (25). Thirdly, the progressio c l i n i c a l disease states remains obscure and speculative. The v a r i a b i l i t y of biochemical and c l i n i c a l manifestations in Gaucher's is i l l u s t r a t i v e of this gap in our knowledge. The range of elevated glucocerebroside in the l i v e r of patients with Gaucher's disease may vary over 100-fold (Table V). Often l i t t l e correlation was observed between the degree of glucocerebroside accumulation and the severity of c l i n i c a l manifestations. Accordingly, a given level of clearance induced by enzyme supplementation may show considerable variation in terms of c l i n i c a l benefits. F i n a l l y , i t has sometimes been t a c i t l y assumed that an enzymatically induced reversal of lysosomal blockage w i l l restore normality to the effected tissues. As already discussed, the reality of biochemically defined corrections has been well established with various models. However, descriptions of morphological correlates to biochemical correction are rather sparce in the literature. The classic experiments of Cohn et a l . {7) reporting a visual observation of the reversal of lysosomal swelling in the sucrose-loaded macrophages upon the uptake by the cells of invertase remains to date the most descriptive accounting of this phenomena. The complexities of biochemical, morphological and physiological relationships in organisms are enormous. The present status of our knowledge is not sufficient to evaluate potentially corrective or simply preventive roles for enzyme replacement. In conclusion, the r e a l i s t i c application of enzyme replacement as a therapeutic measure for the treatment of lysosomal storage disorders has yet to be established. Although the present state of the art is at a delicate embryonic stage of development, nature has extended a helpful hand in providing intracellular homing mechanisms. The continued creative and persistent efforts of investigators can l i k e l y complement this advantage towards a meaningful realization of this form of therapy.


158


REFERENCES 1.

Brady, R., Metabolism 26: 329 (1977).

2. 3.

DeDuve,C.;Fed. Proc. 23, 1045 (1964). Lagunoff, D., Nicol, D., Prilzl, P., Lab. Invest. 29: 449 (1973).

4.

Huijing, F., Waltuck, B., Whelan, W., in Birth Defects Origi nal Article Series, Enzyme Therapy in Genetic Diseases, Vol. IX, No. 2, p. 191, (1973). Daniel Bergsma, Ed.

5.

Pentchev, P., Kusiak, J., Barranger, J., Furbish, F., Rapoport,S., Massey, J., Brady, R., in Enzymes of Lipid Metabolism, Plenum Press i Press 6. Schlesinger, P., Rodman, J., Frey, M., Lang, S., Stahl, P., Arch. Biochem. and Biophys. 172: 606 (1976). 7.

Cohn, Z., Ehrenreich, B., J. Exp. Med. 129: 201 (1969).

8.

Neufeld, L., Shapiro, L., Ann. Rev. Biochem. 44: 357 (1975).

9.

Porter, M., Fluharty, Α., Kihara, H., Science 172: 1263 (1971).

10. Kyriakides, E., Paul, B., Balint, J., J. Lab. Clin. Med. 80: 810 (1972). 11. Dawson, G., Matalon, R., Li, Y., Pediat. Res. 7: 684 (1973). 12. Cantz, M., Kresse, H., J. Biochem. 47: 581 (1974). 13. Jacques, P., Bruns, G., Abs. 2nd Meet. Fed. Eur. Biochem. Soc. A36 (1978) 14. Colley, M., Ryman, Β., Bioch. and Biophys. Acta. 451: 417 (1976). 15. Brady, R., N. Engl. J. Med. 275: 312 (1966). 16. Pentchev, P., Brady, R., Hibbert, S., Gal, Α., Shapiro, D., J. Biol. Chem. 248: 5256 (1973). 17. Brady, R., Pentchev, P., Gal, Α., Fed. Proc. 34: 1310 (1975). 18. Furbish, F., Blair, H., Shiloach, J., Pentchev, P., Brady, R. Proc. Natl. Acad. Sci. USA 74: 3560 (1977).


8. PENTCHEV ET AL.

Lysosomal Blockage

159

19. Belchetz, P., Crawley, J., Braidman, I., Gregoriadis, G. Lancet ii, 116 (1977). 20. Beutler, E., Dale, G., Guinto, E., Kuhl, W. Proc. Natl. Acad. Sci. USA, 74: 4620 (1977). 21. Furbish, F., Steer, C., Barranger, J., Jones, J., Brady, R. Biochem. Biophys. Res. manuscript submitted. 22. Brady, R., King, F. In Lysosomes and Storage Diseases (1973) H. G. Hers and F. Van Hoof, Editors, Academic Press, New York, p. 381. 23. Brady, R., Pentchev, P., Gal, Α., Hibbert, S., Dekaban, Α., Ν. Engl. J. Med. 291: 989 (1974). 24. Daems, W., in Enzym p. 3, (1974), J. M. Tager, G. S. Hooghwinkel and W. Daems, Editors, North-Holland Publishing Company. 25. Wold, F., in Birth Defects, Original Article Series, Vol. IX, No. 2, Enzyme Therapy in Genetic Diseases, p. 46. (1973). Daniel Bergama, Editor, Williams and Wi1 king Publishers, Baltimore, Md. RECEIVED March 15, 1978.


9 Blood Coagulation—Initiation and Regulation by Limited Proteolysis WALTER KISIEL Department of Biochemistry, University of Washington, Seattle, WA 98195

During the coagulation of blood, a number of plasma glycoprotein zymogens are converted to serine proteases in the presence of calcium ions and phospholipid. These activation reactions do not occur randomly but rather as a series of consecutive zymogen activation reactions where the product of one reaction is the catalyst for the next. The focal point of this cascade-like reaction is the formation of thrombin, the serine protease which converts fibrinogen to fibrin. In vivo, the formation of fibrin is thought to occur on the surface of platelets concurrent with their response to an injury in the vessel wall. Indeed, platelets constitute the primary defense in arresting the bleeding of ruptured blood vessels by their rapid adhesion to extravascular surfaces. The platelet adhesion reaction is followed by a morphological change in the platelet membrane which provides a catalytic phospholipid surface for optimal generation of thrombin. The ensuing fibrin serves to stabilize the hemostatic platelet thrombus by virtue of its mesh-like properties. Following tissue repair and vessel wall regeneration, the hemostatic plug is destabilized by the degradation of the fibrin network by plasmin arising from the fibrinolytic system. The common names, as well as the Roman numeral designations, of the glycoproteins participating in coagulation are listed in Table I. Prothrombin, factor VII, factor IX, factor X, factor XI, factor XII, and prekallikrein exist in plasma as precursors to serine proteases. Also listed in Table I are the coagulation disorders associated with the deficiency of a particular coagulation factor. In the past decade, enormous progress has been made in the isolation and characterization of most of these proteins. The availability of homogeneous preparations of each zymogen has made is possible to establish unambiguously the mechanism whereby it is activated, and its proteolytic specificity following activation. The results of these studies have proven invaluable in our understanding of the molecular events in coagulation, and 0-8412-0452-7/78/47-080-160$05.00/0 ©

1978 A m e r i c a n C h e m i c a l Society


9.

KISIEL

Blood

161

Coagulation

TABLE I The

P r o t e i n s o f Blood

Coagulation

Roman Numeral Des i g n a t i o n

Associated

Common Name

Disorder

Fibrinogen

Factor 1

Afibrinogenemia

Prothrombi n

F a c t o r 11

Prothrombin d e f i c i e n c y

Proaccelerin

Factor V

Parahemophi1ia

Proconvertin

F a c t o r VI1

ProSPCA d e f i c i e n c y

Factor

Hemophi1ia A

Tissue

Factor

Antihemophi1ic Christmas

Factor

F a c t o r IX

Christmas disease ( h e m o p h i l i a B)

Factor

Factor X

Stuart factor deficiem

Plasma T h r o m b o p l a s t i n A n t e c e d e n t (PTA)

F a c t o r XI

PTA

Hageman

F a c t o r XI1

Hageman

Factor

Factor XIII d e f i c i e n c y

Stuart

Factor

VIII

Factor

Fibrin-stabi1izing Factor Prekal1ikrein Factor)

XIII

deficiency trait

(Fletcher

HMW K i n i n o g e n (high molecular weight k i n i nogen, Fitzgerald factor)

Fletcher factor Fitzgerald

disease

trait



162

have g r e a t l y a s s i s t e d t h e r e s e a r c h e r i n f o r m u l a t i n g a more I n t e l l i g e n t approach t o the understanding o f the k i n e t i c s o f the overa l l c o a g u l a t i o n p r o c e s s in vivo. F u r t h e r m o r e , f r o m such s t u d i e s has emerged t h e c o n c e p t t h a t t h e p r i n c i p a l mechanism f o r t h e i n i t i a t i o n , p r o p a g a t i o n and r e g u l a t i o n o f t h e c o a g u l a t i o n process i n v o l v e s t h e s e l e c t i v e e n z y m a t i c c l e a v a g e o f u n i q u e p e p t i d e bonds. In t h i s a r t i c l e , t h e p r i m a r y e m p h a s i s w i l l be p l a c e d on more r e c e n t i n f o r m a t i o n r e g a r d i n g t h e mechanism o f a c t i v a t i o n o f e a c h c o a g u l a t i o n zymogen l e a d i n g t o f i b r i n f o r m a t i o n . In a d d i t i o n , c e r t a i n a c t i v a t e d c o a g u l a t i o n f a c t o r s have been o b s e r v e d t o c a r r y o u t s e c o n d a r y p r o t e o l y t i c r e a c t i o n s i n t h e t e s t t u b e and t h e s e w i l l be p r e s e n t e d and d i s c u s s e d i n r e l a t i o n t o t h e i r p o t e n t i a l r e g u l a t o r y r e l e v a n c e in vivo. I I.

M o l e c u l a r Events i n Blood

Coagulation

Two pathways a p p a r e n t l the s e r i n e p r o t e a s e w h i c pathways have been r e f e r r e d t o a s t h e i n t r i n s i c and e x t r i n s i c c o a g u l a t i o n p a t h w a y s . The i n t r i n s i c pathway i n c l u d e s t h o s e r e a c t i o n s t h a t l e a d t o f a c t o r Xa f o r m a t i o n u t i l i z i n g f a c t o r s p r e s e n t o n l y i n p l a s m a . These f a c t o r s i n c l u d e f a c t o r X I I , f a c t o r X I , f a c t o r IX and f a c t o r V I M . P r e s e n t l y , i t i s not c l e a r whether h i g h m o l e c u l a r w e i g h t k i n i n o g e n and p r e k a l 1 i k r e i n a r e a l s o r e q u i r e d f o r t h i s pathway inasmuch a s p l a s m a l a c k i n g t h e s e p r o t e i n s c l o t s p o o r l y upon e x p e r i m e n t a l l y i n i t i a t i n g t h e i n t r i n s i c s y s t e m . The e x t r i n s i c pathway o f f a c t o r Xa g e n e r a t i o n i n v o l v e s t h e p a r t i c i p a t i o n o f p l a s m a f a c t o r V I I and t i s s u e f a c t o r , a s p e c i f i c l i p o p r o t e i n r e l e a s e d f r o m t i s s u e s u r r o u n d i n g t h e damaged b l o o d vessel w a l l . These pathways c l e a r l y c a n be d i f f e r e n t i a t e d i n t h e t e s t t u b e d e p e n d i n g on t h e i n i t i a t i n g s t i m u l u s p r o v i d e d . Factor Xa formed by e i t h e r t h e i n t r i n s i c o r e x t r i n s i c pathway a p p e a r s biochemically indistinguishable. Following i t s formation, factor Xa t h e n t r i g g e r s a s e q u e n c e o f r e a c t i o n s w h i c h r e s u l t s i n t h e formation of insoluble f i b r i n . I t i s perhaps noteworthy t o m e n t i o n t h a t e a c h o f t h e s e pathways I s t h o u g h t t o be t r i g g e r e d by t h e i r i n i t i a l i n t e r a c t i o n w i t h e l e m e n t s o f t h e v a s c u l a r subendothelium. The r a t e and i n t e n s i t y o f f a c t o r Xa f o r m a t i o n in vivo c o n c e i v a b l y i s d i c t a t e d by t h e pathway f o l l o w e d w h i c h i n t u r n i s r e l a t e d t o t h e s e v e r i t y o f t h e v a s c u l a r trauma. Thus, v a s c u l a r trauma w h i c h i n v o l v e s a b r e a k i n t h e c o n t i n u i t y o f t h e v e s s e l with concomitant release o f t i s s u e f a c t o r necessitates immediate f o r m a t i o n o f f a c t o r Xa w h i c h p r e s u m a b l y o c c u r s v i a e x t r i n s i c clotting. On t h e o t h e r h a n d , r e l a t i v e l y m i l d v a s c u l a r trauma i n w h i c h t h e v e s s e l does n o t l o s e i t s c o n t i n u i t y , may c a l l f o r a r e l a t i v e l y s l o w , more c o n t r o l l e d f o r m a t i o n o f f a c t o r Xa. Under t h e s e c i r c u m s t a n c e s , f a c t o r Xa c o u l d be formed v i a t h e i n t r i n s i c pathway i n i t i a t e d by t h e i n t e r a c t i o n o f f a c t o r X I I w i t h a s u b endothelial collagen surface.


9.

A.

KISIEL

Blood

163

Coagulation

The A c t i v a t i o n o f F a c t o r X by t h e

I n t r i n s i c Pathway

The s e q u e n c e o f e v e n t s w h i c h l e a d t o t h e f o r m a t i o n o f f a c t o r Xa by t h e i n t r i n s i c pathway o f b l o o d c o a g u l a t i o n is schematically r e p r e s e n t e d i n F i g u r e 1, The f i r s t s t e p i n t h e s e c a s c a d i n g r e a c t i o n s i s the a c t i v a t i o n o f f a c t o r X I I . F a c t o r X I I has been i s o l a t e d f r o m human and b o v i n e p l a s m a and c h a r a c t e r i z e d i n r e g a r d t o i t s s i z e , s t r u c t u r e and c o m p o s i t i o n (J_,.2). F a c t o r XII i s o l a t e d f r o m e i t h e r s o u r c e a p p e a r s t o be a s i n g l e - c h a i n glycop r o t e i n w i t h a m o l e c u l a r w e i g h t o f a b o u t 75,000. F a c t o r XI l a h a s a l s o been i s o l a t e d f r o m b o v i n e p l a s m a by F u j i k a w a e t a l . ( 3 ) . B o v i n e f a c t o r XI l a i s composed o f a heavy and a l i g h t c h a i n h e l d t o g e t h e r by d i s u l f i d e bonds. The l i g h t c h a i n h a s a m o l e c u l a r w e i g h t o f 28,000 and c o n t a i n s an a m i n o - t e r m i n a l s e q u e n c e o f Val-Val-Gly-Gly ( 3 ) , The a m i n o - t e r m i n a l s e q u e n c e o f the heavy c h a i n (mol wt 46,000) i s T h r - P r o - P r o - T r p - L y s - and i s i d e n t i c a l t o t h a t o b s e r v e d f o r the s i n g l e - c h a i n The c a r b o x y l - t e r m i n a l residu T h u s , the c o n v e r s i o n o f b o v i n e f a c t o r X I I t o f a c t o r XI l a i s due t o t h e c l e a v a g e o f a s i n g l e i n t e r n a l a r g i n y l - v a l i n e p e p t i d e bond. F a c t o r XI l a e x h i b i t e d a m i d a s e a c t i v i t y t o w a r d arginine-containing s u b s t r a t e s and i s r e a d i l y i n h i b i t e d by t h e s e r i n e p r o t e a s e i n h i b i t o r , d i i s o p r o p y l p h o s p h o f l u o r i d a t e (DFP). T h i s i n h i b i t o r i s bound e x c l u s i v e l y t o t h e l i g h t c h a i n o f f a c t o r XI l a i n d i c a t i n g t h a t the a c t i v e s i t e s e r i n e r e s i d u e i s l o c a t e d i n t h i s c h a i n . Furthermore, the a c t i v e s i t e region i n t h e l i g h t c h a i n o f f a c t o r XI l a c o n t a i n s an A s p - A l a - C y s - G l n - G l y - A s p - S e r - G l y - G l y - s e q u e n c e w h i c h i s t y p i c a l o f the o t h e r p l a s m a s e r i n e p r o t e a s e s ( 2 ) . The mechanism whereby f a c t o r X I I i s a c t i v a t e d p h y s i o l o g i c a l l y i s p r e s e n t l y n o t c l e a r . M o r e o v e r , the p r o t e a s e ( s ) w h i c h c a r r i e s o u t t h i s r e a c t i o n has n o t been i d e n t i f i e d . The a c t i v a t i o n o f f a c t o r X I I h a s been t h o u g h t t o o c c u r in vivo f o l l o w i n g i t s i n t e r a c t i o n with a charged s u r f a c e which presumably i s s u b e n d o t h e l i a l collagen. The a c t i v a t i o n o f f a c t o r X I I has been i n v e s t i g a t e d i n the t e s t tube u t i l i z i n g g l a s s o r k a o l i n as the n e g a t i v e l y charged surface. I n c u b a t i o n o f s i n g l e - c h a i n f a c t o r XII w i t h k a o l i n prod u c e s a [ f a c t o r X l l - k a o l i n ] c o m p l e x w h i c h does n o t have e n z y m a t i c a c t i v i t y towards small s u b s t r a t e s . In a d d i t i o n , t h i s c o m p l e x i s not i n h i b i t e d by DFP s u g g e s t i n g t h a t f a c t o r X I I i s n o t c o n v e r t e d t o an a c t i v e enzyme s i m p l y by i t s i n t e r a c t i o n w i t h t h e k a o l i n surface. However, t h e [ f a c t o r X l l - k a o l i n ] c o m p l e x w i l l react w i t h f a c t o r XI and p r e k a l 1 i k r e i n c o n v e r t i n g t h e s e zymogens t o a c t i v e enzymes ( 3 ) . Whether f a c t o r X I I was c o n v e r t e d t o f a c t o r XI l a d u r i n g t h i s i n c u b a t i o n i s n o t known. G r i f f i n and C o c h r a n e (4) and Chan et^ aj_. (5) have r e p o r t e d t h a t human f a c t o r X I I i s a c t i v a t e d i n t h e p r e s e n c e o f k a i l i k r e i n , HMW k i n i n o g e n and k a o l i n . These s t u d i e s s u g g e s t t h a t f a c t o r X I I i n t e r a c t s s t o i c h i o m e t r i c a l l y w i t h HMW k i n i n o g e n on t h e k a o l i n s u r f a c e and i s s u b s e q u e n t l y a c t i v a t e d by k a l l i k r e i n . From t h i s i n f o r m a t i o n , i t i s c o n c e i v a b l e t h a t upon i n t e r a c t i o n w i t h a


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c h a r g e d s u r f a c e i n the p r e s e n c e o f HMW k i n i n o g e n , f a c t o r X\\ u n d e r g o e s a c o n f o r m a t i o n a l change w h i c h p e r m i t s i t s a c t i v e s i t e t o r e a c t s l o w l y and p r e f e r e n t i a l l y w i t h p r e k a l 1 i k r e i n . The r e l a t i v e l y s m a l l amount o f k a l l i k r e i n formed can r e c i p r o c a l l y a c t i v a t e s u b s t a n t i a l amounts o f f a c t o r X I f . Once formed i n s u f f i c i e n t l e v e l s , f a c t o r XI l a c a n * t h e n c a t a l y z e t h e a c t i v a t i o n o f t h e n e x t zymogen i n t h e c a s c a d e , namely f a c t o r X I . F a c t o r XI i s c o n v e r t e d t o f a c t o r X l a by f a c t o r XI l a , a r e a c t i o n a p p a r e n t l y augmented by HMW k i n i n o g e n and k a o l i n ( 6 ) . F a c t o r XI l a i s p r e s e n t l y t h e o n l y c o a g u l a t i o n p r o t e a s e known w h i c h can a c t i v a t e f a c t o r XI i n t h e t e s t t u b e . The a p p a r e n t o b l i g a t o r y r o l e o f f a c t o r XI l a i n the a c t i v a t i o n o f f a c t o r XI i s i n t e r e s t i n g i n v i e w o f the f a c t t h a t p a t i e n t s w i t h f a c t o r X I I d e f i c i e n c y have no d e m o n s t r a t i v e b l e e d i n g t e n d e n c i e s , y e t p a t i e n t s w i t h f a c t o r XI d e f i c i e n c y o f t e n b l e e d c o n s i d e r a b l y f o l l o w i n g i n j u r y or minor s u r g e r y . On possibl explanatio fo thi phe nomenon i s t h a t f a c t o r X I l - d e f i c i e n l e v e l s o f f a c t o r XII t coagulation g coagu l a t i o n p r o c e s s i s p r o p a g a t e d by an a m p l i f i c a t i o n mechanism, the s m a l l e s t l e v e l o f enzyme r e q u i r e d w o u l d be i n t h e i n i t i a l r e actions. An a l t e r n a t i v e e x p l a n a t i o n i s t h a t o t h e r h e r e t o f o r e unr e c o g n i z e d f a c t o r s may p l a y a m a j o r r o l e i n the p h y s i o l o g i c a l a c t i v a t i o n of f a c t o r XI, In t h i s c a s e , p r o t e o l y t i c c l e a v a g e o f f a c t o r XI may be a s e c o n d a r y r e a c t i o n c a t a l y z e d by f a c t o r XI l a and t h a t o t h e r p r o t e i n s s u c h as p r e k a 1 1 i k r e i n and p l a s m i n o g e n p r o a c t i v a t o r c o n s t i t u t e more s p e c i f i c p r i m a r y s u b s t r a t e s for f a c t o r XI l a . F a c t o r XI has been i s o l a t e d f r o m human and b o v i n e p l a s m a . Human f a c t o r XI i s a g l y c o p r o t e i n w i t h a m o l e c u l a r w e i g h t o f a b o u t 124,000. I t i s composed o f two i d e n t i c a l p o l y p e p t i d e c h a i n s (mol wt a b o u t 60,000) h e l d t o g e t h e r by d i s u l f i d e bonds. D u r i n g t h e a c t i v a t i o n o f f a c t o r XI an i n t e r n a l p e p t i d e bond i n e a c h o f the two c h a i n s i s c l e a v e d by f a c t o r XI l a ( 7 ) . T h i s g i v e s r i s e t o two heavy c h a i n s (mol wt 35,000) and two l i g h t c h a i n s (mol wt 25,000) in f a c t o r X l a . The a m i n o - t e r m i n a l amino a c i d s e q u e n c e o f the l i g h t chains in f a c t o r Xla i s I l e - V a l - G l y - G l y . T h i s sequence shows homology w i t h o t h e r p l a s m a s e r i n e p r o t e a s e s i n v o l v e d i n coagulation. The l i g h t c h a i n s a l s o c o n t a i n the a c t i v e s i t e s e r i n e w h i c h r e a c t s q u a n t i t a t i v e l y w i t h DFP. Thus, f a c t o r X l a i s a s e r i n e p r o t e a s e c o n t a i n i n g two a c t i v e s i t e s per m o l e c u l e o f enzyme, and r e p r e s e n t s t h e f i r s t c a s e o f a s e r i n e p r o t e a s e w i t h two c a t a l y t i c s i t e s ( 7 ) . The n e x t zymogen t o be a c t i v a t e d i n t h e i n t r i n s i c pathway i s f a c t o r IX. Human and b o v i n e f a c t o r IX have been i s o l a t e d and w e l l c h a r a c t e r i z e d i n t h e p a s t few y e a r s (8^,9). B o v i n e f a c t o r IX i s a s i n g l e - c h a i n g l y c o p r o t e i n w i t h a m o l e c u l a r w e i g h t o f 55,000. Human f a c t o r IX i s a l s o a g l y c o p r o t e i n w i t h a m o l e c u l a r w e i g h t o f 57,000 and p o s s e s s e s an a m i n o - t e r m i n a l amino a c i d s e q u e n c e n e a r l y i d e n t i c a l t o t h a t o f b o v i n e f a c t o r IX ( 9 ) .


9.

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Coagulation

165

F a c t o r IX i s one o f t h e f o u r c o a g u l a t i o n f a c t o r s w h i c h r e q u i r e v i t a m i n K f o r i t s b i o s y n t h e s i s . The o t h e r f a c t o r s i n c l u d e p r o t h r o m b i n , f a c t o r X and f a c t o r Vitamin K p a r t i c i p a t e s i n the b i o s y n t h e s i s o f these p r o t e i n s i n t h e post-ribosoma1 carboxyl a t i o n o f s p e c i f i c g l u t a m i c a c i d r e s i d u e s p r e s e n t i n t h e aminoterminal p o r t i o n o f these p r o t e i n s (10). The new amino a c i d , y-carboxyglutamic a c i d , i s e s s e n t i a l f o r c a l c i u m i o n b i n d i n g by t h e s e p r o t e i n s and t h e i r s u b s e q u e n t I n t e r a c t i o n w i t h a c i d i c p h o s p h o l i p i d membranes. The y - c a r b o x y g l u t a m i c a c i d residues i n these p r o t e i n s tend t o o c c u r as a d j a c e n t r e s i d u e s w h i c h f a c i l i t a t e t h e c o o r d i n a t i o n o f one c a l c i u m i o n p e r p a i r o f y - c a r b o x y g l u t a m i c a c i d r e s i d u e s (JJ_). F a c t o r IX i s a c t i v a t e d by f a c t o r X l a i n t h e p r e s e n c e o f c a l cium ions i n a two-step process. In t h e f i r s t s t e p , s i n g l e - c h a i n f a c t o r IX i s c o n v e r t e d t o a t w o - c h a i n m o l e c u l e by t h e c l e a v a g e o f an i n t e r n a l A r g - A l a p e p t i d a c t i o n has no e n z y m a t i (mol wt 16,000) w i t h an a m i n o - t e r m i n a l sequence o f T y r - A s n - S e r G l y , and a heavy c h a i n (mol wt 38,000) w i t h an a m i n o - t e r m i n a l sequence o f A l a - G l y - T h r - I l e . T h i s i n t e r m e d i a t e i s then c o n v e r t e d t o f a c t o r IXa i n a r a t e - l i m i t i n g s t e p by l i m i t e d p r o t e o l y s i s . In t h i s r e a c t i o n , a s p e c i f i c A r g - V a l bond i n t h e a m i n o - t e r m i n a l r e g i o n o f t h e h e a v y c h a i n i s c l e a v e d by f a c t o r X l a w i t h t h e c o n c o m i t a n t r e l e a s e o f an a c t i v a t i o n p e p t i d e . T h u s , t h e f a c t o r IXa formed i n t h i s r e a c t i o n i s composed o f two c h a i n s h e l d t o g e t h e r by d i s u l f i d e bonds. The heavy c h a i n (mol wt 27,300) c o n t a i n s t h e active s i t e serine residue and c o n t a i n s an a m i n o - t e r m i n a l s e quence o f V a l - V a l - G l y - G l y . The l i g h t c h a i n (mol wt 1 6 , 6 0 0 ) , w h i c h o r i g i n a t e s f r o m t h e a m i n o - t e r m i n u s o f t h e zymogen, c o n t a i n s the y-carboxyglutamic a c i d r e s i d u e s and t h e a m i n o - t e r m i n a l s e quence o f T y r - A s n - S e r - G l y ( l _ 2 ) . F a c t o r IXa i s t h e s e r i n e p r o t e a s e w h i c h c o n v e r t s f a c t o r X t o f a c t o r Xa by l i m i t e d p r o t e o l y s i s i n t h e i n t r i n s i c pathway. In t h i s r e a c t i o n , f a c t o r IXa f o r m s a m a c r o m o l e c u l a r c o m p l e x w i t h an a d d i t i o n a l p r o t e i n , f a c t o r V I I I , i n the presence o f p h o s p h o l i p i d and c a l c i u m i o n s . F a c t o r V I I I i s t h e plasma p r o t e i n t h a t i s a b s e n t i n i n d i v i d u a l s w i t h c l a s s i c h e m o p h i l i a , t h e most p r e v a l e n t c o a g u l a t i o n d i s o r d e r known. F a c t o r V I I I has been i s o l a t e d f r o m human and b o v i n e plasma and e x h i b i t s a m o l e c u l a r w e i g h t o f a b o u t one m i l l i o n (l_3,]_4). These p r e p a r a t i o n s p o s s e s s potent coagulant a c t i v i t y a s w e l l as von W i l l e b r a n d p l a t e l e t a g g r e g a t i n g a c t i v i t y . Treatment o f t h e bovine f a c t o r V I I I p r e p a r a t i o n w i t h c a t a l y t i c amounts o f b o v i n e t h r o m b i n r e s u l t s i n a 2 5 - f o l d i n c r e a s e i n f a c t o r V I I I procoagulant a c t i v i t y i n t h e presence o f CaCl2. Gel f i l t r a t i o n o f t h i s thrombin-treated preparation o f f a c t o r V I M r e s u l t s i n t h e s e p a r a t i o n o f f a c t o r V I M c o a g u l a n t a c t i v i t y and the p l a t e l e t a g g r e g a t i n g a c t i v i t y (l_5). However, no d e t e c t a b l e p r o t e i n was a s s o c i a t e d w i t h t h e f a c t o r V I M c o a g u l a n t a c t i v i t y . T h i s has l e d t o s p e c u l a t i o n t h a t t h e f a c t o r V I M r e p r e s e n t s o n l y


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GLYCOLIPIDS IN

DISEASE PROCESSES

a small f r a c t i o n o f the t o t a l p r o t e i n i n the f a c t o r V M t prepar a t i o n s d e s c r i b e d (15). The r o l e o f f a c t o r V I M in the a c t i v a t i o n o f f a c t o r X appears t o be one o f a r e g u l a t o r y n a t u r e . Conceivably, factor VMI could augment t h i s r e a c t i o n by p r o m o t i n g t h e f o r m a t i o n o f a more r e a c t i v e c o n f o r m a t i o n i n e i t h e r f a c t o r IXa o r i t s s u b s t r a t e , f a c t o r X. The f i n a l s t e p i n t h i s pathway i s t h e a c t i v a t i o n o f f a c t o r X, F a c t o r X i s a g l y c o p r o t e i n w i t h a m o l e c u l a r w e i g h t o f 55,000 and one o f t h e most w e l l c h a r a c t e r i z e d p r o t e t n s i n b l o o d coagulation. I n d e e d , t h e t o t a l amino a c i d s e q u e n c e o f b o v i n e f a c t o r X has been completed (16). i t c o n s i s t s o f a h e a v y c h a i n (mol wt 39,300) and a l i g h t c h a i n (mol wt 16,500) h e l d t o g e t h e r by d i s u l f i d e b o n d s . The h e a v y c h a i n c o n t a i n s 307 amino a c i d r e s i d u e s w i t h two g l y c o s y l m o i e t i e s l i n k e d t o Asn-35 and T h r - 3 0 0 ( 1 6 ) . The heavy c h a i n a l s o c o n t a i n s the a c t i v e s i t serin located I p o s i t i o 233 Th l i g h t c h a i n c o n t a i n s th acid residues. The c o n v e r s i o n o f b o v i n e f a c t o r X t o f a c t o r Xa by f a c t o r IXa o c c u r s as a r e s u l t o f t h e c l e a v a g e o f a s i n g l e p e p t i d e bond between Arg-51 and M e - 5 2 , T h i s r e s u l t s i n t h e r e l e a s e o f an a c t i v a t i o n p e p t i d e (mol wt 10,500) f r o m t h e a m i n o - t e r m i n a l r e g i o n o f the heavy c h a i n a n a l o g o u s t o t h a t o b s e r v e d in the a c t i v a t i o n o f f a c t o r IX. F a c t o r Xa i s a s e r i n e p r o t e a s e w h i c h , i n t h e p r e s e n c e o f f a c t o r V, p h o s p h o l i p i d and c a l c i u m i o n s , c a t a l y z e s t h e c o n version of prothrombin to thrombin. B.

The

Activation

of

F a c t o r X by

the

Extrinsic

Pathway

The p r o p o s e d s e r i e s o f r e a c t i o n s w h i c h r e s u l t i n t h e a c t i v a t i o n o f f a c t o r X by t h e e x t r i n s i c pathway a r e d e p i c t e d i n F i g u r e 2. In t h e s e r e a c t i o n s , p l a s m a f a c t o r V I I , i n t h e p r e s e n c e o f c a l c i u m i o n s , f o r m s a c o m p l e x w i t h a l i p o p r o t e i n r e l e a s e d f r o m damaged tissue. T h i s c o m p l e x s u b s e q u e n t l y c o n v e r t s f a c t o r X t o f a c t o r Xa which appears i n d i s t i n g u i s h a b l e from t h a t observed f o l l o w i n g i n t r i n s i c a c t i v a t i o n o f f a c t o r X (T7). Present evidence suggests that f a c t o r VII i s c o n v e r t e d to f a c t o r V i l a in o r d e r to c a t a l y z e this reaction. The l i p o p r o t e i n s e r v e s as a h i g h m o l e c u l a r w e i g h t c o f a c t o r which i s a b s o l u t e l y e s s e n t i a l f o r the e x p r e s s i o n o f factor Vila proteolytic activity. F a c t o r VII i s a t r a c e plasma g l y c o p r o t e i n . I t s low c o n c e n t r a t i o n has v i r t u a l l y p r e c l u d e d t h e i s o l a t i o n o f t h i s p r o t e i n f r o m human p l a s m a . F a c t o r V I I has been i s o l a t e d f r o m b o v i n e plasma as a s i n g l e - c h a i n p r o t e i n w i t h a m o l e c u l a r w e i g h t o f 45,500 (l_8,l_9). In the t e s t t u b e , s i n g l e - c h a i n f a c t o r V I I can be c o n v e r t e d t o a t w o - c h a i n p r o t e i n w h i c h e x h i b i t s 30-40 t i m e s t h e c o a g u l a n t a c t i v i t y o f t h e s i n g l e - c h a i n f o r m . The a c t i v a t i o n o f f a c t o r V I I , t h a t i s , t h e f o r m a t i o n o f t w o - c h a i n f a c t o r V I I , has been o b s e r v e d f o l l o w i n g t h e i n c u b a t i o n o f f a c t o r V I I w i t h f a c t o r XI l a ( 2 0 ) , f a c t o r X l a ( 2 0 , k a l l i k r e i n ( 2 J J , f a c t o r Xa ( 2 2 ) , and


9.

KISIEL

Blood

167

Coagulation

t h r o m b i n (22). F a c t o r V M a i s composed o f a h e a v y c h g i n (mol wt 34,000) and a l i g h t c h a i n (mol wt 23,000) h e l d t o g e t h e r by d i s u l f i d e bonds. The a c t i v e s i t e s e r i n e i s l o c a t e d In t h e heavy c h a i n which c o n t a i n s t h e a m i n o - t e r m i n a l sequence o f I l e - V a l - G l y Gly (20). The l i g h t c h a i n c o n t a i n s t h e y - c a r b o x y g l u t a m i c a c i d r e s i d u e s and a r i s e s f r o m t h e a m i n o - t e r m i n a l p o r t i o n o f t h e s i n g l e c h a i n zymogen ( 2 0 ) , The c a r b o x y l - t e r m i n a l residue o f the l i g h t chain i s arginlne. Thus, f a c t o r V t l a Is a s e r i n e p r o t e a s e a r i s ing from t h e p r o t e o l y s i s o f a s i n g l e i n t e r n a l A r g - l l e p e p t i d e bond. F a c t o r V i l a has an a b s o l u t e r e q u i r e m e n t f o r t i s s u e f a c t o r i n o r d e r t o c l e a v e t h e A r g - 5 1 - M e - 5 2 p e p t i d e bond In t h e heavy c h a i n o f f a c t o r X, The p h y s i o l o g i c a l mechanism f o r t h e a c t i v a t i o n o f f a c t o r V I I i s p r e s e n t l y n o t known. W h i l e s e v e r a l s e r i n e p r o t e a s e s t h a t p a r t i c i p a t e i n t h e I n t r i n s i c pathway w i l l c o n v e r t f a c t o r V M t o f a c t o r V i l a in vitvo i t i c i p a t e i n theactivatio factor X i stheonly protei t h e p r o t e o l y t i c r a n g e o f f a c t o r V M a has n o t been Investigated, W h i l e c o n s i d e r a b l e i n f o r m a t i o n h a s a c c u m u l a t e d on t h e m o l e c u l a r p r o p e r t i e s o f f a c t o r V M , c o m p a r a t i v e l y l i t t l e i s known r e g a r d i n g i t s i n t e r a c t i o n w i t h p u r i f i e d t i s s u e f a c t o r and t h e i n t e r a c t i o n o f f a c t o r X w i t h t h i s [ f a c t o r V l l - t i s s u e f a c t o r ] complex. The t i s s u e f a c t o r a p o p r o t e i n , t h e p r o t e i n component o f t h e t i s s u e f a c t o r l i p o p r o t e i n , h a s been i s o l a t e d f r o m s e v e r a l t i s s u e s . The most h i g h l y c h a r a c t e r i z e d a p o p r o t e i n p r e p a r a t i o n a p p e a r s t o be o b t a i n e d f r o m human b r a f n m i c r o s o m e s ( 2 3 ) , T i s s u e f a c t o r apop r o t e i n prepared i n t h i s study i s a s i n g l e - c h a i n glycoprotein c o n t a i n i n g a b o u t 6% c a r b o h y d r a t e and e x h i b i t e d a m o l e c u l a r w e i g h t o f 52,000 by SDS g e l e l e c t r o p h o r e s i s . The t i s s u e f a c t o r a p o p r o t e i n e x h i b i t s no c o a g u l a n t a c t i v i t y i n t h e a b s e n c e o f phospholipid. A d d i t i o n o f mixed p h o s p h o l i p i d s t o t h e a p o p r o t e i n , however, p a r t i a l l y restores the tissue factor coagulant a c t i v i t y . The r o l e o f t i s s u e f a c t o r a p p e a r s t o be s i m i l a r t o t h a t o f f a c t o r V I M i n t h a t i t s e r v e s t o enhance t h e l i m i t e d p r o t e o l y s i s o f f a c t o r X by f a c t o r V i l a s e v e r a l o r d e r s o f m a g n i t u d e . No e n z y m a t i c a c t i v i t y has been o b s e r v e d a s s o c i a t e d w i t h homogeneous preparations o f tissue factor. Moreover, i n c u b a t i o n o f pure bovine f a c t o r VII w i t h c r u d e , h i g h l y a c t i v e p r e p a r a t i o n s o f t i s s u e f a c t o r does n o t r e s u l t i n t h e f o r m a t i o n o f t w o - c h a i n , a c t i v a t e d f a c t o r V I I (21_). y

C.

Prothrombin A c t i v a t i o n

by A c t i v a t e d

Factor X

P r o t h r o m b i n i s c o n v e r t e d t o t h r o m b i n by f a c t o r Xa i n t h e p r e s e n c e o f c a l c i u m i o n s , p h o s p h o l i p i d and f a c t o r Va ( F i g u r e 3 ) . P r o t h r o m b i n and i t s mechanism o f a c t i v a t i o n has been i n t e n s i v e l y i n v e s t i g a t e d o v e r t h e l a s t two d e c a d e s , and t h e d e t a i l s o f t h i s complex r e a c t i o n a r e r e a s o n a b l y c l e a r (24). Prothrombin i s a g l y c o p r o t e i n with a molecular weight o f


168


SURFACE HMW KININOGEN PREKALLIKREIN

FACTOR X I I

FACTOR

XII

FACTOR

IX

FACTOR CA

[FACTOR

2 +

,

IX

A

PLj FACTOR

IX -FACTOR 3

VIII

A

2 +

VIII -CA A

-PL]

Figure 1. Abbreviated mechanisms for the activation of factor X by the intrinsic pathway

FACTOR

XII

FACTOR

XI

; ]

; L

KALLIKREIN FACTOR

X

A

THROMBIN

FACTOR V I I

FACTOR ?

+

CA-

Figure 2. Abbreviated mechanisms for the activation of factor X by the extrinsic pathway

[FACTOR V I I

; 1

VII

-CA

T ISSUE 2 +

FACTOR

-TISSUE

FACTOR X

FACTOR X

; L

|

FACTOR

FACTOR]

X

1

:L

C A , PL j FACTOR V 2 +

;L

[FACTOR X^-FACTOR V ,-CA -PL] ;

2+

FACTOR XIII

PROTHROMBIN

Figure 3. Mechanisms for the formation of cross-linked fibrin following the activation of factor X

FIBRINOGEN j

2+

FACTOR XII I

A

FIBRIN (CROSS-LINKED)


9.

KISIEL

Blood

169

Coagulation

70,000. I t i s o n e o f t h e f o u r v i t a m i n K-dependent c l o t t i n g f a c t o r s a n d c o n t a i n s 10 y - c a r b o x y g l u t a m f c acid residues, I t s c o n c e n t r a t i o n i n plasma f a r exceeds t h a t o f any o t h e r c o a g u l a t i o n zymogen. The amino a c i d s e q u e n c e o f b o v i n e p r o t h r o m b i n h a s been d e t e r m i n e d (25) and t h e s e q u e n c e o f human p r o t h r o m b i n Is n e a r i n g completion. B o v i n e p r o t h r o m b i n c o n t a i n s 582 amino a c i d r e s i d u e s w i t h 12 d i s u l f i d e b r i d g e s . The t h r e e c a r b o h y d r a t e c h a i n s o f bovine prothrombin a r e N - g l y c o s f d i c a l l y l i n k e d t o asparagine r e s i d u e s a t p o s i t i o n s 7 7 , 101 and 376 ( 2 5 ) , The c o n v e r s i o n o f p r o t h r o m b i n t o t h r o m b i n o c c u r s a s a t w o s t e p p r o c e s s on t h e s u r f a c e o f a m a c r o m o l e c u l a r a g g r e g a t e commonly r e f e r r e d t o a s t h e " p r o t h r o m b i n a s e c o m p l e x . " T h i s c o m p l e x c o n s i s t s o f f a c t o r X a , f a c t o r V a , p h o s p h o l i p i d and c a l c i u m i o n s . P r e - t r e a t m e n t o f f a c t o r Xa w i t h DFP o b v i a t e s t h e c o n v e r s i o n o f p r o t h r o m b i n t o t h r o m b i n i n d i c a t i n g t h a t f a c t o r Xa i s t h e a c t i v e p r i n c i p a l i n t h i s complex f a c t o r , appears t o f u n c t i o cofactor in this reaction. Its a b i l i t y to potentiate this r e a c t i o n i s i n c r e a s e d s e v e r a l f o l d by p r e - t r e a t m e n t w i t h f a c t o r Xa or thrombin (26). The f i r s t s t e p i n t h e a c t i v a t i o n o f p r o t h r o m b i n i sthe cleavage o f a p e p t i d e bond between A r g - 2 7 4 and T h r - 2 7 5 by f a c t o r X a . T h i s r e a c t i o n r e l e a s e s a l a r g e I n a c t i v e g l y c o p e p t i d e fragment (mol wt 3 3 , 5 0 0 ) f r o m t h e a m i n o - t e r m i n a l r e g i o n o f p r o t h r o m b i n and y i e l d s a s i n g l e - c h a i n t h r o m b o g e n i c p r o t e i n c a l l e d i n t e r m e d i a t e 11, T h i s p r o t e i n h a s no c o a g u l a n t a c t i v i t y a n d h a s a m o l e c u l a r w e i g h t of 39,000. I n t e r m e d i a t e II i s s u b s e q u e n t l y c l e a v e d by f a c t o r Xa a t a p e p t i d e bond between A r g - 3 2 3 a n d l l e - 3 2 4 t o y i e l d two-chain thrombin. T h r o m b i n I s composed o f a heavy c h a i n (mol wt 3 2 , 0 0 0 ) and a l i g h t c h a i n (mol wt 5 , 7 0 0 ) h e l d t o g e t h e r by a d i s u l f i d e bond. The heavy c h a i n o f t h r o m b i n a l s o c o n t a i n s t h e a c t i v e s i t e serine residue. D.

Formation

of Cross-linked

Fibrin

The c o n v e r s i o n o f f i b r i n o g e n t o f i b r i n monomer by t h r o m b i n I s t h e p e n u l t i m a t e s t e p i n t h e b l o o d c o a g u l a t i o n scheme. Fibrinogen i s a l a r g e g l y c o p r o t e i n (mol wt 3 ^ 0 , 0 0 0 ) composed o f t h r e e p a i r s of non-identical polypeptide chains. These c h a i n s , d e s i g n a t e d a , 8 and y , a r e h e l d t o g e t h e r by s e v e r a l d i s u l f i d e bonds. The m o l e c u l a r w e i g h t s f o r t h e a , 8 and y c h a i n s o f human f i b r i n o g e n a r e 6 3 , 5 0 0 , 5 6 , 0 0 0 and 4 7 , 0 0 0 , r e s p e c t i v e l y (2]_). T h u s , t h e s u b u n i t s t r u c t u r e f o r f i b r i n o g e n may be e x p r e s s e d a s a282Y2. During t h econversion o f f i b r i n o g e n t o f i b r i n , four s p e c i f i c a r g i n y l - g l y c i n e p e p t i d e bonds i n t h e a and 8 c h a i n s a r e c l e a v e d by t h r o m b i n w i t h t h e c o n c o m i t a n t r e l e a s e o f f o u r f i b r i n o p e p t i d e s . Two f i b r i n o p e p t i d e s A a r e r e l e a s e d f r o m t h e a m i n o - t e r m l n u s o f t h e a c h a i n s a n d two f i b r i n o p e p t i d e s B a r e r e l e a s e d f r o m t h e amino-terminal end o f t h e 8 c h a i n s . Subsequent t o t h e r e l e a s e o f t h e s e f i b r i n o p e p t I d e s , t h e f i b r i n monomers r a p i d l y a g g r e g a t e


GLYCOPROTEINS AND

170

GLYCOLIPIDS IN DISEASE PROCESSES

i n an o r d e r l y p r o c e s s t o f o r m t h e " s o l u b l e " f i b r i n p o l y m e r . This f i b r i n polymer i s s o l u b l e i n d e n a t u r a n t s such as urea o r g u a n i d i n e - h y d r o c h l o r i d e , and i s r e a d i l y a t t a c k e d by t h e f i b r i n o l y t i c protease, plasmin. C o n v e r s i o n o f t h e " s o l u b l e " f i b r i n p o l y m e r t o an " I n s o l u b l e " f i b r i n c l o t i s t h e terminal r e a c t i o n i n blood c o a g u l a t i o n . This r e a c t i o n i s c a t a l y z e d by a c t i v a t e d f a c t o r X I I I i n t h e p r e s e n c e o f calcium ions. In t h i s r e a c t i o n , i n t e r m o l e c u l a r p e p t i d e c r o s s l i n k a g e s a r e formed by f a c t o r XI I l a between an amino g r o u p f r o m a s p e c i f i c l y s i n e r e s i d u e on o n e f i b r i n monomer and a g l u t a m i n y l r e s i d u e o f an a d j a c e n t f i b r i n monomer ( 2 8 ) . Two e ( y - g l u t a m y l ) l y s i n e p e p t i d e bonds a r e r a p i d l y formed between i n t e r m o l e c u l a r y chains. Upon l o n g e r i n c u b a t i o n , c r o s s l i n k a g e s a r e a l s o o b s e r v e d between i n t e r m o l e c u l a r a c h a i n s . The t o t a l number o f c r o s s l i n k a g e s a p p e a r s t o be a b o u t s i x p e r mole o f f i b r i n , two of which i n v o l v e cross 1inkage w i t chain wit the a c h a i n s . The p r e s e n c f i b r i n polymer which i g q u i t e r e s i s t a n t t o plasmin d i g e s t i o n . F a c t o r XI I l a , t h e t r a n s g l u t a m i n a s e w h i c h c a t a l y z e s t h e c r o s s l i n k i n g r e a c t i o n s i n f i b r i n , c i r c u l a t e s i n plasma i n a p r e c u r s o r form. In t h e p r e s e n c e o f c a l c i u m i o n s , f a c t o r X I I I i s c o n v e r t e d by t h r o m b i n t o f a c t o r XI I l a by l i m i t e d p r o t e o l y s i s . F a c t o r X I I I i s a g l y c o p r o t e i n composed o f two A c h a i n s (mol wt 7 5 , 0 0 0 ) and two B c h a i n s (mol wt 8 8 , 0 0 0 ) . The n a t i v e p r o t e i n i s a t e t r a mer w i t h a m o l e c u l a r w e i g h t o f 3 2 0 , 0 0 0 ( 2 9 ) . Factor XIII i s a c t i v a t e d by t h r o m b i n i n a t w o - s t e p p r o c e s s . In t h e f i r s t s t e p , t h r o m b i n c l e a v e s an a r g i n y l - g l y c i n e bond i n t h e a m i n o - t e r m i n a l r e g i o n o f t h e A c h a i n s , g i v i n g r i s e t o an i n a c t i v e t e t r a m e r i c i n t e r m e d i a t e and two p e p t i d e s (mol wt 4 , 0 0 0 ) . In t h e n e x t s t e p , which r e q u i r e s c a l c i u m i o n s , t h e t e t r a m e r i c intermediate d i s s o c i a t e s i n t o a c a t a l y t i c d i m e r ( A ' 2 ) and an i n a c t i v e d i m e r o f B chains (30). A l t h o u g h t h e A' c h a i n s a r e p r e s u m a b l y i d e n t i c a l , t h e a c t i v e c y s t e i n e r e s i d u e i s f o u n d i n o n l y one o f t h e c a t a l y t i c subunits. T h u s , f a c t o r XI I l a a p p e a r s t o be an e x a m p l e o f t h e " h a l f - o f - t h e - s i t e r e a c t i v i t y " enzymes ( 3 1 ) . E.

Mechanism lation

Involving P r o t e o l y s i s i n theTermination

o f Coagu-

Under normal p h y s i o l o g i c a l c o n d i t i o n s , t h e a c t i v a t i o n o f a zymogen t h r o u g h p r o t e o l y s i s i s e s s e n t i a l l y an i r r e v e r s i b l e p r o cess. A c c o r d i n g l y , s e v e r a l c o n t r o l mechanisms a r e a v a i l a b l e t o r e g u l a t e and t e r m i n a t e c o a g u l a t i o n f r o m s p r e a d i n g t h r o u g h o u t t h e circulation. P e r h a p s t h e p r i n c i p a l mechanism f o r t h e r e g u l a t i o n o f t h e a c t i v a t e d c o a g u l a t i o n f a c t o r s i s t h e i r n e u t r a l i z a t i o n by s e r i n e protease i n h i b i t o r s found i n t h e blood. Indeed, about 10% o f t h e p r o t e i n i n p l a s m a i s s e r i n e p r o t e a s e i n h i b i t o r s ( 3 2 ) . A n o t h e r i m p o r t a n t mechanism f o r t h e c o n t r o l o f c o a g u l a t i o n i n v o l v e s t h e i n a c t i v a t i o n o f s p e c i f i c c l o t t i n g f a c t o r s by l i m i t e d


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proteolysis. Although the d e t a i l s o f these reactions are j u s t b e g i n n i n g t o emerge, i t a p p e a r s t h a t t h r o m b i n i s c e n t r a l t o t h i s process. E x c e s s t h r o m b i n f o r m a t i o n i s c a r e f u l l y c o n t r o l l e d by several negative feedback r e a c t i o n s e i t h e r i n i t i a t e d o r d i r e c t l y c a t a l y z e d by t h r o m b i n . As p o i n t e d o u t e a r l i e r , p r o t e o l y t i c m o d i f i c a t i o n o f f a c t o r V and f a c t o r VI If by t h r o m b i n e n c h a n c e s t h e coagulant a c t i v i t y o f these p r o t e i n s several f o l d . Continued i n c u b a t i o n o f these p r o t e i n s w i t h t h r o m b i n , however, r e s u l t s i n t h e loss o f t h e i r coagulant a c t i v i t y (15,33). Thrombin a l s o c a t a l y z e s t h e r a p i d c l e a v a g e o f a p e p t i d e bond between A r g - 1 5 6 and Ser-157 i n prothrombin g i v i n g r i s e t o a non-thrombogenic fragment (mol wt 23,000) and a t h r o m b o g e n i c i n t e r m e d i a t e I (mol wt 5 5 , 0 0 0 ) . Intermediate I i s loosely associated with the "prothrombinase c o m p l e x " and c o n s e q u e n t l y c l e a v e d by f a c t o r Xa a t a much s l o w e r r a t e than p r o t h r o m b i n . Hence, t h e o v e r a l l e f f e c t i s t o reduce thrombin f o r m a t i o n as a R e c e n t l y , a new v i t a m i c o v e r e d and t e n t a t i v e l y i d e n t i f i e d a s P r o t e i n C ( J 4 J , Protein C i s a g l y c o p r o t e i n composed o f a heavy c h a i n (mol wt 40,000) and a l i g h t c h a i n (mol wt 22,000) h e l d t o g e t h e r by d i s u l f i d e bonds. Incubation o f P r o t e i n C with thrombin r e s u l t s i n the cleavage o f a s i n g l e p e p t i d e bond between A r g - 1 4 and l l e - 1 5 i n t h e heavy chain w i t h the concomitant formation o f a c t i v a t e d P r o t e i n C (35). A c t i v a t e d P r o t e i n C i s a s e r i n e p r o t e a s e and t h e a c t i v e s i t e s e r i n e r e s i d u e i s l o c a t e d i n t h e heavy c h a i n . The amino a c i d sequence s u r r o u n d i n g t h e a c t i v e s i t e s e r i n e r e s i d u e i n a c t i v a t e d P r o t e i n C i s homologous t o t h e a c t i v e s i t e r e g i o n s o f t h e s e r i n e proteases involved i n blood coagulation. Activated Protein C m a r k e d l y p r o l o n g s t h e c l o t t i n g t i m e o f p l a s m a , and t h i s a n t i c o a g u l a n t e f f e c t was t o t a l l y o b v i a t e d by p r i o r i n c u b a t i o n o f t h e enzyme w i t h DFP. R e c e n t e v i d e n c e i n d i c a t e s t h a t a c t i v a t e d P r o t e i n C, i n t h e p r e s e n c e o f c a l c i u m i o n s and p h o s p h o l i p i d , i s a p o t e n t i n a c t i v a t o r o f f a c t o r V and f a c t o r V I M ( 3 5 , 3 6 ) » and t h a t t h e s e i n a c t i v a t i o n r e a c t i o n s were a b s o l u t e l y d e p e n d e n t on t h e e n z y m a t i c a c t i v i t y o f a c t i v a t e d P r o t e i n C. T h u s , i t a p p e a r s t h a t the l e v e l o f t h r o m b i n a c t i v i t y i s c a r e f u l l y r e g u l a t e d by n e g a t i v e feedback r e a c t i o n s which i n v o l v e the l i m i t e d p r o t e o l y s i s o f prot h r o m b i n , f a c t o r V and f a c t o r V I M e i t h e r d i r e c t l y c a t a l y z e d by thrombin o r i n d i r e c t l y through the formation o f a c t i v a t e d P r o t e i n C.

Literature Cited 1. 2. 3.

Griffin, J . H., and Cochrane, C. G. (1976) Methods Enzymol. 45, 56-65. Fujikawa, K., Walsh, K. A . , and Davie, E. W. (1977) Biochemistry 16, 2270-2278, Fujikawa, K., Kurachi, K., and Davie, E. W. (1977) Biochemistry 16, 4182-4188.


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4.

Griffin, J . H., and Cochrane, C. G. (1976) Proc. Natl. Acad. Sci.

5. 6. 7.

USA 73, 2554-2558.

Chan, J . Y. C., Habal, K. M., Burrowes, C. E . , and Movat, H. Z. (1976) Thromb. Res. 9, 423-430. Wiggins, R. C., Bouma, B. N . , Cochrane, C. G., and Griffin, J. H. (1977) Proc. Natl. Acad. Sci. USA 74, 4636-4640. Kurachi, K., and Davie, E. W. (1977) Biochemistry 16, 58315839.

8. 9. 10. 11.

DiScipio, R. G., Hermodson, M. A . , Yates, S. G., and Davie, E. W. (1977) Biochemistry 16, 698-706. Fujikawa, K., Thompson, A. R., Legaz, M. E . , Meyer, R. G., and Davie, E. W. (1973) Biochemistry 12, 4938-4945. Stenflo, J., Fernlund, P., Egan, W., and Roepstorff, P. (1974) Proc. Natl. Acad. Sci. USA 71, 2730-2733. Furie, B. C., Mann, K. G., and Furie, B. (1976) J. Biol. Chem. 251, 3235-3241

12. 13.

14. 15.

16.

Fujikawa, K., Legaz Biochemistry 13, 4508-4516 McKee, P. A . , Anderson, J . C., and Switzer, M. E. (1975) Ann. N.Y. Acad. Sci. 240, 8-33. Legaz, M. E . , Weinstein, M. J., Heldebrant, C. M., and Davie, E. W. (1975) Ann.N.Y.Acad. Sci. 240, 43-61. Vehar, G. A . , and Davie, E.W.(1977)Science 197, 374-376. Titani, K., Fujikawa, K., Enfield, D. L., Ericsson, L. H . , Walsh, K. A . , and Neurath, H. (1975) Proc. Natl. Acad. S c i . USA 72, 3082-3086.

17. 18.

Fujikawa, K., Coan, M. H . , Legaz, M. E . , and Davie, E. W. (1974) Biochemistry 13, 5290-5299. K i s i e l , W., and Davie, E. W. (1975) Biochemistry 14, 49284934.

19.

Radcliffe, R., and Nemerson, Y. (1975) J. Biol . Chem. 250, 388-395.

20.

21. 22.

K i s i e l , W., Fujikawa, K., and Davie, E. W. (1977) Biochemistry 16, 4189-4194. Kisiel, W., unpublished data. Radcliffe, R., and Nemerson, Y. (1976) J. Biol. Chem. 251 4797-4802.

23. 24.

Bjorklid, E . , and Storm, E. (1977) Biochem. J. 165., 89-96. Suttie, J . W., and Jackson, C. M. (1977) Physiol. Reviews 57,

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1-70.

Magnusson, S., Peterson, T. E . , Sottrup-Jonsen, L . , and Claeys, H. (1975) in Proteases and Biological Control, eds. Reich, E . , Rifkin, D. B . , and Shaw, E. (Cold Spring Harbor Laboratory, New York), Vol. 2, pp. 123-149. Smith, C. M., and Hanahan, D. J. (1976) Biochemistry 15, 1830-1838.

27. 28.

McKee, P. A . , Rogers, L. A . , Marler, E . , and Hill, R. L. (1966) Arch. Biochem. Biophys. 116, 271-279. Lorand, L . , Downey, J., Gotoh, T., Jacobsen, A . , and Tokura, S. (1968) Biochem. Biophys. Res.Commun.31, 222-230.


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Schwartz, M. L., Pizzo, S, V., Hill, R, C., and McKee, P. A. (1973) J. Biol. Chem. 248, 1395-1407. Chung, S. I . , Lewis, M. C., and Folk, J. E. (1974) J. Biol. Chem. 249, 940-950. Levitzki, Α., Stallcup, W. Β., and Koshland, D. E. (1971) Biochemistry 10, 3371-3378. Heimburger, Ν., Haupt, Η., and Schwick, H. (1971) in Proc. Int. Res. Conf. Proteinase Inhibitors, eds. Fritz, H., and Tschesche, H. (W. de Gruyter, Berlin), pp. 1-21. Colman, R. W. (1969) Biochemistry 8, 1438-1445. Stenflo, J . (1976) J. Biol. Chem. 251, 355-363. Kisiel, W., Canfield, W. Μ., Ericsson, L. H., and Davie, E. W. (1977) Biochemistry 16, 5824-5831. Vehar, G., unpublished results.

RECEIVED

March 15, 1978.


10 Biosynthesis of Murine Leukemia Virus Membrane Proteins and their Assembly into Virus Particles R. B. ARLINGHAUS, E. C. MURPHY, JR., R. B. NASO, L. J. ARCEMENT, and W. L. KARSHIN Biology Department, University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, Houston, TX 77030

The cellular plasma membrane forms the surface layer of C-type RNA tumor virus particles. The c e l l ' s plasma membrane is also involved in one of the important phases of the l i f e cycle of RNA tumor viruses, that is the budding of the complete virus particles from the infected c e l l . C-type RNA tumor viruses contain a core structure surrounded by a l i p i d envelope through which spikes protrude. These spikes are believed to be composed, in mouse systems, of the v i r a l glycoprotein gp69/71 (1). Two non -glycosylated proteins termed p12(E) and p15(E) are also associated with the virion envelope. Their roles are not definitely known but recent studies suggest that gp69/71 and p15(E) exist as a complex and this complex forms the basic unit of the protruding spike (1, 2, 3). The core of these viruses is composed of four internal proteins termed p30, p15, pp12 (phosphoprotein) and p10 (4). The latter two have been shown to be associated with the v i r a l genomic RNA (5, 6). Viral p30 is thought to be the capsid protein of the core (4) whereas v i r a l protein p15 (a hydrophobic protein) could be involved in the association of the core with the l i p i d envelope. The virus also contains a DNA polymerase in its core. This polymerase can readily copy RNA into DNA with an appropriate p r i mer. It is present in virions in amounts estimated to be 50-100 molecules per virus particle (7) whereas the internal structural proteins are thought to be present in approximately 5,000 copies per virion (8). The envelope proteins [gp69/71, p15(E)] are also initially present in relatively large amounts but gp69/71 appears to be lost from the virion probably because of proteolytic degradation that releases the mature glycoprotein into the surrounding medium (9). 0-8412-0452-7/78/47-080-180$05.25/0

© 1978 American Chemical Society


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These v i r a l p r o t e i n s are encoded by three d i f f e r e n t genes w i t h i n the 35S (-3 x 10^ daltons o r 10,000 bases) v i r a l genome. Each v i r a l p a r t i c l e i s thought to c o n t a i n two i d e n t i c a l copies of the RNA genome. The gene coding f o r the four i n t e r n a l core prot e i n s has been termed g a g o r group antigens; that coding f o r the v i r a l RNA-directed DNA polymerase i s termed p o l o r reverse t r a n s c r i p t a s e (RT); the envelope p r o t e i n s are coded w i t h i n the envelope gene or e n v (10). The purpose o f t h i s r e p o r t i s to summarize the events l e a d ing to the formation of the mature v i r a l envelope p r o t e i n s and t h e i r assembly i n t o v i r i o n s . We a l s o w i l l propose a model f o r v i r i o n assembly and budding of the v i r u s p a r t i c l e from the c e l l . f

f

f

f

f

f

The V i r i o n P r o t e i n s of Rauscher Murine Leukemia V i r u s Rauscher murine leukemia v i r u s (R-MuLV) coded p r o t e i n s can be r e s o l v e d by e l e c t r o p h o r e s i denaturing c o n d i t i o n s ( F i g can be seen are p30, p l 5 , pl2(E) and plO; minor amounts o f gp69/71 and pl5(E) are a l s o present i n the v i r i o n . A s u b s t a n t i a l amount o f an uncleaved precursor to the core p r o t e i n s , termed Pr65§ S, i s a l s o present i n v i r u s . The v i r a l phosphoprotein ppl2 i s not s o l u b i l i z e d by t h i s procedure but i t i s r e a d i l y seen i n v i r u s denatured w i t h 6 M quanidine-HCl and f r a c t i o n a t e d by molecu l a r s e i v e chromatography i n 6 M guanidine. The r a d i o a c t i v e v i r u s , whose p r o t e i n s are sljown i n F i g . 1, was prepared by i n c u b a t i n g i n f e c t e d c e l l s w i t h ^ C-arginine and l y s i n e f o r a 48 hr i n t e r v a l . Under these c o n d i t i o n s much of the v i r a l gp69/71 i s l o s t from the v i r u s p a r t i c l e (9). This l o s s of v i r a l gp69/71 may correspond to the low amount of pl5(E) that i s present i n mature v i r u s p a r t i c l e s (see below). Murine C-type v i r u s e s appear to c o n t a i n a complex made up of gp69/71 and pl5(E) (1, 2, 3). The gp69/71-pl5(E) complex can be d i s s o c i a t e d w i t h s u l f h y d r i l reagents such as mercaptoethanol (1, _2, 3) . This complex may be the b a s i c u n i t of the surface s t r u c ture of the v i r u s . V i r a l gp69/71 has been shown to be on the surface of the v i r u s by s e v e r a l methods. One i n v o l v e s l a c t o peroxidase-catalyzed i o d i n a t i o n of the v i r i o n s u r f a c e components (11, 12). Another i n v o l v e s sodium borohydride r e d u c t i o n f o l l o w ing galactose oxidase treatment (13). A t h i r d procedure concerns the use of immunoelectromicroscopy using monospecific a n t i s e r a r a i s e d against p u r i f i e d gp70 (14), and most important, anti-gp70 can n e u t r a l i z e i n f e c t i v i t y (15, 16). The surface l o c a t i o n o f pl5(E) i s i n d i c a t e d by the f i n d i n g that a n t i s e r a to pl5(E) can p r e c i p i t a t e i n t a c t v i r u s (17, 18, 19) u n l i k e a n t i s e r a prepared against v i r a l i n t e r n a l components. Thus, i t i s q u i t e evident that both pl5(E) and gp69/71 are present i n the same place i n the v i r i o n which i s c o n s i s t e n t w i t h t h e i r being complexed together. a


182

Figure


1.

Preparative sodium dodecyl sulfate-polyacrylamide mature viral proteins

gel electrophoresis of

Rauscher murine leukemia virus (R-MuLV) was grown in a Balh/C spleen-thymus cell line (JLS-V5 cells) in the presence of C-arginine and lysine for 48 hr. Virus was purified as described (30), and viral proteins were separated by electrophoresis on a 11.25% polyacrylamide slab gel. The radioactive virus was distributed among the three sample wells after boiling in electrophoresis sample buffer (20). The radioactive bands were detected by fluorography using pre-flashed x-ray film so as to obtain a linear response to the amount of radiactivity (26). 14


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I n t r a c e l l u l a r V i r a l S p e c i f i c Glycoproteins V i r u s i n f e c t e d and u n i n f e c t e d NIH Swiss mouse embryo f i b r o b l a s t s (JLS-V16 c e l l s ) were incubated f o r 5-1/2 hr w i t h C glucosamine to detect v i r a l g l y c o p r o t e i n s . The cytoplasmic ext r a c t , t r e a t e d w i t h NP-40 and sodium deoxycholate detergents (20), was mixed w i t h an optimal c o n c e n t r a t i o n of antiserum r a i s e d i n r a b b i t s against p u r i f i e d R-MuLV that had been d i s r u p t e d by the above detergents (21). Figure 2 shows the r e s u l t s of f r a c t i o n a t i o n of the immunoprecipitates on a 10% sodium dodecyl s u l f a t e (SDS) polyacrylamide g e l . Figure 2 lane A shows the r a d i o a c t i v e g l y c o p r o t e i n s were not observed i n u n i n f e c t e d c e l l s i n the area where two broad bands termed g P r 9 0 and gp69/71 were seen i n i n f e c t e d c e l l s ( F i g . 2, lane B). These r e s u l t s i n d i c a t e that two major g l y c o p r o t e i n s are present i n v i r a l i n f e c t e d c e l l s but not i n u n i n f e c t e d c e l l s . One of the g l y c o p r o t e i n s corresponds i n molecular weight to the other we have shown to b gp69/71 and the non-glycosylated p r o t e i n pl5(E) (see below and references 22, 23). Since both gp69/71 and g P r 9 0 r e a d i l y incorporate radioact i v e glucosamine, i t was o f i n t e r e s t to t e s t r a d i o a c t i v e l a b e l i n g w i t h other sugars. Of p a r t i c u l a r i n t e r e s t i s fucose s i n c e i t i s o f t e n a t e r m i n a l sugar i n g l y c o p r o t e i n s (24). Fucosyl tranferases are a l s o present i n plasma membranes o f the c e l l (25) thereby r e s t r i c t i n g f u c o s y l a t i o n to the l a t t e r stages of g l y c o p r o t e i n proc e s s i n g . With these p o i n t s i n mind, we then incubated i n f e c t e d c e l l s w i t h % - l a b e l e d fucose and p r e c i p i t a t e d the v i r a l glycoprot e i n s e i t h e r w i t h anti-RLV serum ( F i g . 2, lane C) or anti-gp69/71 ( F i g . 2, lane D). The r e s u l t s showed that g P r 9 0 d i d not i n corporate fucose whereas the mature v i r a l g l y c o p r o t e i n (gp69/71) d i d . Lanes G and H of F i g . 2 a l s o show that v i r i o n gp69/71 a l s o i n c o r p o r a t e s r a d i o a c t i v e fucose as w e l l as glucosamine. Thus, gPr90 appears to c o n t a i n glucosamine but l a c k s fucose and d i s t i n g u i s h e s i t c l e a r l y from the mature gp69/71. In a p a r a l l e l experiment v i r u s - i n f e c t e d c e l l s were incubated w i t h r a d i o a c t i v e glucosamine f o r 5-1/2 hr and the cytoplasmic ext r a c t was d i v i d e d i n t o two p a r t s . One p o r t i o n was immunoprecipit a t e d w i t h anti-gp69/71 ( F i g . 2, lane E) and the other p o r t i o n was i s o l a t e d by anti-R-MuLV immunoprecipitation ( F i g . 2, lane F ) . Again the major v i r a l glucosamine-containing g l y c o p r o t e i n s recogn i z e d bv e i t h e r sera were gp69/71 and g P r 9 0 . We note that n ti~R-MuLV sera a l s o p r e c i p i t a t e d a minor glucosamine-containing g l y c o p r o t e i n that migrates j u s t slower than g P r 9 0 ( F i g . 2, lanes B and F ) . This g l y c o p r o t e i n may be r e l a t e d to gag' gene products because i t i s not recognized by anti-gp69/71 but i t i s seen w i t h anti-RLV. Anti-RLV serum contains a n t i b o d i e s t o g a g and 'env' determinants but not to ' p o l determinants (26). I n f r e q u e n t l y another p r o t e i n was a l s o detected i n the anti-R-MuLV p r e c i p i t a t e from glucosamine l a b e l e d i n f e c t e d c e l l s . I t comi1 4

e n v

e n V

e n v

e n v

a

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DISEASE PROCESSES

a

grates w i t h Pr2008 S~P°l which i s the precursor to the v i r i o n r e verse t r a n s c r i p t a s e (26, 27, 28). Kabat and co-workers have r e ported t h a t F r i e n d leukemia v i r u s i n f e c t e d c e l l s c o n t a i n 'gag gene p r o d u c t - r e l a t e d g l y c o p r o t e i n s of 95,000 and 200,000 mol. wt. (29). The l a t t e r were found i n a d d i t i o n to the v i r a l 'env' gener e l a t e d g l y c o p r o t e i n s . The r o l e of the 'gag' gene-related g l y c o p r o t e i n s i n the v i r u s l i f e c y c l e i s unknown. To show the s p e c i f i c i t y of immunoprecipitation, R-MuLV i n f e c t e d c e l l s were p u l s e - l a b e l e d w i t h -^C-amino a c i d s f o r 10 min and p a r a l l e l c u l t u r e s were p u l s e - l a b e l e d f o r 10 min and then i n cubated i n complete growth medium f o r 60 min (chase). Cytoplasmic e x t r a c t s were p r e c i p i t a t e d w i t h monospecific antiserum d i r e c t e d a g a i n s t R-MuLV gp69/71 and p30 (22). Anti-gp69/71 p r e c i p i tated g P r 9 0 almost e x c l u s i v e l y i n p u l s e - l a b e l e d c e l l s ( F i g . 3, lane A) and both g P r 9 0 and gp69/71 i n chase incubated c e l l s ( F i g . 3, lane B). Anti-p30 serum, however, d i d not recognize gPr90 i n pulse-labele mainly precursors Pr80g A f t e r chase i n c u b a t i o n ( F i g . 3, lane D), a n a l y s i s of the anti-p30 p r e c i p i t a t e showed t h a t Pr80S § disappeared and P r 6 5 was r e duced i n i t s i n t e n s i t y w h i l e p30 appeared as a major band. No gp69/71 or g P r 9 0 were p r e c i p i t a t e d by a n t i s e r a d i r e c t e d a g a i n s t 30. The k i n e t i c s of v i r a l g l y c o p r o t e i n s y n t h e s i s were s t u d i e d by p u l s e - l a b e l i n g c e l l s w i t h -^C-amino a c i d s and chasing r e p l i c a t e c u l t u r e s f o r 2, 4 and 6 hr p e r i o d s . The r e s u l t s c l e a r l y showed that gp69/71 was not l a b e l e d i n a 10 min p u l s e - l a b e l i n g ( F i g . 4, lane B) but became r a d i o a c t i v e between 60 and 120 min of the chase i n c u b a t i o n ( F i g . 4, lane E) w h i l e a t the same time there was a s l i g h t decrease i n the i n t e n s i t y of g P r 9 0 . Chase incubat i o n s of up to 6 hr s t i l l showed a s i g n i f i c a n t amount of g P r 9 0 v but the r a t i o of gp69/71 to g P r 9 0 reached a value greater than one whereas a f t e r 2 hr of chase i t was l e s s than one. As p r e v i o u s l y r e p o r t e d , p r e c i p i t a t i o n of c e l l u l a r e x t r a c t s w i t h antiserum to d i s r u p t e d v i r u s revealed other v i r a l p r o t e i n s (e.g. p30 and p l 5 ) t h a t appeared upon chase at the expense of Pr80gag and Pr65§ 8 (20, 21, 2_2, 30) ( F i g . 4, lanes A, D, G, and J ) . V i r a l pl2(E) a l s o appeared during long chase periods (2 to 6 hr) whereas only pl5(E) was seen i n short chase periods (1 h r ) . 1

e n v

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e n v

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Presence of gp69/71 and p!5(E) Peptide Sequences i n g P r 9 Q e n V

e n V

We have p r e v i o u s l y shown t h a t g P r 9 0 and gp69/71 c o n t a i n many t r y p t i c peptides that comigrate on i o n exchange columns (22). However, the p a t t e r n s obtained were q u i t e complex, to s i m p l i f y the complexity of the t r y p t i c d i g e s t p r o f i l e s and the subsequent comparisons of the peptide sequences, we e l e c t e d to tag the v i r a l p r o t e i n s w i t h r a d i o a c t i v e t y r o s i n e (23, 30). F i g u r e 5 shows i o n exchange p r o f i l e s i n which l ^ C - t y r o s i n e - l a b e l e d g P r 9 0 tryptic peptides were mixed w i t h t r y p t i c peptides of % - t y r o s i n e l a b e l e d e n v



Figure 2.

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Pohjacrylamide gel electrophoresis of R-MuLV glycoproteins from infected cells and virus.

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specific

Cytoplasmic extracts from uninfected (lane A) and R-MuLV-infected NIH Swiss mouse embryo fibroblasts (JLS-V16 cells) (lane B) were labeled for 5V2 hr with C-glucosamine and the anti-R-MuLV immunoprecipitates were analyzed as in Figure 1. Cytoplasmic extracts from R-MuLV-infected JLS-V15 cells were labeled with H-fucose were immunoprecipitated with anti-gp69/71 serum (lane C) or anti-R-MuLV serum (lane D). R-MuLVinfected cells were labeled for 5 /2 hr with C-glucosamine and the antigp69/71 immunoprecipitate (lane E) or the anti-R-MuLV immunoprecipitate (lane F) also were analyzed. Columns G and H are H-fucose and C-glucosa7nine-labeled virus, respectively, obtained form growth medium from R-MuLV-infected JLS-V16 cells following a SV2 hr labeling period. 14

3

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14

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14

Figure 3. Analysis of intracellular RMuLV specific proteins precipitated by anti-gp69/71 and anti-p30 sera. Cytoplasmic extracts from R-MuLV-infected JLS-V16 cells labeled with C-labeled amino acids were prepared and immunoprecipitation was performed with either anti-gp69/71 or anti-p30 serum. Electrophoresis on 10% gels andfluorographywere as in Figure 1. Column A, anti-gp69/71 immune precipitate from a 10-min pulse labeling; column B, antigp69/71 immune precipitate from a 10-min pulse labeling with a 60 min chase; column C, anti-p30 immune precipitate from a 10min pulse labeling; column D, anti-p30 immune precipitate of a 10-min pulse labeling with a 60-min chase incubation. 14



Figure 4. Formation chase experiment Cytoplasmic extracts from infected JLS-V16 cells were prepared, and immunoprecipitation was performed as described. Electrophoresis on 6-12% gradient gels and fluorography were carried out as in Figure 1. Columns Vi are C-labeled amino acid marker virus; columns A, B, and C, 10 min pulse with Clabeled amino acid protein hydrolysate; columns D, £, and F, 2-hr chase incubation of the 10-min pulse labeling; columns G, 77, and 7, 4-hr chase; columns J, K, and L, 6-hr chase; columns A, D, G, and J, anti-R-MuLV precipitated; columns B, E, 77, and K, anti-gp69/71 precipitated; and columns C, F, 7, and L, precipitated with anti-MuLV absorbed with excess R-MuLV proteins. 14

14

Figure 5. Ion exchange chromatography of tryptic digests of tyrosine-labeled gFr90 and gp69/71. The arrows show the two additional tyrosine-containing tryptic peptides in gPr90 that are not found in gp69/71. €nv

env


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gp69/71. The r e s u l t s c l e a r l y show that g P r 9 0 shares many t y r o s i n e - c o n t a i n i n g t r y p t i c peptides w i t h gp69/71. Figure 5 a l s o shows that there are at l e a s t two a d d i t i o n a l t y r o s i n e - c o n t a i n i n g t r y p t i c peptides i n g P r 9 0 t h a t are not found i n gp69/71 (see arrows i n F i g . 5). An a n a l y s i s of a -*- C-tyrosine-labeled pl5(E) t r y p t i c d i g e s t mixed w i t h a ^ H - t y r o s i n e - l a b e l e d g P r 9 0 digest ( F i g . 6) c l e a r l y showed that these two a d d i t i o n a l t r y p t i c peptide f r a c t i o n s are d e r i v e d from p l 5 ( E ) . We conclude from these r e s u l t s that pl5(E) and gp69/71 are cleavage products o f g P r 9 0 . e n v

e n v

4

e n v

e n v

The Gene Order of the P r o t e i n s W i t h i n g P r 9 Q

e n V

e t l V

The order of gp69/71 and pl5(E) w i t h i n g P r 9 0 was examined by i n c u b a t i n g i n f e c t e d c e l l s w i t h r a d i o a c t i v e amino a c i d s i n the presence and absence of pactamycin, a drug that s e l e c t i v e l y i n h i b i t s the i n i t i a t i o n of t r a n s l a t i o n at 5 x 10""^ M but not e l o n g a t i o n (31, 32). In th t i a l l y l a b e l the C-termina In the case of g P r 9 0 , we could determine which p r o t e i n i s a t the C-terminus. R e s u l t s of such s t u d i e s (23) showed that pactamycin treatment f o r 30 to 60 seconds before and during p u l s e l a b e l i n g reduced the amount of r a d i o a c t i v e gp69/71 formation i n a chase i n c u b a t i o n , whereas r a d i o a c t i v e pl5(E) was increased r e l a t i v e to that found i n the absence of the drug. The r e s u l t s were q u a n t i t a t e d y i e l d i n g a r a t i o of pl5(E) to gp69/71 of 0.39 i n the c o n t r o l and 0.76 i n the presence of pactamycin. These r e s u l t s suggest that pl5(E) i s C-terminal i n g P r 9 0 . e n v

fenv

V i r a l P r o t e i n p!2(E) We have i d e n t i f i e d a new v i r a l p r o t e i n , termed p l 2 ( E ) , i n v i r u s p a r t i c l e s . I t can be d i s t i n g u i s h e d from phosphoprotein p l 2 (ppl2) on s e v e r a l grounds. F i r s t pl2(E) i s not phosphorylated (23), i t migrates i n the v o i d volume f r a c t i o n of a 6 M guanidineHC1 column whereas ppl2 e l u t e s i n the 12,000 mol. wt. range (23), and t h i r d l y i t has a peptide map much d i f f e r e n t from ppl2 (23); and l a s t l y , pl2(E) and ppl2 can be r e s o l v e d by 2-dimensional g e l s c o n s i s t i n g of i s o e l e c t r i c f o c u s i n g i n the f i r s t dimension and sodium dodecyl s u l f a t e (SDS) polyacrylamide g e l e l e c t r o p h o r e s i s (PAGE) i n the second (23). We emphasize the p o i n t that i s o l a t i o n of pl2(E) from v i r u s by SDS-PAGE o f t e n y i e l d s p l 2 ( E ) contaminated w i t h fragments of p l 5 and p30. We have found i t necessary to use a two-step procedure i n v o l v i n g guanidine-HCl agarose column chromatography f o l l o w e d by SDS-PAGE i n order to o b t a i n a r e l a t i v e l y pure p r e p a r a t i o n of p l 2 ( E ) . The r o l e of pl2(E) i n v i r u s s t r u c t u r e i s not known. I t i s tempting to propose that i t i s a degradation product of pl5(E) and i s the r e s u l t of a p r o t e o l y t i c cleavage of the gp69/71-pl5(E) complex r e s u l t i n g i n the l o s s o f gp69/71 from the v i r i o n (4, 9) and the generation of an uncomplexed pl2(E) remaining i n the



0--0

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H-1yrosine labeled g P r 9 0

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Fraction Number

Figure 6. Ion exchange chromatography of tryptic digests of tyrosine-labeled gPr90 and pl5(E). The pl5(E) used in this experiment was purified from virus by sodium dodecyl sulfatepolyacrylamide gel electrophoresis as described in the legend to Figure 1. env


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v i r u s envelope. We propose that t h i s p r o t e o l y t i c cleavage occurs a f t e r the v i r u s i s r e l e a s e d from the c e l l i n t o the c u l t u r e medium. We have shown t h a t as R-MuLV p a r t i c l e s age i n the c u l t u r e medium i n which they were r e l e a s e d , such p a r t i c l e s show a steady l o s s o f gp69/71 (Arlinghaus, unpublished r e s u l t s ) . C e l l - F r e e Synthesis of an Unglycosylated P15(E)

Precursor t o gp69/71 and

C e l l - f r e e t r a n s l a t i o n o f genomic RNAs from murine and avian RNA tumor v i r u s e s has shown that the f u l l - l e n g t h 35S RNA can code f o r the core p r o t e i n precursor P r 6 5 S 8 (28, 33, 34, 35. 36, 37) as w e l l as f o r a l a r g e r p o l y p r o t e i n c o n t a i n i n g both 'gag' and p o l gene products (_28, 34, 3 9 ) . The e n v gene product has never been observed to be coded f o r by f u l l l e n g t h v i r i o n genomic RNA although the i n f o r m a t i o n f o r t h i s gene product i s d e f i n i t e l y encoded i n th In order t o determin could be synthesized from i n t r a c e l l u l a r RNA from v i r a l i n f e c t e d c e l l s , we i s o l a t e d 18-25S RNA and 25-35S RNA from a t o t a l RNA ext r a c t by sucrose d e n s i t y gradient c e n t r i f u g a t i o n . These RNA c l a s s e s were again f r a c t i o n a t e d on separate sucrose g r a d i e n t s and i n d i v i d u a l f r a c t i o n s were analyzed i n a mRNA-dependent c e l l - f r e e amino a c i d i n c o r p o r a t i o n system (28^, 34) . The r e s u l t s o f t h i s experiment, d i s p l a y e d i n F i g . 7 (lanes E and F) showed that only RNA of about 20-22S i n s i z e , can be t r a n s l a t e d i n t o a polypeptide prec i p i t a b l e by antiserum to gp69/71. This polypeptide had an apparent molecular weight of 68,000 to 70,000. I n some experiments t h i s polypeptide migrated as two bands (a d o u b l e t ) . Polypeptides both l a r g e r and s m a l l e r than the 68,000 t o 70,000 were a l s o recogn i z e d by anti-gp69/71 serum. However, the smaller polypeptides were found i n a l l RNA f r a c t i o n s and the l a r g e r ones were found i n c o n s i d e r a b l y s m a l l e r q u a n t i t i e s than the 68,000 t o 70,000 mol. wt. polypeptide. The i d e n t i t y o f these s m a l l e r and l a r g e r polypept i d e s i s unknown. Immunoprecipitation o f the c e l l - f r e e t r a n s l a t i o n product of the 25S t o 35S RNA c l a s s w i t h anti-gp69/71 i n d i cated t h a t the 68,000 t o 70,000 mol. wt. polypeptide was absent ( r e s u l t s not shown). Hence, a 20-22S RNA seems to be the s o l e mRNA f o r an envelope-related p r o t e i n i n R-MuLV i n f e c t e d c e l l s . The t r a n s l a t i o n product o f each o f the RNA f r a c t i o n s i n the 18-25S and 25-30S s i z e c l a s s e s was a l s o t e s t e d by immunoprecipit a t i o n w i t h anti-p30 serum. The r e s u l t s showed that the g a g o r core p r o t e i n precursor Pr658 § and the RT precursor Pr200§ 8"P are synthesized e x c l u s i v e l y from RNA which i s greater than 28S i n s i z e ( r e s u l t s not shown). T r y p t i c peptide a n a l y s i s o f t h i s 65,000 mol. wt. product showed that i t had methionine-containing t r y p t i c peptides c h r a c t e r i s t i c of a u t h e n t i c P r 6 5 ^ ^ . We have p r e v i o u s l y e s t a b l i s h e d that P r 6 5 8 8 i s the major product and Pr2008 8"" i s a minor product i n c e l l - f r e e p r o t e i n s y n t h e s i z i n g systems o f 35S R-MuLV genomic RNA t r a n s l a t e d (34). a

f

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o1

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Figure 7. Sodium dodecyl sulfate slab gel electrophoresis of the cell-free product produced from 20-22S mRNA from R-MuLV infected JLS-V5 cells. Total cellular nucleic acid was extracted from RMuLV-infected JLS-V5 cells as described (28), and RNA was selectively obtained by washing the nucleic acid alcohol precipitate with 3M LiClSmM EDTA = (pH 6) which removes DNA.The RNA was fractionated by velocity sedimentation on a sucrose gradient, and the 18S-to-25S RNA fraction was pooled and again separated on a sucrose gradient. Aliquots of the fractions (1 ^g of RNA) were analyzed in a mRNA-dependent protein synthesizing system (28,34). The protein products were immunoprecipitated with anti-gp69/71 serum and analyzed on a 6-12% gradient slab-gel of polyacrylamide, as in Figure 1. Lane A is a pulse-chase experiment from R-MuLV-infected JLS-V16 cells showing authentic viral specific protein precursors obtained with anti-R-MuLV serum (20). Lanes B-G represent aliquots across a gradient in which 18S ribosomal RNA sediments to the middle of the centrifuge tube (lane D). Lane B represents the top two fractions, lane G represents the bottom two fractions, and fractions C, D, E, and F represent the in-between fractions.


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Peptide Maps o f the Envelope-related P r o t e i n Synthesized From I n t r a c e l l u l a r 20-22S mRNA I f the 68,000 t o 70,000 mol. wt. polypeptide coded f o r by 20-22S mRNA i s r e l a t e d t o R-MuLV envelope precursor g P r 9 0 , i t should possess t r y p t i c peptides c h a r a c t e r i s t i c o f gp69/71 and p l 5 ( E ) . Therefore, 3 5 - e t h i o n i n e - l a b e l e d 68,000 t o 70,000 mol. wt. p o l y p e p t i d e product of 20-22S mRNA was digested w i t h t r y p s i n and mixed w i t h a u t h e n t i c g P r 9 0 t r y p t i c peptides l a b e l e d w i t h 3 -methionine. The mixture was f r a c t i o n a t e d by i o n exchange chromatography ( F i g . 8 ) . A n e a r l y p e r f e c t c o - e l u t i o n of t r y p t i c methionine-containing peptide was observed. The one exception being a minor peak ( F r a c t i o n 45) i n the i n v i t r o synthesized product that was n o t observed i n the a u t h e n t i c g P r 9 0 . The pept i d e peak e l u t i n g a t F r a c t i o n 76 i s c h a r a c t e r i s t i c of pl5(E) (20, 22) as i s the peptide peak a t F r a c t i o n 83 i f the t r y p t i c peptides are not o x i d i z e d w i t h performi c h a r a c t e r i s t i c o f gp69/7 s u l t s ) . We conclude from these experiments that the 68,000 t o 70,000 mol. wt. p o l y p e p t i d e termed P r 6 8 , i s i d e n t i c a l t o gPr90 i n o v e r a l l peptide content and P r 6 8 probably r e p r e sents the u n g l y c o s y l a t e d form of that molecule. e n v

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e n v

e n v

e n v

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T r a n s l a t i o n of F r a c t i o n a t e d R-MuLV Genomic RNA e n v

Since a 20-22S i n t r a c e l l u l a r mRNA species codes f o r P r 6 8 , i t was o f i n t e r e s t to determine whether RNA from v i r u s p a r t i c l e s had a s i m i l a r coding c a p a c i t y . V i r a l RNA p r e p a r a t i o n s obtained from v i r u s , t h a t has remained 24 h r i n the c u l t u r e f l u i d , have i n a d d i t i o n t o 35S RNA a range o f fragments o f the f u l l l e n g t h genome (35S) whose sedimentation r a t e v a r i e s down to 4S. T r a n s l a t i o n of v a r i o u s s i z e c l a s s e s o f v i r i o n RNA f r a c t i o n a t e d on a sucrose grad i e n t i n d i c a t e d that a s i g n i f i c a n t amount o f a polypeptide o f the size of P r 6 8 and p r e c i p i t a b l e by anti-gp69/71 i s coded f o r by v i r a l RNA of l e s s than f u l l l e n g t h s i z e . I t i s necessary t o obt a i n the t r y p t i c peptide map o f t h i s p o l y p e p t i d e to determine that i t i s a e n v gene product and not a g a g gene r e l a t e d p r o t e i n that i s a r t i f a c t u a l l y seen i n the anti-gp69/71 p r e c i p i t a t e . e n v

f

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Model f o r the Synthesis and P r o c e s s i n g o f R-MuLV P r o t e i n s Our r e s u l t s and those o f others (40, 41, 42, 43) i n d i c a t e that there a r e a t l e a s t two s i z e c l a s s e s of mRNAs that code f o r R-MuLV p r o t e i n s . The f u l l - l e n g t h genome s i z e 35S R-MuLV contains genes termed gag* o r core p r o t e i n s , p o l o r the DNA polymerase and e n v o r the envelope p r o t e i n s . However, only the g a g and p o l genes appear t o be t r a n s l a t e d from 35S genomic RNA (28^, 34, 39). Our f i n d i n g s i n d i c a t e that a s m a l l e r s i z e c l a s s of mRNA (-22S) codes f o r the envelope p r o t e i n s . Studies i n the a v i a n system i n d i c a t e the gene order w i t h i n the genome of non-defective ?

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192

GLYCOPROTEINS AND


avian sarcoma v i r u s e s (another C-type RNA tumor v i r u s ) i s : 5'gag-pol-env-src -poly(A)-3 (44). Given the same gene order i n the R-MuLV genomic RNA (e.g. 'gag-pol-env'), our r e s u l t s i n d i c a t e t h a t only the f i r s t two genes are t r a n s l a t e d from v i r i o n RNA. Furthermore, our r e s u l t s suggest t h a t only one ribosome o f 15 t o 25, that has completed the t r a n s l a t i o n o f the 'gag gene, proceeds i n t o the ' p o l ' gene (26, 28, 34) ( F i g . 9 ) . This l a t t e r process we have termed read-through (28, 34) and the product o f t h i s read-through event i s a 200,000 mol. wt. p o l y p r o t e i n termed Pr200§ ° . I t contains peptide sequences and a n t i g e n i c determinants o f the 'gag' p r o t e i n and the v i r a l DNA polymerase (26, 27, 28, 34). We have shown i t to be the precursor o f the v i r i o n reverse t r a n s c r i p t a s e (26, 27). The d e t e c t i o n o f both R T - s p e c i f i c and core p r o t e i n - s p e c i f i c a n t i g e n i c determinants i n a s i n g l e precursor p o l y p r o t e i n ( P r 2 0 0 S " ) r a i s e d the f o l l o w i n g i s s u e concerning the t r a n s l a t i o n o f the v i r a l genome gene products proceeds b precursor o n l y , then t h i s would e n t a i l the s y n t h e s i s o f equimolar amounts o f g a g and ' p o l ' gene products. V i r u s p a r t i c l e s c o n t a i n much l e s s polymerase than s t r u c t u r a l p r o t e i n s 07, 8 ) . Moreover, measurement of the steady s t a t e amount o f R T - s p e c i f i c sequences i n v i r u s - i n f e c t e d c e l l s has i n d i c a t e d t h a t these sequences are present i n much s m a l l e r amounts than the v i r a l s t r u c t u r a l p r o t e i n s C7, 45). This i n d i c a t e s t h a t i f RT polypeptide sequences are i n deed made i n equimolar amounts compared t o the core p r o t e i n s , they must be q u i c k l y degraded. We have been able t o shed more l i g h t on t h i s p o i n t by comparing the amount o f RT precursors t o the amount of core p r o t e i n precursors a f t e r p u l s e - l a b e l i n g o f i n f e c t e d c e l l s (26, 27). These c o n d i t i o n s should more c l e a r l y approximate the r a t e o f s y n t h e s i s o f these p o l y p e p t i d e s . Our measurement revealed that 1/25 t o 1/10 as much R T - s p e c i f i c precursors are present i n the c e l l , a f t e r a p u l s e - l a b e l i n g , as core p r o t e i n precursors (26) . Moreover, intermediate RT precursors appear to be formed by c l e a v age of preformed Pr2008 8""P°l, w h i l e formation o f core p r o t e i n precursors i s only compatible w i t h t h i s mechanism i f very f a s t o r nascent chain cleavage o f Pr2008 S~P°l takes p l a c e , which i s not supported by our s t u d i e s w i t h amino a c i d analogs o r protease i n h i b i t o r s (26). This makes i t u n l i k e l y t h a t the pathways of synt h e s i s o f 'gag' and ' p o l ' gene products only i n v o l v e the cleavage of a common precursor. Three p o s s i b l i t i e s are t h e o r e t i c a l l y compatible w i t h our f i n d i n g s . F i r s t , the mRNA (35S i n s i z e ) coding f o r P r 2 0 0 8 S ~ P i s a minor RNA species compared t o a s m a l l e r s i z e mRNA i n v o l v e d i n the s y n t h e s i s o f 'gag' gene products. Second, there i s only one species o f mRNA coding f o r both 'gag' and ' p o l ' , but a t r a n s l a t i o n a l c o n t r o l mechanism a l l o w s the s y n t h e s i s on the mRNA of more 'gag' than ' p o l ' gene products. A t h i r d p o s s i b i l i t y i s that two c l a s s e s o f s i m i l a r s i z e mRNA (e.g. 35S) e x i s t i n the i n f e c t e d c e l l . One codes f o r 'gag' and the other codes f o r 'gag-pol'. A ,

?

T

1

ag P

a g

p O

f

T

a

a

a


o1

10.

Murine


Figure 8. gPrQQcnv

Leukemia

Virus

Membrane

Proteins

193

Ion exchange chromatography of tryptic digests of authentic i 68,000 mol wt polypeptide (pr68 ) made from ~ 22S mRNA from R-MuLV-infected JLS-V5 cells.

anc

t

n

cnv

e

Pr68 was labeled with S-methionine in the cell-free mRNA-dependent protein synthesizing system (as in Figure 7). gPr90 " was isolated from R-MuLV-infected JLS-V16 cells labeled with H-methionine. The proteins were purified by immunoprecipitation with anti-gp69/71 and sodium dodecyl sulfate gel electrophoresis. The bands were digested with TPCK-trypsin and fractionated on a cation exchange column (30). env

35

c

v

3

Translotionol control

t

35SI mole P r 8 0 Pr65

Pol

Q>

Poly (A) -vw\

T

0'

gPr90

fl0g

H—-1

20S ,nv

viral mRNA

— I mole

-

,afl

4

\

Pr 27

Pr 42

4

4 \

pi5

\

gp 69/71

iZT gag-pol

\

pl2E

pl2 p30 plO

1/8 - 1/15 mole Pr 200

pl5E

1 pol

- Pr 145

I = initiation T = termination

•

Polymerase

Figure 9.

A model illustrating the overall steps involved in the synthesis and processing of Rauscher murine leukemia viral proteins


GLYCOPROTEINS AND

194


c o n s i d e r a t i o n o f other a v a i l a b l e i n f o r m a t i o n favors the second o r t h i r d p o s s i b i l i t y . Thus, the major species o f v i r a l mRNA i n murine and a v i a n RNA tumor v i r u s - i n f e c t e d c e l l s has a s i z e o f 35S, but species o f 21S and 16S have a l s o been detected (40, 41, ^ 2 , 43). Moreover, f u r t h e r s t u d i e s i n d i c a t e that the 35S RNA i s the main species present i n polyribosomes p r e c i p i t a b l e w i t h anti-p30 mono-specific serum (46). Studies i n a v i a n systems a l s o suggest that the f u l l - l e n g t h genomic RNA i s the mRNA f o r 'gag and ' p o l ' gene products (38, 47). These observations make i t u n l i k e l y that there i s a s m a l l s i z e mRNA (-16S) on which the m a j o r i t y o f the 'gag gene products are synthesized and d i s f a v o r the p o s s i b i l i t y that a l a r g e s i z e species (-35S) o f v i r a l mRNA, such as one able to code f o r P r 2 0 0 , c o n s t i t u t e a minor p o p u l a t i o n . I t i s d i f f i c u l t t o e l i m i n a t e the p o s s i b i l i t y that there are indeed two c l a s s e s o f 35S o r f u l l - l e n g t h genomic s i z e RNAs, a major one coding f o r 'gag' gene products and a minor one coding f o r 'gag' and ' p o l ' gen that s t o r e d 35S v i r a l RN gene a t the expense o f the 'gag' gene precursors than f r e s h batches 35S v i r a l RNA (Murphy and A r l i n g h a u s , unpublished r e s u l t s ) . This r e s u l t i s c o n s i s t e n t w i t h the idea that some s t r u c t u r a l a l t e r a t i o n ^ ) of 35S RNA genomic i s r e s p o n s i b l e f o r P r 2 0 0 g g ~ P s y n t h e s i s . This a l t e r a t i o n could be brought i n t o p l a y by i n t e r a c t i o n o f the 35S v i r a l RNA w i t h a second molecule. A candidate f o r t h i s second molecule would be a RNA s i n c e pronease-treated v i r a l 35S RNA does not a l t e r the frequency o f P r 2 0 0 ~ P synt h e s i s over non-treated RNA (Murphy and A r l i n g h a u s , unpublished results). Our r e s u l t s i n d i c a t e t h a t the envelope gene i s probably never t r a n s l a t e d from f u l l length genomic s i z e RNA but that a -22S i n t r a c e l l u l a r mRNA serves as a mRNA f o r the envelope p r o t e i n . The p o l a r i t y o f t h i s mRNA i s unknown but a s i m i l a r v i r a l envelope mRNA from a v i a n sarcoma v i r u s i n f e c t e d c e l l s has the same p o l a r i ty as v i r a l genomic RNA (47, 48, 49). Therefore, we would assume that -22S mRNA composed o f sequences from the 3' o n e - t h i r d o f R-MuLV genomic RNA i s the mRNA f o r the envelope p r o t e i n s . We would a l s o assume that t h i s RNA i s capped and contains some unt r a n s l a t e d sequences found a t the 5' end o f the 35S genomic RNA (48). 1

1

g a g p

o

a

g a g

o1

o 1

Model f o r the Synthesis and P r o c e s s i n g o f V i r a l Envelope P r o t e i n s As the model i n F i g . 10 shows, the i n i t i a l t r a n s l a t i o n product of the -22S 'env' mRNA i s a 68,000 mol. wt. non-glycosylated polypeptide that contains peptide sequences o f the major g l y c o p r o t e i n (gp69/71) and non-glycosylated p l 5 ( E ) . The next step i s shown to be the g l y c o s y l a t i o n o f the p o l y p r o t e i n producing g P r 9 0 . A l t e r n a t e l y , i t i s possible that inside infected c e l l s g l y c o s y l a t i o n occurs p r i o r t o formation o f the complete 'env' gene product which would r e s u l t i n the immediate formation o f e n v


10.


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Leukemia

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e n v

g P r 9 0 . I f t h i s were the case, P r 6 8 would only be seen under c o n d i t i o n s o f c e l l - f r e e t r a n s l a t i o n and not i n whole c e l l s . However, i t has not been e s t a b l i s h e d whether or not some P r 6 8 i s formed i n i n f e c t e d c e l l s . Cleavage and f u r t h e r g l y c o s y l a t i o n (fucose residues are added) of g P r 9 0 v y i e l d s gp69/71 and pl5(E) which probably e x i s t s as a complex held together by d i s u l f i d e bonds and p o s s i b l y a d d i t i o n a l non-covalent bonds. The b a s i c u n i t of the p r o t r u d i n g s p i k e - g l y c o p r o t e i n knob i s b e l i e v e d to be t h i s gp69/71-pl5(E) complex. The a s s o c i a t i o n of such b a s i c u n i t s would then r e s u l t i n formation of the spike-knob s t r u c t u r e that t r a v e r s e s and p r o j e c t s through the l i p i d envelope of the v i r u s ( F i g . 11). The r o l e of v i r a l p r o t e i n pl2(E) i n the s t r u c t u r e of the v i r i o n i s unknown. Since, i t does not appear to form a complex w i t h gp69/71 (50), we assume that i t may be an a r t i f a c t of aged v i r u s r e s u l t i n g from the p r o t e o l y s i s o f the spike-knob s t r u c t u r e which i n t u r n leads to a s m a l l p o r t i o n of the e n v

en

V i r u s Assembly Cleavage of v i r a l p o l y p r o t e i n s c o n s t i t u t e s a predominant f e a t u r e i n the assembly of many animal and b a c t e r i a l v i r u s e s ( f o r reviews see 51, J52, J53, 54) . RNA tumor v i r u s e s are no exception. We have shown that actinomycin D reduced the r a t e of cleavage of Pr65gag (55) and have suggested that the r a t e o f cleavage o f Pr65S £ to the mature v i r a l - p r o t e i n s i s c a t a l y z e d by the v i r a l genomic RNA. Recent experiments have a l s o shown that the c l e a v age r a t e of Pr200g "~ i s a l s o a f f e c t e d by i n h i b i t i n g v i r a l RNA s y n t h e s i s (Kopchick and A r l i n g h a u s , unpublished r e s u l t s ) . We note that the cleavage of Pr80S S to P r 6 5 g i s not a f f e c t e d under these c o n d i t i o n s nor i s the cleavage of g P r 9 0 . However, cleavages of Pr65gag and P r 2 0 0 " " P do occur (but at a slower r a t e ) i n the absence of v i r a l RNA and, i n t e r e s t i n g l y , the reverse t r a n s c r i p t a s e can be incorporated i n t o v i r i o n s that are d e f i c i e n t of v i r a l RNA (56). Based on a number of s t u d i e s from my own labor a t o r y (55), we have proposed a model f o r the assembly of R-MuLV p a r t i c l e s ( F i g . 11). In t h i s model, P r 6 5 g g r a t h e r than the mature v i r a l p r o t e i n s c o n s t i t u t e the i n i t i a l assembly u n i t . U n i t s of P r 6 5 g g assemble, presumably at the c e l l membrane, i n t o a s t r u c t u r e that remains open on the i n s i d e , due to the e l e c t r o s t a t i c r e p u l s i o n of the b a s i c p o r t i o n (plO) of P r 6 5 g g , which i s then cleaved to form the v i r a l p r o t e i n s i n c l u d i n g plO. V i r a l plO i n t u r n i s found i n the v i r i o n i n a s s o c i a t i o n w i t h the v i r a l RNA (5, 57). This model a l s o proposes that P r 2 0 0 g ~ i s incorpor a t e d i n t o the v i r i o n by means of i t s s i m i l a r i t y i n s t r u c t u r e t o P r 6 5 g . In f a c t , P r 2 0 0 g g " * contains a l l the a n t i g e n i c d e t e r minants and peptide sequences that are found i n P r 6 5 g g plus those of the reverse t r a n s c r i p t a s e . Thus, Pr200g g~P°l would s u b s t i t u t e f o r s m a l l number (30-50) o f the P r 6 5 molecules r e s u l t i n g a

ag

p

a

g a

e n v

gag

o1

a

a

a

g a

a g

a

p o 1

po1

a

a

g a g


196

GLYCOPROTEINS AND GLYCOLIPIDS IN DISEASE PROCESSES gp69/71

20-22S

mRNA

-p15(E) -...poly (A)

Cap.

3'

Translation

Pr68

,

env

p15(E)

K

prvrvn

1

s Translation and / Glycosylation

Glycosylation

gPr90

LTTl

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|

V

f

l a E I

COOH

Carbohydrate Chains Cleavage and Fucosylation

gp69/71 -p15(E) Complex

gp69/71

p15(E)

s-s

Viral Spike and Knob Assembly Figure 10.

Synthesis of Rauscher murine leukemia virus envelope proteins

9a9 f>0

^-Pr200 - ' vRNA

-Unc leaved Pr65W

| Cleavage of Pr65W and Pr200W-i>

01

Mature core proteins Budding virus

Cell membrane Figure 11. A model showing the assembly and budding of Rauscher murine leukemia virus particles


10.


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i n the i n c l u s i o n o f RT i n the v i r i o n . _ A s u b s t a n t i a l amount o f cleavage o f P r 6 5 fend P r 2 0 0 ° ) i s proposed i n the model i n order to a l l o w v i r u s r e l e a s e . V i r a l RNA, due t o charge n e u t r a l i z a t i o n , c a t a l y z e s the cleavage o f Pr65§ and P r 2 0 0 " . T h i s r e s u l t s i n a more r a p i d r a t e o f assembly of RNA-containing v i r a l c o r e s , and, i f p r e c u r s o r c l e a v age i s viewed to p l a y a p a r t i n the budding process, i n a f a s t e r r a t e o f r e l e a s e o f RNA-containing v i r u s p a r t i c l e s . We f u r t h e r propose that host c e l l RNA can s u b s t i t u t e t o a c e r t a i n degree f o r v i r a l RNA i n c a t a l y z i n g the cleavages of P r 6 5 and P r 2 0 0 " P , but host c e l l u l a r RNA i s packaged to a much l e s s e r extent than v i r a l RNA s i n c e v i r u s i s o l a t e d a f t e r actinomycin D treatment exh i b i t e d a lower r a t i o o f RNA to p r o t e i n than c o n t r o l p r e p a r a t i o n s of v i r u s (55). T h i s suggests the e x i s t a n c e of s p e c i f i c r e c o g n i t i o n by the assembled v i r a l s t r u c t u r e s o f v i r a l genomic RNA. Whether the b a s i s f o r t h i s r e c o g n i t i o n l i e s i n the s p e c i f i c i n t e r a c t i o n between some o f the v i r a v i r a l RNA (6) remains t In the above mentioned model of assembly, we have p o s t u l a t e d t h a t cleavages o f P r 6 5 and P r 2 0 0 8 ~ P i s probably r e q u i r e d f o r v i r u s budding and r e l e a s e . There i s a p o s s i b i l i t y , however, that Pr658 8 and P r 2 0 0 ~ may be cleaved not d u r i n g budding but a f t e r r e l e a s e o f the v i r u s from the c e l l . For many y e a r s , C-type p a r t i c l e s have been observed to undergo a p o s t - r e l e a s e maturation process i n which the core center of the v i r i o n c o l lapses i n t o a condensed s t r u c t u r e that i s c l e a r l y d i f f e r e n t from the organized c o n c e n t r i c c o i l s observed i n the core of budding p a r t i c l e s (58, 59, 60). The s i g n i f i c a n c e o f t h i s maturation process remains u n c e r t a i n . The recent r e s u l t s of Yoshinaka and L u f t e g (61) suggest that there i s a p o s i t i v e c o r r e l a t i o n between the amount of P r 6 5 8 8 i n v i r a l cores and the amount o f immature cores. However, i t remains to be shown whether o r not the c l e a v age o f Pr658 8 can occur i n r e l e a s e d v i r i o n s and i f so that the changes i n r e l e a s e d v i r i o n s (immature to mature) a r e r e l a t e d t o precursor cleavage. A number o f _ l i n e s evidence suggest that cleavage o f P r 6 5 8 8 and P r 2 0 0 ° occur mainly p r i o r t o v i r a l r e l e a s e (62) and p r i o r t o the immature t o mature v i r u s t r a n s i t i o n that takes p l a c e i n the c u l t u r e f l u i d . 1

g a g

ag

g a g

P

p o

g a g

g a g

a

g a g

g a g

a g

g a g

o 1

o 1

P

a

a

a

g a g

P

Literature Cited 1. Pinter, A. and Fleissner, E. Virology (1977) 83, 417. 2. Leamnson, R. N., Shander, M. H. M. and Halpern, M. S. Virology (1977) 76, 437. 3. Witte, O. N., Tsukamoto-Adey, A. and Weissman, I . L. Virology (1977) 76, 539. 4. Bolognesi, D. P . , Montelaro, R. C., Frank, H. and Schafer, W. Science (1978) 199, 183. 5. Davis, J., Scherrer, M., Tsai, W. P . , and Long, C. J . Virol. (1976) 18, 709.


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Proteins

199

32. Butterworth, B. E . , and Rueckert, R. R. J . Virol. (1972) 9, 823. 33. Naso, R. B . , Arcement, L. J., Wood, T. G., Saunders, T. E . , and Arlinghaus, R. B. Biochim. Biophys. Acta (1975) 383, 195. 34. Murphy, J r . , E. C., and Arlinghaus, R. B. Virology (1978) In press. 35. Kerr, I. M., Olshevsky, U . , Lodish, H . , and Baltimore, D. J. Virol. (1975) 18, 627. 36. Pawson, T., Martin, G. S., and Smith, A. E. J . Virol. (1976) 19, 950. 37. Von Den Helm, K . , and Duesberg, P. H. Proc. Natl. Acad. Sci. (1975) 72, 614. 38. Paterson, B. M., Marciani, D. J., and Papas, T. S. Proc. Natl. Acad. Sci. USA (1977) 74, 4951. 39. Philipson, L., Andersson, P., Olshevsky, U . , Weinberg, R., and Baltimore, D. Cell (1978) 13 189 40. Fan, H. and Baltimore 41. Gielkens, A. L. J., Bloemendal, H. Proc. Natl. Acad. Sci. USA (1976) 73, 356. 42. Shanmugam, G., Bhadure, S. and Green, M. Biochim. Biophys. Res. Commun. (1974) 56, 697. 43. Shincarial, A. and Joklik, W. Virology (1973) 56, 532. 44. Duesberg, P. H . , Wang, L. H . , Mellan, P . , Mason, W. S., and Vogt, P. K. "ICN-UCLA Symposia on Molecular and Cellular Biology" Vol. IV, pp 107. Edited by D. Baltimore, A. S. Huang, and C. F. Fox. Academic Press, Inc., New York, New York. 1976. 45. Chen, J . H. and Hanafusa, H. J . Virol. (1974) 13, 340. 46. Mueller-Lantsch, N. and Fan, H. Cell (1976) 9, 579. 47. Haywood, W. S. J . Virol. (1977) 24, 47. 48. Mellon, P. and Duesberg, P. H. Nature (1977) 270, 63. 49. Stacey, D. W., Allfrey, V. G., and Hanafusa, H. Proc. Natl. Acad. Sci. USA (1977) 270, 631. 50. Montelaro, R. C., Sullivan, S. J., and Bolognesi, D. P. Virology (1978) In press. 51. Casjens, S. and King, J . Annu. Rev. Biochem. (1975) 44, 555. 52. Hershko, A. and Fry, M. Annu. Rev. Biochem. (1975) 44, 775. 53. Korant, B. D. "Cold Spring Harbor Symposium on Control of Cell Proliferation" Vol. 21, p. 621. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1975. 54. Showe, M. H. and Kellenberg, E. "Control Processes in Virus Multiplication" p. 407. Cambridge University Press, Cambridge, England. 1975. 55. Jamjoom, G. A . , Naso, R. B . , and Arlinghaus, R. B. J . Virol. (1976) 19, 1054. 56. Levin, J . G., Grimley, P. M., Ramseur, J . M., and Berezesky, I. K. J . Virol. (1974) 14, 152. 57. Fleissner, E. and Tress, E. J . Virol. (1973) 12, 1612. 58. Fleissner, E . , Ikeda, H . , Tung, J . S., Vitetta, E . , Tress, E., Hard, J r . , W., Stockert, E . , Boyse, E. A . , Pincus, T., and



200

O'Donnell, P. Cold Spring Harbor Symp. Quant. Biol. (1975) 39, 1057.

59. Dalton, A. J. and Haguenau, F. "Ultrastructure of Animal Viruses and Bacteriophages" pp. 255. Eds. A. J. Dalton and F. Haguenau. Academic Press, New York, New York. 1973. 60. deHarven, E. Adv. Virus Res. (1974) 19, 221. 61. Yoshinaka, Y. and Luftig, R. B. Proc. Natl. Acad. Sci. USA (1977) 74, 3445.

62. Jamjoom, G. A . , Ng, V. L., and Arlinghaus, R. B. J . Virol. (1978) 25, 408. RECEIVED

March 15, 1978.


11 Glycoprotein Inhibitor of Lipoprotein Lipase from Aortic Intima—Its Possible Role in Atherosclerosis PREMANAND V. WAGH Veterans Administration Hospital, 300 East Roosevelt Road, Little Rock, AR 72206

Early observations th f glycoprotein i th mammalian arterial wal -Spreer et a l , 1960; Berenso , 1962) Althoug siderable research interest is centered upon the relationship of glycoproteins in the aortic wall to atherosclerosis and ageing, little is known in regard to the structure and function of these macromolecules. Several soluble glycoproteins have been purified from bovine aorta after extraction of the tissue with neutral buffers (Radhakrishnamurthy et a l , 1964, Maier and Buddecke, 1971). The concentration of arterial glycoproteins in atherosclerosis and in ageing is a subject of question and is being actively investigated. The wet weight of the aorta has been found to be increased in atherosclerosis and among the aged (Manley and Mullinger, 1967). Our data (Wagh et a l , 1973), shown in Table I, provide evidence that the glycoprotein concentration increased significantly in atherosclerotic tissue as expressed by an increase in total neutral sugars, hexosamine and sialic acid. These results suggest that the increased glycoprotein concentration may have a functional role in the formation of atherosclerotic plaque. Several functions have been ascribed to arterial glycoproteins such as transplantation rejection and maintenance of structural integrity (Anderson, 1976). Furthermore, Ishii (1971) demonstrated that preparations of crude glycoprotein obtained from various organs including the aorta of dog inhibited dextran sulfate released lipoprotein lipase (LPL) activity of human plasma. LPL catalyzes the hydrolysis of the triglyceride moiety of chylomicrons and very low density lipoproteins (Eisenberg and Levi, 1975), and is normally located on the surface of endothelial cells where it is physiologically active in the normal clearance of lipoprotein-bound triglycerides (Robinson, 1970). Since the surface of the intimal layer of aortic wall is susceptive to continuous hemodynamic insult and thus may be i n volved in the genesis of atherosclerosis and other physiological events, we focussed our attention here for the purpose of isolation of glycoprotein. One of the shortcomings in the isolation 201 This chapter not subject to U . S . copyright Published 1978 A m e r i c a n C h e m i c a l Society


202


of p u r i f i e d g l y c o p r o t e i n from the i n t i m a l region has been to separate i t from the r e s t of the a o r t i c w a l l . A new procedure (dermatome procedure) was developed i n our l a b o r a t o r y (Roberts et a l , 1974) that r a p i d l y and r e l i a b l y separates the intima i n s u f f i c i e n t q u a n t i t i e s from both porcine and human a o r t a . Studies on Porcine I n t i m a l G l y c o p r o t e i n ( L i p o l i p i n ) . We r e ported on the p u r i f i c a t i o n and c h a r a c t e r i z a t i o n of a novel g l y c o p r o t e i n from the i n t i m a l r e g i o n of porcine aorta (Wagh and Roberts 1972). Swine a o r t a was chosen because i n t h i s s p e c i e s , the aorta i s r e l a t i v e l y f r e e from a t h e r o s c l e r o t i c l e s i o n s and the m a t e r i a l i s r e a d i l y a v a i l a b l e . The p u r i f i c a t i o n procedure involved e x t r a c t i o n of i n t i m a l t i s s u e with n e u t r a l b u f f e r , (Nlfy^SO^ p r e c i p i t a t i o n between 40-90% s a t u r a t i o n and two DEAE-cellulose chromatographic steps. The p u r i f i e d g l y c o p r o t e i n was homogeneous upon a n a l y t i c a l u l t r a c e n t r i f u g a t i o n (4.86 S) and by polyacrylamide disc-gel electrophoresis p r o t e i n was obtained fro Due to the presence of equimolar glucose and galactose and the absence of hydroxylysine i n the molecule, i t was suggested that t h i s g l y c o p r o t e i n was unique i n i t s c h a r a c t e r i s t i c s and t h e r e f o r e of a new type. The i s o e l e c t r i c p o i n t of the p u r i f i e d glycoprot e i n was 4.3 as observed by a n a l y t i c a l i s o e l e c t r i c focusing (Baig and Ayoub, 1976). Subsequent s t u d i e s (Roberts and Wagh, 1976) revealed that sodium dodecyl s u l f a t e - polyacrylamide g e l e l e c t r o p h o r e s i s of the n a t i v e and i t s S-carboxyamidomethyl d e r i v a t i v e a t d i f f e r e n t polyacrylamide concentrations d i d not a f f e c t the molecular weight (72,000 daltons) i n d i c a t i n g the absence of subunits. The carboxyt e r m i n a l amino a c i d was found to be s e r i n e . Attempts to determine the i d e n t i t y of the amino a c i d i n d i c a t e d that the amino group was not f r e e . The g l y c o p r o t e i n d i d not contain an a l k a l i - l a b i l e (0g l y c o s i d i c ) carbohydrate-peptide linkage as tested by $-eliminat i o n r e a c t i o n . The r e l e a s e of monosaccharides from the i n t a c t g l y c o p r o t e i n as a f u n c t i o n of time was studied employing m i l d a c i d h y d r o l y s i s (0.5 M HC1, 80°C) and a l s o by the use of neuraminidase, a-D- and $-D-glucosidases and $-D-N-acetylglucosaminidase. From the observations on the r e l e a s e of monosaccharides and analogy with standard features determined by other i n v e s t i g a t o r s on s o l u b l e a o r t i c g l y c o p r o t e i n s (Radhakrishnamurthy e t a l , 1964; Radhakushnamurthy and Berenson, 1966; Klemer and Nager, 1967; Maier and Buddecke, 1971), a p r e d i c t i o n was made as to the general features of the carbohydrate moiety of the g l y c o p r o t e i n ( F i g . 1 ) . This p o s t u l a t e d s t r u c t u r e must s t i l l withstand r e s u l t s of f u t u r e i n v e s t i g a t i o n s i n c l u d i n g methylation s t u d i e s , o x i d a t i o n with p e r i o date and the use of other s p e c i f i c g l y c o s i d a s e s . The p u r i f i e d porcine i n t i m a l g l y c o p r o t e i n was tested f o r i t s LPL i n h i b i t o r y a c t i v i t y by employing post-heparin dog plasma as the source of enzyme and E d i o l ( s t a b i l i z e d coconut o i l emulsion) as the t r i g l y c e r i d e s u b s t r a t e . Because i t i n h i b i t e d post-heparin


11.

WAGH

Glycoprotein

Inhibitor

of Lipoprotein

203

Lipase

TABLE I C o n c e n t r a t i o n of Component Sugars of Normal and A t h e r o s c l e r o t i c Aortae Component

Sclerotic

Normal

2 Wet weight Xmg/cm ) DDAF (mg/cm ) T o t a l carbohydrate (mg/cm ) Hexose (ug/cm ) Hexosamine (ug/cm ) Uronic a c i d (ug/cm^) S i a l i c a c i d (ug/cm^) T o t a l carbohydrate (ug/m Hexose (ug/mg DDAF) Hexosamine (ug/mg DDAF) Uronic a c i d (ug/mg DDAF) S i a l i c a c i d (ug/mg DDAF)

250.4 (a) 36.9 (b) 2.30 (a) 978 (a) 698 (c) 308 (NS) 317 (b)

195.7 39.3 2.10 870 651 297 285

2

(a) 19.4 (a) 8.5 (NS) 8.8 (a)

17.0 7.9 7.5

The c o n c e n t r a t i o n o f components are expressed as weight per u n i t area and weight per u n i t dry, d e f a t t e d , a s h - f r e e r e s i d u e (DDAF). A l l v a l u e s represent averages from 15 samples of aortae obtained from i n d i v i d u a l s of 7 0 . 3 + 2 . 8 (mean + S.E.M.) years of age. P a i r e d t t e s t : (a) = P100 000 IgM, IgD H-2K, H-2D, I a Synaptic g l y c o p r o t e i n s Synaptic g l y c o p r o t e i n s Nicotinic acetylcholine receptor

f

Band I I I Band I I I + g l y c o p h o r i n Band I I I Band I I I + 2 other glycoprote ins Glycophorin Glycophorin A A s i a l o g l y c o p h o r i n (T-Ag) Major g l y c o p r o t e i n Glycoproteins Glycoprote i n s HLA antigens Component 5.1 T-25, T-200 Major antigen H-2K, H-2D, I a IgM IgD Glycoproteins: MW 20,000-80,000

Material Purified

Table 1:

Mu lymphocytes Mu B lymphocytes Mu lymphocytes Rat b r a i n Rat b r a i n Rat b r a i n o r e l e c t r i c e e l

TX-100 TX-100 TX-100 NaDOC SDS TX-100

PA-^CA^CAj48 Seph WGA, BPA-Seph 48 PA-Seph 48 PA-Seph 48 LCA-, WGA-Seph 49 Con A-, UEA-Seph 50,51 Con A, WGA-Seph, 52 RCAj-glass beads

TX-100

Mu lymphocytes

36 37 38 40 41 42 43 44 45 46 47 47

36

RCAj-Seph WGA-Seph WGA-Seph PNA-Seph RCAj-Seph Con A-Seph LCA-Seph LCA-Seph Con A-Seph Con A, PA-Seph LCA-Seph Con A-Seph Con A-Seph

TX-100 TX-100 SDS DDG (Empigen BB) Emulphogene NaDOC NaDOC NaDOC TX-100 NP-40, NaDOC NaDOC NP-40 NP-40

34 34 35

Ref.

Con A-Seph LCA-Seph Con A-Seph

Affinity Adsorbent

HuRBC

TX-100 TX-100 DTAB

Membrane S o l u b i l i z i n g Agent

HuRBC HuRBC HuRBC + NANase Bovine RBC P i g lymphocytes P i g lymphocytes Hu lymphocytes Ra thymocytes Mu thymocytes Rat thymocytes Mu thymocytes Mu B lymphocytes

HuRBC HuRBC HuRBC

Source o f Membrane

ISOLATION AND CHARACTERIZATION OF MEMBRANE GLYCOPROTEINS USING IMMOBILIZED LECTINS


Rat spermatozoa Enveloped v i r u s e s ( i n f l u e n z a , Sendai) Mu mammary tumor v i r u s D i c t y o s t e l i u m discoidum

Con A-receptors V i r a l glycoprotein

TX-100 NaDOC

Membrane S o l u b i l i z i n g Agent NaDOC CTAB LIS LIS LIS SDS DDG NaDOC

Affinity Adsorbent Con A-Seph Con A-Seph Con A-Seph WGA-Seph Con A-Seph WGA-Seph RCAj-Seph RCAj-, Con A-, WGA, SBA-Seph Con A-Seph LCA-Seph 61 62

Ref. 53 54 55 56 57 58 59 60

Serdox NNP10 Con A-Seph Major g l y c o p e p t i d e s 63 Membrane-bound enzymes NaDOC Con A-Seph 64 (cf, 5'-nucleotidase, a l k a l i n e phosphatase) A b b r e v i a t i o n s : BPA, Bauhinia purpurea a g g l u t i n i n ; CTAB, cetyltrimethylammonium bromide; DDG, d i m e t h y l dodecylglycine (Empigen BB); DTAB, dodecyltrimethy1ammonium bromide; Emulphogene, a l k o x y p o l y (ethyleneoxy)ethanol; Ham, hamster; Hu, human; HuRBC, human r e d blood c e l l s ; Mu, murine; NaDOC, sodium deoxycholate; NP-40, Nonidet P-40, p o l y o x y e t h y l e n e g l y c o l ( 6 - 7 ) - p - t - o c t y l p h e n o l ; Ra, r a b b i t ; SDS, sodium dodecyl s u l f a t e ; Seph, Sepharose; Serdox NNP10, a n o n i o n i c detergent, Servo, Delden, The Netherlands; TX-100, T r i t o n X-100, p o l y o x y e t h y l e n e g l y c o l ( 9 - 1 0 ) - p - t - o c t y l p h e n o l .

Source o f Membrane Bovine b r a i n Bovine r e t i n a Hu p l a t e l e t s Mu LI210 lymphoma Mu L-929 c e l l s Hu HeLa c e l l s Ham embryo f i b r o b l a s t s (NIL) Mu E h r l i c h a s c i t e s carcinoma

OF MEMBRANE GLYCOPROTEINS USING IMMOBILIZED LECTINS

Material Purified Tissue f a c t o r Rhodopsin Major g l y c o p r o t e i n WGA-receptors Con A-receptor WGA-receptors G a l a c t o p r o t e i n s a and b Glycoproteins

Table 1 ( c o n ' t . ) : ISOLATION AND CHARACTERIZATION

GLYCOPROTEINS AND

262

GLYCOLIPIDS IN

DISEASE PROCESSES

g l y c o p r o t e i n (glycophorin) i n h i b i t o r of WGA, Agaricus bisporus a g g l u t i n i n and PHA. The RCAj-Sepharose column adsorbed s e v e r a l g l y c p r o t e i n s c o n t a i n i n g b i n d i n g s i t e s f o r RCAj and Abrus p r e c a t o r i u s a g g l u t i n i n , while most of the g l y c o p r o t e i n receptors f o r A. bisporus a g g l u t i n i n , PHA, LCA and WGA were not r e t a i n e d by the column. P u r i f i c a t i o n of g l y c o p h o r i n i n a one-step p r e p a r a t i v e procedure was obtained by Kahane et a l . (37) a f t e r SDS s o l u b i l i z a t i o n of HuRBC ghosts i n the presence of r e l a t i v e l y high s a l t and a d s o r p t i o n to WGA-Sepharose. The r e c e p t o r f o r peanut a g g l u t i n i n was i s o l a t e d from neuraminidase-treated, dimethyl d o d e c y l g l y c i n e s o l u b i l i z e d HuRBC by chromatography on PNA-polyacrylhydrazidoSepharose (38). In t h i s l a s t procedure c e l l surface galactose residues on i n t a c t neuraminidase-treated HuRBC were r a d i o l a b e l e d by o x i d a t i o n w i t h galactose oxidase f o l l o w e d by r e d u c t i o n w i t h N a B H t p r i o r to t h e i r p u r i f i c a t i o n . The use of p o l y a c r y l h y d r a zido-Sepharose a f f o r d e d a high c a p a c i t y charge-free and nonl e a c h i n g supporting m a t r i a d s o r p t i o n and high r e c o v e r i e PNA-polyacrylhydrazido-Sepharose w i t h l a c t o s e c o n s i s t e d of two g l y c o p r o t e i n s (MW ^82,000 and ^46,000, r e s p e c t i v e l y ) which resemb l e d a s i a l o g l y c o p h o r i n i n e l e c t r o p h o r e t i c m i g r a t i o n and amino a c i d composition. L i k e a s i a l o g l y c o p h o r i n , the PNA receptor formed p r e c i p i t i n bands w i t h SBA and RCA-j- but f a i l e d to p r e c i p i t a t e w i t h Con A, Dolichos b i f l o r u s a g g l u t i n i n and WGA (38). In an e f f o r t to determine the degree to which the carbohydrate s i d e chains of HuRBC membrane g l y c o p r o t e i n s vary between s p e c i e s , Emerson and K o r n f e l d (40) used non-ionic detergent s o l u b i l i z e d bovine e r y t h r o c y t e membranes as a source of g l y c o p r o t e i n s . RCAjSepharose adsorbed the 230,000 mol. wt. major g l y c o p r o t e i n of bovine e r y t h r o c y t e membrane which contains ^80% carbohydrate, mostly l i n k e d o - g l y c o s i d i c a l l y . V i r t u a l l y a l l the carbohydrate of the membrane (excluding g l y c o l i p i d s ) was borne by t h i s g l y c o p r o t e i n . L e c t i n A f f i n i t y P u r i f i c a t i o n of Lymphoid C e l l Membrane Components Lymphoid c e l l membranes c o n t a i n a wide v a r i e t y of g l y c o p r o t e i n molecules compared to RBC; t h e r e f o r e , l e c t i n a f f i n i t y columns are g e n e r a l l y u s e f u l f o r p a r t i a l p u r i f i c a t i o n or enrichment of g l y c o p r o t e i n s . For example, almost a l l the g l y c o p r o t e i n s of p i g lymphocyte plasma membrane s o l u b i l i z e d i n sodium deoxycholate (NaDOC) were adsorbed on columns of Con A-Sepharose (41) or LCA-Sepharose (42). The l a t t e r immobilized l e c t i n was much more e f f i c i e n t i n p u r i f y i n g the s o l u b i l i z e d membrane g l y c o p r o t e i n s . Con A-Sepharose bound 20% of the a p p l i e d m a t e r i a l i n a n o n s p e c i f i c manner, and the y i e l d of g l y c o p r o t e i n s o f f the Con A-Sepharose column was low ( 5 % ) , whereas LCA-Sepharose y i e l d e d more than twice the amount obtained w i t h Con A-Sepharose, and the recovery was 95%. SDS-polyacrylamide g e l e l e c t r o p h o r e s i s (SDS-PAGE) of the s a c c h a r i d e - e l u t e d LCA-Sepharose f r a c t i o n s revealed a t l e a s t ten p r o t e i n bands t h a t s t a i n e d f o r carbohydrate w i t h p e r i o d a t e - S c h i f f reagent. P u r i f i e d LCAreceptors i n h i b i t e d LCA-induced lymphocyte t r a n s f o r m a t i o n 10 times 3

+


14.

LOTAN AND NICOLSON

Membrane

Glycoproteins

263

more e f f e c t i v e l y compared t o the u n f r a c t i o n a t e d membrane g l y c o p r o t e i n s , and a t l e a s t one o f the LCA-receptor g l y c o p r o t e i n s was a l s o capable o f i n h i b i t i n g PHA-induced lymphocyte s t i m u l a t i o n . I n the above s t u d i e s f u r t h e r c h a r a c t e r i z a t i o n was not performed t o e l u c i d a t e the nature and f u n c t i o n o f the l e c t i n r e a c t i v e m a t e r i a l s ; however, i n other r e p o r t s defined components have been i s o l a t e d by l e c t i n a f f i n i t y chromatography. For example, the h i s t o c o m p a t i b i l i t y antigens o f human lymphocytes (HLA) were s o l u b i l i z e d i n sodium deoxycholate and then p u r i f i e d by chromatography on LCA-Sepharose (43) . Improvements have been made i n l e c t i n a f f i n i t y chromatography techniques t o increase s p e c i f i c i t y and y i e l d as w e l l as reduce nons p e c i f i c a d s o r p t i o n . In an attempt t o increase the s e l e c t i v i t y o f lectin-Sepharose chromatography, S c h m i d t - U l l r i c h e t a l . (4£) adsorbed T r i t o n X - 1 0 0 - s o l u b i l i z e d r a b b i t thymocyte membrane g l y c o p r o t e i n s onto Con A-Sepharos i othe procedures th e l u t i o n was performed i c e n t r a t i o n s o f the hapte methy a-glucoside procedur lowed p r e f e r e n t i a l e l u t i o n o f receptors according t o t h e i r r e l a t i v e b i n d i n g a f f i n i t i e s t o Con A. Using these procedures a s i a l o g l y c o p r o t e i n t h a t has been designated component 5.1 because o f i t s e l e c t r o p h o r e t i c m o b i l i t y i n SDS-PAGE was e l u t e d i n a homogeneous form. This Con A receptor has an apparent MW o f 55,000 d a l t o n s but tends t o aggregate i n s o l u t i o n . When t h i s p r e p a r a t i o n was examined f u r t h e r , two molecules were found t h a t d i f f e r i n t h e i r carbohydrate side chains, and these could be recognized by cross immune e l e c t r o p h o r e s i s . Trowbridge e t a l . (45) reported t h a t specific I - l a b e l e d surface antigens (T25 and T200) on mouse thymocytes could be p u r i f i e d from NaDOC-solubilized membranes by a d s o r p t i o n t o e i t h e r Con A- o r PA-Sepharose. Upon a n a l y s i s by SDS-PAGE these components separated i n t o s e v e r a l s p e c i e s , p o s s i b l y due to heterogeneity o f t h e i r carbohydrate s i d e chains. Fabre and W i l l i a m s (46) i s o l a t e d a major antigen from the surfaces o f r a t thymocyte membranes by s p e c i f i c a d s o r p t i o n on LCA-Sepharose, and t h i s behavior demonstrated t h a t the antigen i s a g l y c o p r o t e i n . N i l s s o n and Waxdal (47) a l s o l a b e l e d p r o t e i n s on the surface o f murine lymphocytes by l a c t o p e r o x i d a s e - c a t a l y z e d I - i o d i n a t i o n or by c u l t u r i n g the c e l l s i n media c o n t a i n i n g H - l e u c i n e o r H fucose. C e l l membranes were s o l u b i l i z e d i n NP-40 before p a s s i n g the s o l u b i l i z e d g l y c o p r o t e i n s through Con A-Sepharose. The m a t e r i a l s p e c i f i c a l l y e l u t e d from Con A-Sepharose was c h a r a c t e r i z e d by imm u n o p r e c i p i t a t i o n w i t h s p e c i f i c a n t i s e r a , and i t was found t h a t Con A-receptors i n c l u d e d antigens coded by the h i s t o c o m p a t i b i l i t y - 2 complex such as H-2K, H-2D and l a . I n a d d i t i o n , the immobilized Con A a l s o adsorbed immunoglobulins M and D from s o l u b i l i z e d B lymphocyte membranes. E s s e n t i a l l y the same r e s u l t s were obtained r e c e n t l y by Iwata e t a l . (48) who e x t e n s i v e l y f r a c t i o n a t e d murine lymphoid c e l l surface components on a b a t t e r y o f lectin-Sepharose columns (see Table 1). This l a s t study demonstrated t h a t the use of s e v e r a l lectin-Sepharose columns f o r f r a c t i o n a t i o n o f c e l l 1 2 5

1 2 5

3


3

GLYCOPROTEINS AND

264

GLYCOLIPIDS IN

DISEASE PROCESSES

membrane components narrows the range of i s o l a t e d components. For example, PLA-, LCA- and RCAj-Sepharose s p e c i f i c a l l y bound g l y c o p r o t e i n s of MW i n the range 20,000-80,000 d a l t o n s , whereas g l y c o p r o t e i n s i n the higher MW (>100,000) range t h a t were not adsorbed on the l a t t e r columns were p r e f e r e n t i a l l y bound by WGA- and BPASepharose (48). L e c t i n A f f i n i t y Chromatography of Nerve C e l l Components L e c t i n a f f i n i t y chromatography has been e s p e c i a l l y u s e f u l i n the p u r i f i c a t i o n of carbohydrate-containing macromolecules from nervous t i s s u e . G l y c o p r o t e i n s present i n NaDOC-solubilized syna p t i c plasma membranes prepared from r a t b r a i n s t h a t were i n j e c t e d w i t h H-fucose 16 hr e a r l i e r have been f r a c t i o n a t e d by chromatography on LCA- or WGA-Sepharose (49). In these s t u d i e s LCA-Sepharose adsorbed 40-45% of the a p p l i e d r a d i o a c t i v i t y and bound most of the 7-8 g l y c o p r o t e i n s known to be present i n the s y n a p t i c membranes; whereas WGA-Sepharose bound o n l y 25-30% of the r a d i o a c t i v i t y suggesting t h a LCA-Sepharose were not boun and Mahler (49) decided t o use both of these a f f i n i t y columns i n tandem such t h a t the sample a p p l i e d onto LCA-Sepharose was allowed to pass i n t o the WGA-Sepharose column. A f t e r washing both columns and s e q u e n t i a l l y e l u t i n g w i t h the appropriate monosaccharide i n h i b i t o r s f o r each l e c t i n , four f r a c t i o n s were obtained: LCA-negative, WGA-negative; LCA-negative, WGA-positive; L C A - p o s i t i v e , WGA-negat i v e ; and L C A - p o s i t i v e , WGA-positive. When analyzed by SDS-PAGE, each of the f r a c t i o n s had d i s t i n c t p a t t e r n s w i t h some o v e r l a p of molecular weight c l a s s e s . Zanetta e t a l . (50,51) have f r a c t i o n a t e d r a t b r a i n s y n a p t i c membranes d i s s o l v e d i n SDS on Con A- or UEASepharose. E l u t i o n from the former a f f i n i t y column was w i t h methyl a-glucoside; however, L-fucose was unable to r e l e a s e bound m a t e r i a l but c o u l d i n h i b i t b i n d i n g t o the UEA-Sepharose. I n c r e a s i n g the SDS c o n c e n t r a t i o n i n the e l u t i o n b u f f e r (without saccharides) r e leased the adsorbed m a t e r i a l , and subsequent PAGE a n a l y s i s revealed a l a r g e number of carbohydrate c o n t a i n i n g components. P a r t of the heterogeneity i n g l y c o p r o t e i n s e l u t e d by these procedures was a t t r i b u t e d t o the f a c t t h a t the s y n a p t i c membranes were d e r i v e d from a heterogeneous p o p u l a t i o n of neurons. S p e c i f i c , f u n c t i o n a l g l y c o p r o t e i n s have been i s o l a t e d from s o l u b i l i z e d b r a i n t i s s u e by lectin-Sepharose chromatography. Nicot i n i c a c e t y l c h o l i n e r e c e p t o r , T r i t o n X-100 s o l u b i l i z e d from a crude, p o s t - n u c l e a r membrane f r a c t i o n of r a t c e r e b r a l c o r t e x or from membranes of Torpedo c a l i f o r n i c a e l e c t r o p l a x , was r e t a i n e d on immobilized Con A, WGA or RCAj (52) . In another study p a r t i a l p u r i f i c a t i o n of coagulant arylamidase and a l k a l i n e phosphatase enzymes s o l u b i l i z e d i n NaDOC from bovine b r a i n was achieved by a d s o r p t i o n on Con A-Sepharose (53). L e c t i n A f f i n i t y Chromatography of Components from Other C e l l Types In c o n t r a s t to the m u l t i t u d e of l e c t i n receptors found i n s y n a p t i c membranes, s e v e r a l systems have been described i n which 3


L O T A N AND NICOLSON

14.

Membrane

Glycoproteins

265

a s i n g l e receptor was obtained. For example, s o l u b i l i z a t i o n o f bovine r e t i n a l r o d outer segments w i t h cetyltrimethylammonium bromide f o l l o w e d by chromatography on Con A-Sepharose y i e l d e d a homogeneous rhodopsin (54). The major human p l a t e l e t membrane g l y c o p r o t e i n (MW ^100,000) was s o l u b i l i z e d by l i t h i u m d i i o d o s a l i c y l a t e -phenol e x t r a c t i o n o f I - l a b e l e d membranes and p u r i f i e d on Con ASepharose (55). Using s i m i l a r e x t r a c t i o n methods, four WGA-recept o r s o f MW 40,000-60,000 were i s o l a t e d from L1210 c e l l s (560, and a s i n g l e Con A receptor having an apparent MW o f 100,000 and a t l e a s t f i v e carbohydrate s i d e chains was i s o l a t e d from mouse L929 c e l l s (57J. M u l t i p l e WGA receptors o f MW i n the range 40,000300,000 were i s o l a t e d from S D S - s o l u b i l i z e d membranes o f il a b e l e d HeLa c e l l s (58). E x t r a c t i o n o f hamster embryo f i b r o b l a s t s (NIL) l a b e l e d w i t h galactose-oxidase and NaB^H^ w i t h 8M urea o r s o l u b i l i z a t i o n w i t h d i e m t h y l d o d e c y l g l y c i n e f o l l o w e d by chromatography on RCASepharose allowed p u r i f i c a t i o f galactoprotei (LETS) and g a l a c t o p r o t e i by an a c t i n - l i k e p r o t e i n galactoprotei appeare composi t i o n a l l y heterogeneous. While most o f the above i n v e s t i g a t o r s d i d not r e p o r t problems i n u s i n g lectin-Sepharose columns, an e x t e n s i v e study by Nachbar e t a l . (60) demonstrated t h a t i n c e l l membranes o f E h r l i c h a s c i t e s carcinoma c e l l s s o l u b i l i z e d i n NaDOC there are s e v e r a l carbohydrate c o n t a i n i n g components t h a t adsorb t o Con A-, WGA-, SBA- and RCA-Sepharose columns. Using C-glucosaminel a b e l e d membrane components the r e c o v e r i e s from the v a r i o u s columns were determined, and i t was found t h a t s p e c i f i c e l u t i o n o f the WGAand the Con A-Sepharose columns r e s u l t e d i n r e l e a s e o f only 55-70% of the bound m a t e r i a l . The remaining t i g h t l y bound m a t e r i a l may have been i r r e v e r s i b l y h e l d t o the column by hydrophobic i n t e r a c t i o n s . The r e c e p t o r s f o r the d i f f e r e n t l e c t i n s appeared s i m i l a r on SDS-PAGE, and some immobilized l e c t i n s p u r i f i e d the same g l y c o p r o t e i n r e c e p t o r s . P u r i f i c a t i o n o f g l y c o p r o t e i n s from s o l u b i l i z e d enveloped v i r u s e s (62,63) and o f membrane-associated enzymes on l e c t i n a f f i n i t y columns has a l s o been d e s c r i b e d (Table 1 ) . Although one-step p u r i f i c a t i o n t o homogeneity o f a membrane g l y c o p r o t e i n has only been reported f o r HuRBC g l y c o p h o r i n (37), most s t u d i e s i n d i c a t e t h a t a f f i n i t y chromatography on immobilized l e c t i n columns i s the method o f c h o i c e , a t l e a s t i n p r e l i m i n a r y stages o f membrane g l y c o p r o t e i n p u r i f i c a t i o n . In e a r l i e r s t u d i e s (34,36,41,42) no attempt was made t o r a d i o a c t i v e l y l a b e l c e l l membrane components p r i o r t o s o l u b i l i z a t i o n and chromatography, b u t more recent i n v e s t i g a t i o n s have demonstrated t h a t p r e l a b e l i n g membrane components e i t h e r by l a c t o p e r o x i d a s e - c a t a l y z e d I-iodinat i o n (47,55) o r neuraminidase f o l l o w e d by galactose oxidase and NaB Hi (_38/57) f a c i l i t a t e s the f r a c t i o n a t i o n procedure and i n s u r e s t h a t the i s o l a t e d components o r i g i n a t e from the plasma membrane s u r f a c e . I n a d d i t i o n , the l a b e l e d components may be detected by autoradiography o r fluorography a f t e r s e p a r a t i o n by SDS-PAGE, even i f they are obtained i n s m a l l q u a n t i t i e s . Many o f the saccharides found i n membrane g l y c o p r o t e i n s are 1 2 5

1 2 5

ll+

1 2 5

3

+


266


u b i q u i t o u s ; t h e r e f o r e , o n l y p a r t i a l p u r i f i c a t i o n should be expected when a s i n g l e lectin-Sepharose column i s used. The use o f a hapten g r a d i e n t i n the e l u t i o n step (44) o r combinations o f immobilized l e c t i n s w i t h d i f f e r e n t sugar s p e c i f i c i t i e s (48,49) has proven t o be very powerful i n the s e p a r a t i o n o f membrane g l y c o p r o t e i n s . The choice o f detergent f o r the s o l u b i l i z a t i o n o f membranes and s t a b i l i z a t i o n o f membrane g l y c o p r o t e i n s p r i o r t o a p p l i c a t i o n onto lectin-Sepharose columns seems i n many r e p o r t s t o have been random, or i n the best examples, e m p i r i c a l . This problem has been e l i m i nated i n p a r t by a systematic a n a l y s i s o f the e f f e c t s o f s e v e r a l z w i t t e r i o n i c , c a t i o n i c , a n i o n i c and non-ionic detergents on g l y c o p r o t e i n b i n d i n g t o and s p e c i f i c e l u t i o n from f i v e lectin-Sepharose columns (39). Our r e s u l t s (Figure 3) i n d i c a t e t h a t f o r most l e c t i n s , NaDOC caused marked decreases i n b i n d i n g a f f i n i t y . The z w i t t e r i o n i c detergent dimethyl d o d e c y l g l y c i n e decreased the e f f i c i e n c y o f the Con A- and SBA-Sepharose w h i l e the non-ionic detergents NP-40 and T r i t o b i l i z e d l e c t i n s t e s t e d (39_ n i t y chromatography techniques w i l l be g r e a t l y f a c i l i t a t e d by opt i m i z i n g v a r i o u s l e c t i n s , detergents, s o l u t i o n c o n d i t i o n s , columns and techniques f o r s p e c i f i c e l u t i o n o f bound g l y c o p r o t e i n s . DYNAMICS OF CELL SURFACE LECTIN RECEPTORS One o f the most u s e f u l approaches f o r studying c e l l s u r f a c e g l y c o p r o t e i n dynamics has been the u t i l i z a t i o n o f l e c t i n s as probes f o r f l u o r e s c e n t o r e l e c t r o n microscopy (18,65). Observing the movement and r e d i s t r i b u t i o n o f c e l l surface-bound l e c t i n s has allowed estimates t o be made on the r a t e s o f l a t e r a l d i f f u s i o n and, i n c e r t a i n cases, i n t e r n a l i z a t i o n o f the l e c t i n - r e c e p t o r comp l e x e s (_5) . These experiments have been g e n e r a l l y conducted w i t h f l u o r e s c e i n - l a b e l e d l e c t i n s and UV-fluorescent microscopy o r f e r r i t i n - , hemocyanin- o r p e r o x i d a s e - l a b e l e d l e c t i n s and e l e c t r o n microscopy (65). By l a b e l i n g p r e f i x e d o r u n f i x e d c e l l s u r f a c e s w i t h these probes and then observing the a l t e r a t i o n s i n s u r f a c e d i s t r i b u t i o n s o f the probes under a v a r i e t y o f experimental c o n d i t i o n s , a dynamic p i c t u r e o f l e c t i n r e c e p t o r movements has been obtained i n s e v e r a l b i o l o g i c a l systems. These observations have l e d i n v e s t i g a t o r s t o conclude the f o l l o w i n g : F i r s t , the i n h e r e n t topographic d i s t r i b u t i o n s o f most l e c t i n r e c e p t o r s (as found on p r e f i x e d c e l l s o r c e l l s l a b e l e d a t 0°C) are uniform and random across the e n t i r e c e l l s u r f a c e . Some exceptions have been noted, however, i n s p e c i a l i z e d c e l l s o f h i g h asymmetry such as sperm where WGA-receptors were found t o be l o c a l i z e d o n l y i n c e r t a i n regions o f the sperm head (66) and nerve c e l l s where Con A recept o r s were found t o be r e l a t i v e l y immobile and predominately i n the s y n a p t i c c l e f t (67). Most l e c t i n r e c e p t o r r e d i s t r i b u t i o n s t u d i e s have r e v e a l e d t h a t l i g a n d - r e c e p t o r complexes undergo r a p i d change from random, uniform d i s t r i b u t i o n s t o form c l u s t e r s and patches (68-72). I n some c e l l types the c l u s t e r e d l i g a n d - r e c e p t o r complexes coalesce t o form a s i n g l e p o l a r "cap" o r aggregate o f c r o s s l i n k e d l i g a n d - r e c e p t o r complexes. A f t e r r e d i s t r i b u t i o n the c l u s -


14.


Membrane

Glycoproteins

I. Serum Glycoprotein-Type

LECTIN

267

Oligosaccharide

NANA-Gal-GlcNAc-Man. Man-GlcNAc-GlcNAc-Asn

LPA Gal-GlcNAc-Man*

t Fuc

RCA,SBA,PNA PHA WGA Con A,LCA,PA LTA,UEA I I . Mucin-Type

Oligosaccharide NANA-Gal-GalNAc-Ser(Thr)

LPA Gal-GalNAc-Ser(Thr) PNA

I

1

SBA,RCA Figure 2. Possible binding region for lectins on oligosaccharide side chains of a hypothetical cell membrane glycoprotein. LPA, Limulus polyphemus agglutinin; RCA, Ricinus communis agglutinin; SBA, soybean agglutinin; PNA, peanut agglutinin; PHA, phytohemagglutinin from Phaseolus vulgaris; WGA, wheat germ agglutinin; Con A, concanavalin A; LCA, Lens culinaris agglutinin; PA, pea agglutinin; LTA, Lotus tetragonolobus agglutinin; UEA, Ulex europeus agglutinin.

• RC

60 20

1 t 1 I it 1 J I 11 TXIOOPj,

100 60 20

3

100 60 20 100

a SBA • Con Am WGA

PNA

NP40

100

-D

DDG

1 m 1 ii y DOC

1

60 20 100 60 20

•E

c

i! i 2

0.1

T A B

1.0

DETERGENT CONC. (%)

2.5

Biochemistry

Figure 3. Effect of increasing concentrations of detergents on the glycoprotein binding efficiencies of immobilized lectins (for experimental details see Ref. 39)


268


t e r e d o r capped l i g a n d - r e c e p t o r complexes may be i n t e r n a l i z e d by endocytosis o r shed from the c e l l . The extents and r a t e s o f r e d i s t r i b u t i o n , i n t e r n a l i z a t i o n and shedding appear t o be c o n t r o l l e d by the c e l l type and the biochemical nature o f the l i g a n d and i t s r e c e p t o r s (reviewed i n 4,18,73). I t has been noted t h a t w h i l e a l e c t i n a t one c o n c e n t r a t i o n can induce r e d i s t r i b u t i o n o f i t s r e ceptors i n t o p o l a r caps, a t other c o n c e n t r a t i o n s ( u s u a l l y much h i g h e r ) , i t can i n h i b i t the movement o f d i f f e r e n t c e l l surface r e c e p t o r s (74,751 Q u a n t i t a t i o n o f the r o t a t i o n a l and l a t e r a l m o b i l i t i e s o f c e l l surface g l y c o p r o t e i n s on c e l l surfaces has been achieved by use o f f l u o r e s c e i n i s o t h i o c y a n a t e - l a b e l e d Con A. S h i n i t z k y e t a l . (76) and Inbar e t a l . (77) have s t u d i e d the r o t a t i o n a l m o b i l i t i e s o f Con A receptor complexes on c e l l surfaces by fluorescence p o l a r i z a t i o n . The recent i n t r o d u c t i o n o f the fluorescence photobleaching recovelry technique allowed Jacobso a l (78) d Schles s i n g e r e t a l (79) t o measur the Con A-receptor complexes w i t h i s u r f a c e membranes o s e v e r a c u l t u r e d c e l l s . Such s t u d i e s and others i n d i c a t e d t h a t the m o b i l i t i e s o f c e l l membrane g l y c o p r o t e i n s are determined t o a l a r g e ext e n t by t h e i r i n t e r a c t i o n s w i t h other membrane components and membrane-associated s t r u c t u r e s such as m i c r o f i l a m e n t s and microtubules (4,5^) . I t i s hoped t h a t i s o l a t i o n and c h a r a c t e r i z a t i o n of s p e c i f i c membrane g l y c o p r o t e i n s w i l l increase the understanding of the exact molecular s t r u c t u r e and o r g a n i z a t i o n o f c e l l membranes. Acknowledgments; Supported by PHS NCI Contract CB-74153, PHS NCI Grant CA-15122 and American Cancer S o c i e t y Grant BC-211A t o G.L. Nicolson.


14.


Membrane

Glycoproteins

269

LITERATURE CITED

1. 2. 3. 4. 5.

6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.

Singer, S.J. & Nicolson, G.L. (1972) Science 175, 720. Edidin, M. (1974) Ann. Rev. Biophys. Bioeng. 2, 179. Singer, S.J. (1974) Ann. Rev. Biochem. 43, 805. Nicolson, G.L. (1976) Biochim. Biophys. Acta 457, 57. Nicolson, G.L., Poste, G. & Ji, T.H. (1977) In "Dynamic Aspects of Cell Surface Organization", Vol. 3 of "Cell Surface Reviews" (G. Poste & G.L. Nicolson, eds.), p. 1, NorthHolland, Amsterdam. Bretscher, M.S. (1971) J . Mol. Biol. 59, 351. Segrest, J.P., Kahne, I . , Jackson, R.L. et a l . (1973) Arch. Biochem. Biophys. 155, 167. Morrison, M., Mueller, T.J. & Huber, C.T. (1974) J . Biol. Chem. 249, 2658. Hunt, R.C. & Brown, J.C. (1975) J . Mol. Biol. 97, 413. Gahmberg, C.G. (1977) In"DynamicAspects of Cell Surface Organization", Vol G.L. Nicolson, eds.) Pinto da Silva, P. & Nicolson, G.L. (1974) Biochim. Biophys. Acta 363, 311. Wang, K. & Richards, F.M. (1974) J . Biol. Chem. 249, 8005. Hynes, R.O. & Pearlstein, E.S. (1976) J . Supramol. Struct. 4, 1. Nicolson, G.L. & Painter, R.G. (1973) J . Cell Biol. 59, 395. Ji, T.H. & Nicolson, G.L. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 2212. Elgsaeter, A . , Shotton, D.M. & Branton, D. (1976) Biochim. Biophys. Acta 426, 101. L i s , H. & Sharon, N. (1973) Ann. Rev. Biochem. 43, 541. Nicolson, G.L. (1974) Int. Rev. Cytol. 39, 89. Spiro, R.G. (1973) Adv. Protein Chem. 27, 349. Spiro, R.G. & Bohyroo, V.D. (1974) J . Biol. Chem. 249, 5704. Toyoshima, S., Fukada, M. & Osawa, T. (1974) Biochemistry 11, 4000. Thomas, D.B. & Winzler, R.J. (1969) J . Biol. Chem. 244, 5943. Kornfeld, S. & Kornfeld, R. (1971) In "Glycoproteins in Blood Cells and Plasma" (G.A. Jamieson & T.J. Greenwalt, eds.), p. 50, J.B. Lippincott, Philadelphia. Tomita, M. & Marchesi, V.T. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 2964. Newman, R.A., Glöckner, W.M. & Uhlenbruck, G.G. (1976) Eur. J . Biochem. 64, 373. Finne, J . (1975) Biochim. Biophys. Acta 412, 317. Funakoshi, I . , Nakada, H. & Yamashina, I. (1974) J . Biochem. (Tokyo) 76, 319. Nakada, H., Funakoshi, I . & Yamashina, I. (1975) J . Biochem. (Tokyo) 78, 863. Kornfeld, S. & Kornfeld, R. (1969) Proc. Natl. Acad. Sci. U.S.A. 62, 1439. Kornfeld, R. & Kornfeld, S. (1970) J . Biol. Chem. 245, 2536.


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Kornfeld, S., Rogers, J . & Gregory, W. (1971) J . Biol. Chem. 246, 6581. Akiyama, Y. & Osawa, T. (1972) Hoppe-Seyler's Z. Physiol. Chem. 353, 323. Marchesi, V.T., Furthmayr, H. & Tomita, M. (1976) Ann. Rev. Biochem. 46, 667. Findlay, J.B.C. (1974) J . Biol. Chem. 249 4398. Ross, A.H. & McConnell, H.M. (1977) Biochem. Biophys. Res. Commun. 74, 1318. Adair, W.L. & Kornfeld, S. (1974) J . Biol. Chem. 249 4696. Kahane, I . , Furthmayr, H. & Marchesi, V.T. (1976) Biochim. Biophys. Acta 426, 464. Carter, W.G. & Sharon, N. (1977) Arch. Biochem. Biophys. 180, 570. Lotan, R., Beattie, G., Hubbell, W.L. et a l . (1977) Biochemistry 16, 1787 Emerson, W.A. & Kornfeld Allan, D., Auger, J Crumpton, (1972) 236, 23. Hayman, M.J. & Crumpton, M.J. (1972) Biochem. Biophys. Res. Commun. 47, 923. Snary, D., Goodfellow, P., Hayman, M.J. et a l . (1974) Nature New Biol. 247, 457. Schmidt-Ullrich, R., Wallach, D.F.H. & Hendricks, J . (1975) Biochim. Biophys. Acta 382, 295. Trowbridge, I.S., Nilsen-Hamilton, M., Hamilton, R.T. et a l . (1977) Biochem. J . 163, 211. Fabre, J.W. & Williams, A.F. (1977) Transplantation 23, 349. Nilsson, S.F. & Waxdal, M.J. (1976) Biochemistry 15, 2698. Iwata, M., Ide, H., Terao, T. et a l . (1977) J . Biochem. (Tokyo) 82, 661. Gurd, J.W. & Mahler, H.R. (1974) Biochemistry 13, 5193. Zanetta, J.P., Morgan, I.G. & Gombos, G. (1975) Brain Res. 83, 337. Zanetta, J.P., Reeber, A . , Vincendon, G. et a l . (1977) Brain Res. 138, 317. Salvaterra, P.M., Gurd, J.M. & Mahler, H.R. (1977) J . Neurochem. 29, 345. P i t l i c k , F.A. (1976) Biochim. Biophys. Acta 428, 27. Steinemann, A. & Stryer, L. (1973) Biochemistry 12, 1499. Nachman, R.L., Hubbard, A. & Ferris, B. (1973) J . Biol. Chem. 248, 2928. Jansons, V.K. & Burger, M.M. (1973) Biochim. Biophys. Acta 291, 127. Hunt, R.C., Bullis, C.M. & Brown, J.C. (1975) Biochemistry 14, 109. Kramer, R.H. & Canellakis, E.S. (1977) Proc. Am. Assoc. Cancer Res. 18, 56. Carter, W.G. & Hakomori, S.-I. (1977) Biochem. Biophys. Res. Commun. 76, 299.

32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59.


14.

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Nachbar, S.M., Oppenheim, J.D. & Aull, F. (1976) Biochim. Biophys. Acta 419, 512. Fournier-Delpech, S., Danzo, B . J . & Orgebincrist, M.C. (1977) Ann. Biol. Anim. Biochem. Biophys. 17, 207. Hayman, M.J., Skehel, J . J . & Crumpton, M.J. (1973) FEBS Lett. 29, 185. Westenbrink, F . , Koonstra, W. & Bentvelzen, P. (1977) Eur. J . Biochem. 76, 85. Crean, E.V. & Rossomando, E.F. (1977) Biochem. Biophys. Res. Commun. 75, 488. Nicolson, G.L. (1978) In "Advanced Techniques in Biological Electron Microscopy" (J.K. Koehler, ed.), Vol. 2, SpringerVerlag, New York (in press). Nicolson, G.L., Usui, N . , Yanagimachi, R. et a l . (1977) J . Cell Biol. 74, 950. Kelly, P., Cotman C.W. Gentry C a l (1976) J Cell Biol. 71, 487. de Petris, S. & Rabb, M.C. (1974) Eur. J . Immunol. 4, 130. Rosenblith, J . Z . , Ukena, T.E., Yin, H.H. et al. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 1625. Nicolson, G.L. (1973) Nature New Biol. 243, 218. de Petris, S., Raff, M.C. & Mallucci, L. (1973) Nature New Biol. 244, 275. Goldman, R., Sharon, N. & Lotan, R. (1976) Exp. Cell Res. 99, 408. de Petris, S. (1977) In "Dynamic Aspects of Cell Surface Organization", Vol. 3 of "Cell Surface Reviews" (G. Poste & G.L. Nicolson, eds.), p. 643, North-Holland, Amsterdam. Loor, F., Forni, L. & Pernis, B. (1972) Eur. J . Immunol. 2, 203. Yahara, I. & Edelman, G.M. (1972) Proc. Natl. Acad. Sci. U.S.A. 69, 608. Shinitzky, M., Inbar, M. & Sachs, L. (1973) FEBS Lett. 34, 247. Inbar, M., Shinitzky, M. & Sachs, L. (1973) J . Mol. Biol. 81, 245. Jacobson, K., Wu, E.-S. & Poste, G. (1976) Biochim. Biophys. Acta 433, 215. Schlessinger, J., Koppel, D.E., Axelrod, D. et a l . (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2409.

RECEIVED

April 4, 1978.


15 Masking of Cell-Surface Antigens by Ectoglycoproteins JOHN F. CODINGTON Laboratory for Carbohydrate Research, Massachusetts General Hospital, Boston, MA 02114

The surfaces ofmammalian antigenic activities, tha to individual (1, 2), and tissue (3) of origin. Specific antigens at the cell surface may reflect the stage of embryonic development (4), and antigens may result from environmental factors, such as exposure to viruses (5), chemicals (6), or radiation (7). The impact of such factors can result i n neoplasia, and the surfaces of neoplastic cells then possess antigens which have resulted from these factors (7, 8, 9) or they may possess derepressed embryonic or fetal antigens (10). These antigens, known as tumor associated or tumor specific antigens, cannot usually be detected at the surfaces of normal cells similar to those from which the neoplasm was derived. The effectiveness of an immune surveillance system i n the defense against cancer i n humans has been debated widely (11, 12). An immune response to tumor specific antigens has often proven sufficient to cause rejection of some experimental tumors, although i n established neoplasms the manifestation of a similarly successful defense mechanism is rare (12). A number of explanations have been proposed to explain the capacity of tumor cells to survive and grow progressively i n immunologically hostile environments. Of particular concern i s the survival mechanism(s) employed by metastatic or suspended tumor cells i n the blood, lymph, or other body fluids. Masking of cell-surface antigens to protect them from immune defense factors of the host has been proposed by a number of investigators (13-22). Evidence to support the existence of at least four different types of masking has been presented. These include masking of cryptic antigenic sites, usually galactose terminated, by sialic acid (14, 15); nonspecific coulombic or charge repulsion due to cell-surface s i a l i c acid residues (16); masking by adsorbed exogenous glycoprotein (17, 18) or proteoglycan (19) material, which may be specific Sor particular cell-surface receptors; and masking of nearby antigenic sites by large endogenous glycoprotein molecules (13, .20-22). The present discussion will be limited to evidence 0-8412-0452-7/78/47-080-277$05.00/0 © 1978 American Chemical Society In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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i n support of masking by endogenous glycoproteins and the possible relevance of this phenomenon to human cancer. Occurrence Evidence i n support of antigen masking by large endogenous ectoglycoproteins has been presented, to my knowledge, for only two ascites tumor systems, the mammary adenocarcinoma, TA3, of the s t r a i n A mouse and the IgA-synthesizing plasmacytoma c e l l , 58-8, of the BALB/c mouse. In each case, evidence to support this mechanism has involved comparison with c e l l s derived from the same tumor i n which no evidence f o r masking was found. In the TA3 tumor system several nonstrain-specific sublines were investigated, as w e l l as s i x nonstrain-specific TA3-Ka/A.CA hybrid cloned l i n e s , which had resulted from fusion of c e l l s from the nonstrain-specific TA3-H sublin d l embryoni f i b r o blasts of the A.CA mous the major histocompatibility antige (II-* ) investigated Each nonstrain-specific c e l l l i n e was found to possess at i t s c e l l surface an abundance of rod-like glycoprotein (epiglycanin) molecules of high molecular weight. These molecules are believed to be involved i n masking. Fortuitously, a s t r a i n - s p e c i f i c ascites subline, TA3-St, which had been derived from the same spontaneous s o l i d tumor as the TA3-Ha c e l l , was available f o r comparison. The TA3-St ascites c e l l was not found to possess any epiglycanin at i t s surface. As described below, immunological, chemical, immunochemical, and physical studies support the hypothesis of masking by epiglycanin molecules. In the plasma c e l l tumor, 56-8, which has been less extensively studied than the TA3 tumor, the capacities to absorb anti-H-2 antisera or rabbit anti-58-8 antisera were observed to decrease over f i v e to s i x passages i n the ascites form i n the mouse (passage 8 to passage 13) (23). After passage 13 no absorption of either antibody could be observed. Removal of protein material by b r i e f treatment with pronase, however, restored the capacity to absorb each antibody. Moreover, after incubation of the pronase-treated c e l l s for several hours i n culture at 37°, the c e l l s again resisted absorption of antisera, suggesting resynthesis of a masking glycoprotein. These characteristics, which are closely related to those of nonstrains p e c i f i c TA3-Ha c e l l s , suggest masking by endogenous molecules. To my knowledge, no chemical or imrnunochemical evidence for a masking glycoprotein has been reported. Glycoproteins related i n composition to epiglycanin have been isolated from hepatoma ascites c e l l s by Funakoshi and coworkers (24) and from B-16 melanoma ascites c e l l s by Bhavanandan and Davidson (2_5). To my knowledge, no firm evidence i n support of a masking function for the glycoproteins has been published. a


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Early Chemical and. Imrr.uiiochemical Studies of the TA3 Ascites Sublines" In 1962 Gasic and Gasic reported, on the basis of histochemical studies, that the TA3-Ha c e l l possessed at i t s surface a high concentration of s i a l i c acid-containing glycoprotein material (26). This finding was confirmed by our laboratory, which reported i n 1970 that large amounts of carbohydrate and protein material could be removed by proteolysis from viable TA3Ha ascites c e l l s (27). Scon thereafter we described the i s o l a t i o n of a glycoprotein fraction of high molecular weight, which had been removed from viable ascites c e l l s by proteolysis (28). This material possessed, a high concentration c f serine, threonine, and N-acetylgalactosamine, components associated with glycoproteins with a high proportion of O-glycosyl-linked carbohydrate chains. The cell-surface glycoprotein from which these fragments were derived was l a t e r terme this finding was not recognize s p e c i f i c subline of the same tumor, TA3-St, revealed that the TA3-St ascites c e l l did not possess any detectable amount of this fraction (20, 21). At about t h i s time several laboratories described some s t r i k i n g b i o l o g i c a l differences between the same two ascites sublines, the nonstrain-specific TA3-Ha c e l l and the s t r a i n s p e c i f i c TA3-St subline (21, 30-35). Friberg (30) reported marked differences i n karyotypes of the two c e l l l i n e s , and Hauschka et a l . (32) confirmed a near d i p l o i d chromosome number for the TE"3-Ha c e l l . The work of several groups (30-34) showed c l e a r l y that the TA3-Ka c e l l was s i g n i f i c a n t l y less immunogenic than the TA3-St c e l l and far more immunoresistant. Of particular significance was the finding of Friberg (31) that the TA3-St c e l l possessed the capa.city i n v i t r o to absorb many f o l d more anti-H-2 antibody to the major histocompatibility antigen (H-2 ) than the TA5-Ha c e i l . Sanford et a l . (21) l a t e r confirmed this observation and showed, as did FriEerg and L i l l i h o o k (35), that disruption of the c e l l structures by l y o p h i l i z a t i o n resulted i n a marked increase i n the number of H-^! antigenic sites exposed on the surface of the TA3-Ha c e l l , but t h i s treatment resulted i n no increase i n antigenic s i t e s exposed on the TA3-St c e l l surface. These observations, coupled with the finding that the TA3-Ha, but not the TA3-St, c e l l possessed at i t s surface an abundance of epiglycanin molecules, led us to suggest at that time that a l l o t r a n s p l a n t a b i l i t y and the reduced capacity to absorb anti-H2 antibody was due to masking of surface antigens by epiglycanin molecules (20, 21). Springer et_ a l . (36) demonstrated that epiglycanin was a potent i n h i b i t o r oF hemagglutination by the l e c t i n from V i c i a graminea. seeds, a l e c t i n of high s p e c i f i c i t y for structures at the surfaces of human erythrocytes of N blood group s p e c i f i c i t y . G

a

a

a


280


Later Cooper et a l . (57) developed a method for the quantitation of epiglycanin~T)y the use o f this l e c t i n . Although receptors f o r the V i c i a graminea l e c t i n , which are present i n epiglycanin, are abundant at the TA3-Ha c e l l surface, no receptors for this l e c t i n were detected at the surface of the TA3-St ascites c e l l (20, 38). The Origin of Nonstrain S p e c i f i c i t y i n the TA3 Ascites Cells The TA3-Ha ascites subline was i n i t i a l l y s t r a i n s p e c i f i c (32). Loss o f s t r a i n s p e c i f i c i t y i n the TA3-Ha subline occurred during routine passage as an ascites c e l l i n the syngeneic s t r a i n A mouse (32). Although t h i s event occurred i n ascites c e l l s following passage 200, neither the exact passage (s) nor the circumstances of the occurrence were reported. Although epiglycanin may have appeared at the TA3-Ha c e l l surface at this time, i t s presence was not observed u n t i l after passage 520 by means of chemical method Another opportunit occurred recently i n our laboratory (39, 40). Strain specific TA3-St c e l l s were inoculated intraperitoneally into s t r a i n A mice suffering from incipient acute i n t e r s t i t i a l pneumonia. Later two micoorganisms, Pasteurella pneumotropica and Pasteurella multocida, were isolated from the sick mice. Cells were harvested routinely on day 7 and tested for the presence o f receptors for the l e c t i n from V i c i a graminea seeds at t h e i r surfaces. The determination of l e c t i n receptors was part o f a project i n progress at that time i n collaboration with a graduate student, D. K. M i l l e r , and with the laboratory of Dr. A. G. Cooper. Whereas, TA3-St ascites c e l l s normally demonstrate essentially no l e c t i n receptors, the c e l l s were now able to adsorb a high level of V i c i a graminea l e c t i n (39). I t was shown that the new c e l l l i n e , which was c a l l e d TA3-MM, possessed at i t s surface a high concentration of epiglycanin molecules (40). Similar to the nonstrain s p e c i f i c TA3-Ka ascites c e l l , the TA3-MM c e l l l i n e was able to grow progressively i n mice of foreign strains. The o r i g i n and characteristics of the TA3-MM subline are described i n two papers prepared for publication (39, 40). The new allotransplantable c e l l t i n e , TA3-MM, was approximately tetraploid with an average of about 80 chromosomes, which was 12 more than was possessed by the TA3-St c e l l (approximately 68) and twice the number of the TA3-Ha c e l l (41-42) (39). Although about 20% greater i n diameter than the TA3-Ha ascites c e l l , i t possessed about the same amount (2.3 mg/10 c e l l s ) of epiglycanin per c e l l (40). Comparison of the compositions of p u r i f i e d fractions o f epiglycanin from TA3-Ha and TA3-NM c e l l s are presented i n Table I . Values are almost i d e n t i c a l for the two c e l l l i n e s . One may speculate that nonstrain s p e c i f i c i t y i n the TA3-Ha ascites c e l l l i n e also occurred under the influence of pneumonia associated microorganisms, but no evidence i s available to support 9


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TABLE I Carbohydrate and Amino Acid Compositions of Epiglycanin Isolated from the Ascites F l u i d of Syngeneic Mice Bearing TA3 Ascites Cells TA3-Ha Carbohydrate Components

TA3-M^l

(moles r e l a t i v e to N-acetylgalactosamine)

N-Acetylgalactosamine

1.0

1.0

Galactose

1.4

1.4

N-Acetylglucosamine

0.

N-Acetylneuraminic Acid

0.4

Amino Acid Components

0.6

(residues per 1000 residues) 24

31

Threonine

307

306

Serine

252

223

Glutamic Acid

32

36

Proline

93

107

Glycine

81

92

Alanine

131

131

2o

12

1

11

Leucine

33

49

Lysine

12

n.d.

Aspartic Acid

Valine Isoleucine

a

a

Values were obtained for the TA3-MM/1 subline (40).


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this contention. Moreover, the two c e l l lines demonstrate many different characteristics at this time. I t i s , of course, conceivable that after many passages the TA3-MM c e l l w i l l become similar to the TA3-Ha c e l l , but i t i s also plausible that the two o r i g i n a l s t r a i n s p e c i f i c ascites sublines, TA3-Ha and TA3-St, were dissimilar i n important respects and some of t h e i r d i s s i m i l a r i t i e s have persisted after loss of s t r a i n s p e c i f i c i t y . I t i s clear, however, that i n each subline a l l o t r a n s p l a n t a b i l i t y i s associated with the presence at the c e l l surface of a large number (approximately 4 x 10 ) of molecules of epiglycanin. 6

Topographical Features o f the TA3-Ha and TA3-St Cells Electron microscopy revealed major differences i n the topography of the TA3-Ha and TA3-St ascites c e l l s (41). Scanning electron microscopy showed the surface of the TA3-Ha c e l l to consist of long m i c r o v i l l surface, as shown i n Figur , surface consisted of irregularly-spaced ridges or folds (Figure 2) (41). By dark f i e l d light microscopy, m i c r o v i l l i of viable TA3-Ha c e l l s i n suspension appeared to be of greater length than the diameter of the c e l l s and the m i c r o v i l l i appeared to have a c i l i a l i k e motion (42). By transmission electron microscopy the two c e l l types were hardly distinguishable at a magnification of about 10,000, with the exception of the morphological differences i n the microextensions, those of the TA3-Ha c e l l being more erect, more regularly spaced, and longer than those of the TA3-St c e l l (41). Each c e l l type appeared to contain two d i s t i n c t types of virus particles (41). This observation was consistent with immunologic findings which demonstrated that the TA3-Ha, TA3-St, and TA3-MM ascites c e l l s contained the same three types of virus p a r t i c l e s , the murine mammary tumor v i r u s , the Rauscher leukemia v i r u s , and. the pneumonia virus of mice (39). After glutaraldehyde and osmium tetroxide f i x i n g , high resolution electron microscopy (> 50,000 times magnification) revealed a network of fine filaments extending from the surface of the TA3-Ha ascites c e l l . On occasion these filaments appeared to aggregate into thicker, more rod-like structures, as shown i n Figure 3. These extended 200-400 nm, sometimes 500 nm, from the plasma membrane. By contrast, the TA3-St c e l l , when fixed s i m i l a r l y , possessed no v i s i b l e extracellular coat (41). Evidence that these extracellular structures at the TA3-Ha c e l l surface represented s i a l i c acid-containing glycoproteins, probably epiglycanin, was obtained by the use of pclycationic f e r r i t i n labeling. Anionic groups, labeled with the electron-dense polycationic f e r r i t i n , appeared to exist several hundred nm from the plasma membrane of the TA3-Ma ascites c e l l . By contrast, this reagent appeared to be attached only i n close proximity to the


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plasma membrane of TA3-St c e l l s treated i n a similar way. After p r i o r treatment with neuraminidase to remove the c e l l surface s i a l i c acid r e l a t i v e l y few f e r r i t i n p a r t i c l e s were visualized at the surface of either c e l l (41). I t has been shown that i n the TA3-Ha c e l l epiglycanin contains approximately 60% of the surface s i a l i c acid (43). Isolation of Epiglycanin Epiglycanin was o r i g i n a l l y i d e n t i f i e d as glycopeptide frapients cleaved from viable c e l l s by proteolysis and fractionated by gel f i l t r a t i o n (28). The molecular weight of t h i s material (Fraction I ) , as calculated from sedimentation equilibrium and electron microscopy data, ranged from 80,000 to 460,000 (44). The yields of epiglycanin obtained by t h i s method averaged about 2.3 mg/10 c e l l s for either the TA3-Ha or TA5-NIM ascites sublines (Table I ) . Further fractionatio i n the proteolysis product (44). This v a r i a b i l i t y i n the composition of carbohydrate i s due to variations i n the r e l a t i v e proportion of each of f i v e different types of 0-glycosyl-linked carbohydrate chains (29, 43, 45). We have observed that epiglycanin i s shed from the c e l l surfaces of each nonstrain-specific TA3 c e l l l i n e which we have investigated. I t may be detected i n the growth medium at levels below 5 ng/ml by a hemagglutination i n h i b i t i o n method (37). By this method epiglycanin has been detected i n the serum 6T s t r a i n A mice bearing TA5-Ha ascites c e l l s on day 2 or 3 following i n t r a peritoneal inoculation of the c e l l s . Isolation of epiglycanin from the ascites f l u i d followed precipitation of the bulk of other protein and glycoprotein material by b r i e f exposure to 0.3 M perchloric acid at 0°. Fractionation by passage of the perchloric acid-free solution through a long column of Sspharose 4B gave nearly pure epiglycanin of composition similar to that of proteolysis derived material (43). Yields of epiglycanin by t h i s method averaged about 0.2 to 0.3 mg/10 c e l l s , and the molecular weight of the materials isolated from either TA3-Ha or TA3-MM ascites c e l l s was approximately 500,000, as determined by sedimentation equilibrium or electron microscopy (40). By shadow-casting electron microscopy (H. S. Slayter, personal communication), approximately 60-70% of the material obtained i n t h i s way ranged from 450 to 500 nm i n length. Whether epiglycanin sned into the growtli medium represents the entire native molecule or only the major portion cleaved from the c e l l surface by membrane-bound proteases has not been determined. This material demonstrated l i t t l e tendency to aggregate, i n contrast to epiglycanin isolated after s o l u b i l i z a t i o n with detergents or, a l t e r n a t i v e l y , with lithium diiodosalicylate, followed by phenol extraction (43). By gel f i l t r a t i o n chromatography small yields of non-aggregated 9

9


284


Figure 1. Scanning electron micrograph of the TA3-Ha ascites cell. Long, regularly-spaced microvilli cover the cell surface. X4300. (Courtesy of S. C. Miller and E. D. Hay.)


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Figure 2. Scanning electron micrograph of the TA3-St ascite cell. The cell surface is characterized by numerous irregularly spaced folds or ridges. X4300. ( Courtesy of S. C. Miller and E. D. Hay.)


286


Figure 3. Transmission electron micrograph of the TA3-Ha ascites cell surface. Cells were fixed with glutaraldehyde and osmium tetroxide. Extramembranous structures 200-500 nm in length are observed on the cell surface. X 43,000. (Courtesy of S. C. Miller and E. D. Hay.)


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epiglycanin were obtained by either of these two methods. From gel f i l t r a t i o n effluent volumes and by measurement of the molecular lengths on electron micrographs (II. S. Slayter, personal communication) the molecular size of epiglycanin obtained i n these ways appeared to be s i m i l a r to that o f ascites fluid-derived epiglycanin. I t seems probable, however, that i n the case of a molecule as large as epiglycanin (approximately 1300 amino acids i n an extended chain), the addition of a peptide segment of 20 to 30 amino acids could not be readily detected. Physicocnemical

Characterization of Epiglycanin

The carbonydrate and amino acid compositions of epiglycanin from the TA3-MM and TA3-IIa sub-lines are s i m i l a r (Table I ) . The percentage of carbohydrate, however, appears to be consistently higher (30-90%) i n TA3-MM material tha i TA3-H epiglycani (7^-80%). Five types o from epiglycanin as t h e i N-acetylgalactosaminito , and t h e i r sequences proposed (13, 43, 4o). A pentasaccharide, containing two galactose, one N-acetylglucosamine, one N-acetylgalactosamine, and one s i a l i c acid residue was suggested on the basis of compositional data obtained by gas-liquid chromatography (43). Recent analyses, however, (45) suggest that a hexasaccharide with two s i a l i c acid residues, rather than one, as suggested for the pentasaccharide, may be present. In this chain i t would be expected that the second s i a l i c acid residue i s attached by a 2-H3 linkage to N-acetylgalactosamine. A s i a l i c acid (2-*3) galactose linkage has been previously proposed (29). The number of carbohydrate chains present i n a molecule of 500,000 molecular weight, consisting of approximately 1300 amino acid residues, has been found to be 500-600 (29, 43). A l l samples of epiglycanin whicli we have investigated have contained a small amount of marjiose (approximately 0.3%). I t was not known whether the presence o f this residue indicated that a glycoprotein impurity were present or i f the mannose were a component of an asparagine-linked carbohydrate chain i n epiglycanin. By the use of a f f i n i t y chromatography with a Concanavalin A - Sepharose 4B column i t has recently been possible to obtain from proteolytically-cleaved fragments (Fraction I) a fraction of epiglycanin free of mannose. I t was found, however, that epiglycanin isolated from the ascites f l u i d (molecular weight, 500,000) was completely adsorbed on a s i m i l a r a f f i n i t y column and could be eluted from the column with methyl-a-mannopyranoside. These results suggest that epiglycanin does contain N-glycosyl-linked carbohydrate material. On the basis of 0.3% mannose there could be no more than nine residues of mannose per molecule of epiglycanin, enough for one, possibly two, asparaginelinked carbohydrate chains. I t has not been possible to obtain amino acid sequence data for epiglycanin isolated from the ascites f l u i d , since the amino


288

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DISEASE PROCESSES

group at the N-terminal end of tiie molecule i s blocked. This observation suggests that the N-terminal amino group i s extrac e l l u l a r , and that the carboxy terminal i s rooted i n the c e l l , as i t i s i n the erythrocyte glycoprotein which contains M and N blood group a c t i v i t y (46). By shadow casting electron microscopy a l l samples of epiglycanin have appeared as s i m i l a r rod-like p a r t i c l e s . Widths were consistently 2.J nm, and lengths varied from about 70 nm for the smallest proteolytically-cleaved fragment (44) to 450-500 nm for material isolated without proteolytic digestion (40). Further Evidence for Masking i n the TA3 Tumor The hypothesis of masking was tested i n a series of s i x hybrid c e l l lines which had resulted from fusion i n v i t r o of TA3-Ha c e l l s and normal embryoni fibroblast f tli A.CA (47). These lines had bee i n s t r a i n A mice and possesse y majo histocompatibility complex of the TA3-Ha parent, H-2 (48, 49). Each of the hybrid lines exhibited the capacity to grow progressively i n mice of foreign s t r a i n s ; each showed a reduced capacity, compared to the TA5-St s t r a i n s p e c i f i c c e l l l i n e , to absorb anti-H-2 antibody; and each of the hybrid lines possessed epiglycanin at i t s c e l l surface (22). The r e l a t i v e values for the three parameters, allotransp l a n t a b i l i t y {% of foreign mice k i l l e d by the tumor), resistance to antibody absorption (absorptive capacity plotted i n reverse order of magnitude), and amount (mg) of epiglycanin per 10 c e l l s are plotted i n Figure 4. The results strongly suggest that with increasing amounts of epiglycanin the c e l l s increase t h e i r capacity to grow i n mice of foreign strains and possess a reduced capacity to absorb antibody to H-2 antigen. These results support the hypothesis that i n the TA3 murine tumor system a function of epiglycanin i s the masking of cell-surface antigens (22). a

a

9

a

Relevance to Human Cancer of Masking by Endogenous Glycoproteins Tne discovery of epiglycanin and the correlation of i t s presence at the c e l l surface with a l l o t r a n s p l a n t a b i l i t y was unprecedented. Furthermore, since the i n i t i a l report of i t s i s o l a t i o n (28) i n 1972 and b i o l o g i c a l function (2£, 23) i n 1973 very l i t t l e evidence for the occurrence of a s i m i l a r phenomenon i n other tumor systems has been reported. Three plausible explanations for this may be suggested: (a) I t may be that masking of this type does not often occur i n neoplasia, but i s associated only with certain stages i n the cancer process and occurs only under c e r t a i n conditions, (b) Suitable experimental models for the study of t h i s phenomenon have not been available, (c) Investigators have not always u t i l i z e d experimental procedures


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TA3-H* l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l f l

i iiiiiiiiiiiMiiiiiiiiiiiiiiiiiiiiiiiiHiiiiiiiiiiiiiiiiiiiiiiiiiiuriiiiii

TA3-Ha//LCA/lO

TA3-Ha/*.CA/ll

IIIIIIIIIIIIIIIIIIIIMIIMMIIIMIMIIIHIIIIII

MIIIIIIIIIIIIIIIIMIIIIII

Figure 4. Comparison of three characteristics of six TA3-Ha/A.Ca hybrid, the TA3-Ha, and the TA3-St ascites cell lines. The relative amounts of epiglycanin on the cell surfaces ( ) are plotted with the capacities of the cells to resist absorption of anti-H-^ antibody ( millmi) and their capacities to grow progressively in mice of foreign strains (i J ). The units and scales used in plotting this graph are similar to those described in Ref. 22.


290


suitable for a study o f t h i s type. I t i s my contention that a l l of these explanations may apply to t h i s situation. The appearance of large extended molecules, such as epiglycanin, on tumor c e l l surfaces probably occurs only under c e r t a i n circumstances i n established tumors - with probably disastrous consequences for the host. Furthermore, i t may be d i f f i c u l t to establish "spontaneously-developed" masking-glycoprotein-synthesizing c e l l s as experimental models for study, or i n the past when c e l l s of t h i s type did develop i n experimental systems they may have been overlooked. Loss of s t r a i n s p e c i f i c i t y i n the TA3 tumor system i s known to have occurred only twice, and only on the second occurrence were the circumstances recorded (39, As described i n t h i s report the capacity o f s t r a i n - s p e c i f i c c e l l s to synthesize epiglycanin occurred i n the TA3-St subline i n vivo i n hosts infected by microorganisms Th mechanis B which t h i chang took place i n the TA3-H biochemical event which synthesi epiglycani could occur under the influence o f other, seemingly unrelated factors. During the period o f tumor development i n humans, which i s often of many years duration, the patient may be subjected to many types of environmental influences, among them invasions by viruses and bacteria. Under such circumstances changes i n the character of tumors may possibly occur. Indeed, the occurrence of changes i n the morphology and growth characteristics of human, tumors i s w e l l documented (50). Of p a r t i c u l a r relevance to t h i s discussion may be the appearance o f invasiveness, metastasis, and a more rapid rate o f c e l l p r o l i f e r a t i o n . Indeed, evidence from the TA3 murine mammary carcinoma system suggest that the presence of epiglycanin-like glycoprotein molecules at the surfaces of human tumor c e l l s could possibly explain an increase i n virulence i n some human cancers. Comparison o f the growth rates o f TA3 ascites c e l l s i n the peritoneal cavity o f syngeneic mice revealed that c e l l s bearing epiglycanin at t h e i r surfaces, namely those o f the TA3-Ha and TA3-NM l i n e s , grew more r a p i d l y than those (Ta3-St) without epiglycanin. Friberg (30) attributed differences i n growth rates o f TA3-Ha and TA3-St l i n e s to an i n i t i a l lag period i n TA3-St growth. We have found that during a 7 day growth period the average doubling time f o r the TA3-MM and TA3-Ha c e l l s i s approximately 15 hours, compared to a doubling time of about 20 hours f o r the TA3-St c e l l . A plausible explanation for the increased growth rate i n the epiglycanin-bearing c e l l s may involve masking of tumor associated antigens, although to my knowledge no comparison of the properties o f the tumor-associated antigens i n the TA3 sublines has been reported. Recent reports o f M i l l e r et a l . (51) and Cooper and coworkers (52) describe properties i n TA3"suBlines which are consistent with a role i n metastasis by epiglycanin. I t was shown that the


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appearance of epiglycanin depends upon environmental factors and that, under certain circumstances epiglycanin may become l o s t , or become nondetectable, at the TA3-Ha c e l l surface. After several weeks of growth i n culture epiglycanin could not be detected at the surface of the TA3-Ha c e l l (51). However, after passage i n the mouse i n ascites form, epiglycanin was again present i n abundance at the c e l l surface. It was recently reported by Hagmar and Ryd (53) that the TA3-Ha c e l l was not transplantable when passaged from the ascites form i n the s t r a i n A mouse to a subcutaneous s i t e i n a mouse of a foreign s t r a i n . However, Cooper et a l . (52) have demonstrated that with continuous passage of TA3-Ha ascites c e l l s subcutaneously i n syngeneic mice the concentration of epiglycanin increased, as evidenced by the appearance of an increased concentration of receptors for the V i c i a graminea l e c t i n (38), with the appearance of subcutaneous a l l o t r a n s p i a n t a b i l i t y These circumstances f o r tumor growth may more nearl growing s o l i d tumors i correlaticn between the presence of epiglycanin and allotransp i a n t a b i l i t y i n s o l i d tumors and suggest that masking by endogenous glycoprotein molecules may occur i n s o l i d tumors. A possible relationship between epiglycanin concentration and metastatic properties was also demonstrated by this group (52). Variants derived from TA3-Ha c e l l s by prolonged suEcutaneous growth i n syngeneic mice w ere selected on the basis of t h e i r capacity to grow i n s i t e s distant from the t a i l vein injection s i t e , i . e . , beyond the lung, i n the l i v e r , spleen, of kidney. I t was found that those variants which possessed the greatest concentration of epiglycanin demonstrated the greatest metastatic property. The most "metastatic" of the c e l l s investigated was a TA3-Ha variant which possessed 2-3 times as much epiglycanin at i t s surface as the established TA3-Ha ascites c e l l l i n e . Thus, a c o r r e l a t i o n has been demonstrated between the presence of epiglycanin. and the capacity of c e l l s i n the TA3 murine carcinoma to pass f r e e l y i n the circulatory system to distant sites before establishing f o c i of tumor growth. Any conclusions regarding the relationship between masking by large endogenous ectoglycoproteins and cancer i n humans must be considered as purely speculative at t h i s time. Nevertheless, the investigations of the TA3 mamirary carcinoma i n the mouse described here suggest, as was stated i n 1975 (13), that "similar-type transformations, resulting i n the appearance of epiglycanin-Iike macromolecules, could occur i n other spontaneously derived tumors. Such phenomena may have relevance to human cancers, p a r t i c u l a r l y to the occurrence of metastases or to the recurrence of cancer i n seemingly cured patients." r



292

Acknowledgements This investigation was supported by grants from the National Institutes of Health, U.S. Public Health Service (CA-18600 to J. F. Codington and CA-08418 to R. W. Jeanloz).

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Kahan, B. D., and Reisfeld, R. A . , Eds., "Transplantation Antigens", Academic Press, New York, 1972. Nathenson, S. G., and Cullen, S. E . , Biochim. Biophys. Acta (1974) 344, 1-25. Moscona, A.A., Proc. Soc. Exptl. Biol. Med. (1956) 92, 410416. Moscona, A., Ed., "The Cell Surface in Development", J . Wiley and Sons, Inc., New York 1974 Old, L. J., and Boyse Prehn, R. T., Canad Conf. (l963), 387. Klein, G., in Davis, B. D., and Warren, L., Eds., "The Specificity of Cell Surfaces", pp. 165-180, Prentice-Hall, Inc., New Jersey, 1967. McKhann, C. F . , and Jagarlamoody, S. M . , in Day, S. B . , and Good, R. A . , Eds.,"Membranesand Viruses in Immunopathology", pp. 577-599, Academic Press, New York, 1972. Prehn, R. T., in Nowotny, A . , Ed., "Cellular Antigens", pp. 308-313, Springer-Verlag, New York, 1972. Granatek, C. H . , Hanna, M. G., Jr., Hersh, E. M., Gutterman, J . U . , Mavligit, G. M . , and Candler, E. L., Cancer Res. (1976) 36, 3464-3470. Smith, R. T., and Landy, M., Eds., "Immune Surveillance", Academic Press, New York, 1970. Klein, G., and Klein, E . , Proc. Natl. Acad. S c i . U.S.A. (1977) 74, 2121-2125. Codington, J. F . , in "Cellular Membranes and Tumor Cell Behavior", pp. 399-419, Williams and Wilkins Co., Baltimore, 1975. Jeanloz, R. W., and Codington, J . F . , in Rosenberg, A . , and Schengrund, C. -L., Eds., "Biological Roles of Sialic Acid", pp. 201-238, Plenum Press, New York, 1976. Bekesi, J. G., Roboz, J. P., and Holland, J. F . , Ann. N.Y. Acad. S c i . (1976) 277, 313-331. Apffel, C. A . , and Peters, J . H . , J. Theor. Biol. (1970) 26, 47-59. Wnitehead, R. H . , Roberts, G. P., Thatcher, J., Teasdale, C., and Hughes, L. E., J. Natl. Cancer Inst. (1977) 58, 1573-1576. Dorval, G., Witz, I. P . , Klein, E., and Wigzell, H . , J. Natl. Cancer Inst. (1976) 56, 523-527. Lippman, M., Nature (1968) 219, 33-36. Codington, J. F . , Sanford, B. H . , and Jeanloz, R. W., J. Natl. Cancer Inst. (1973) 51, 585-591.


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21.

Sanford, B. H . , Codington, J. F . , Jeanloz, R. W., and Palmer, P. D., J . Immunol. (1973) 110, 1233-1237. Codington, J. F., Klein, G., Cooper, A. G., Lee, N . , Brown, M. C., and Jeanloz, R. W., J. Natl. Cancer Inst. (1978) in press. Ohno, S., Natsu-ume, S., and Migita, S., J. Natl. Cancer Inst. (1975) 55, 569-578. Funakoshi, I., Nakada, H . , and Yamashina, I., J . Biochem. (1974) 76, 319-533. Bhavanandan, V. P., and Davidson, E. A . , Biochem. Biophys. Res. Commun. (1976) 70, 139-145. Gasic, G., and Gasic, T . , Nature (1962) 196, 170. Codington, J. F . , Sanford, B. H . , and Jeanloz, R. W., J. Natl. Cancer Inst. (1970) 45, 637-647. Codington, J. F . , Sanford, B. H . , and Jeanloz, R. W., Biochemistry (1972) 11 2559-2564 Codington, J. F . , Linsley and Osawa, T., Carbchydr (1975) , Friberg, S., Jr., J . Natl. Cancer Inst. (1972) 48, 1463-1476. Friberg, S., J r . , J . Natl. Cancer Inst. (1972) 48, 1477-1489. Hauschka, T. S., Weiss, L., Holdridge, B. A . , Cudhey, T. L., Zunpft, M., and Planinsek, J. A . , J. Natl. Cancer Inst. (1971) 47, 343-359. Grohsman, J., and Nowotny, A . , J. Immunol. (1972) 109, 10901095. Lippman, M. M., Venditti, J. M . , Kline, I . , and Elam, D. L., Cancer Res. (1973) 33, 679-684. Friberg, S., J r . , ana Lilliehook, B . , Nature New Biol. (1973) 241, 112-113. Springer, G. F . , Codington, J. F . , and Jeanloz, R. W., J. Natl. Cancer Inst. (1972) 49, 1469-1470. Cooper, A. G., Codington, J. F . , and Brown, M. G., Proc. Natl. Acad. S c i . U.S.A. (1974) 71, 1224-1228. Codington, J. F . , Cooper, A. G., Brown, M. C., and Jeanloz, R. W., Biochemistry (1975) 14, 855-859. Cooper, A. G., Codington, J. F., Miller, D. K . , and Brown, M. C., prepared for publication. Codington, J. F . , Cooper, A. G., Miller, D. K . , Slayter, H. S., and Jeanloz, R.W., prepared for publication. Miller, S. C., Hay, E. D., and Codington, J. F . , J. Cell Biol. (1977) 72, 511-529. Codington, J. F . , and Bruns, R. R., unpublished results. Codington, J. F . , Van den Eijnden, D. H . , and Jeanloz, R. W., in Harmon, R. E., Ed., "Cell Surface Carbohydrate Chemistry", pp. 49-66, Academic Press, New York, 1978. Slayter, H. S., and Codinton, J. F . , J. B i o l . Chem. (1973) 248, 3405-3410. Van den Eijnden, D. H . , Evans, N., Codington, J. F . , and Jeanloz, R. W., prepared for publication.

22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45.

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46. Marchesi, V. T., and Furthmayr, H . , Ann. Rev. Biochem. (1976) 45, 667-698. 47. Wiener, G., Klein, G., and Harris, H . , J. Cell S c i . (1971) 8, 681-692. 48. Klein, G., Friberg, S., J r . Wiener, F . , J. Natl. Cancer Inst. (1973) 50, 1259-1268. 49. Friberg, S., Jr., Klein, G., Wiener, F . , J. Natl. Cancer Inst. (1973) 50, 1269-1286. 50. Berenblum, I., i n Florey, H. W., "General Pathology", pp. 645-667, W. B. Saunders Co., Philadelphia, 1970. 51. Miller, D. K . , Cooper, A. G., Brown, M. C., and Jeanloz, R. W., J. Natl. Cancer Inst. (1975) 55, 1249-1252. 52. Cooper, A . , Morgello, S., Miller, and Brown, M., in "Cancer Invasion and Metastasis: Biologic Mechanisms and Therapy", pp. 49-64, Raven Press, New York, 1977. 53. Hagnar, B . , and Ryd W. Transplantation (1977) 23 93-97 RECEIVED

February 27, 1978


16 Glycoconjugate Alterations in Malignant and Inflammatory Disease of the Colon YOUNG S. KIM, JAMES S. WHITEHEAD, BADER SIDDIQUI, and DEAN TSAO Gastrointestinal Research Laboratory, Veterans Administration Hospital and University of California, San Francisco, CA 94121 In the United State dence among all malignan easily detectable and curable skin cancer. As is the case with most tumors, the survival rate and prognosis of patients with colonic cancer depends upon the grade and the extent of malignancy. Low grade, more differentiated colonic tumors as well as disease confined to the mucosa and submucosa are associated with longer patient survival. The prognosis is dismal for those who have a high rate of tumor growth or nonlocalized disease (1). The final outcome rests with a balance between the biological properties of the tumor and the type and extent of the host response. This appears to be reflected in the wide variation in the clinical development of the disease and in the growth rate and changes in biochemical and immunochemical properties of the tumor. In considering development of methods for early diagnosis of colonic cancer in those patients of high risk, such as individuals with inflammatory bowel diseases or intestinal polypoid lesions, and development of rational methods to control colonic neoplasia, it i s important to identify and characterize organ specific tumor markers and to clarify the molecular mechanisms involved in the altered biochemical and biological properties that accompany malignant transformation in the colon. The involvement of c e l l surface components in c e l l - c e l l interaction, in growth regulation of cells and in the process of malignant transformation has been reported by many investigators (2-5). Much of our current knowledge has been obtained from studies on virally transformed murine or avian fibroblasts. Although most human carcinomas, including colonic tumors, are epithelial in type and origin, relatively little i s known about cell surface properties of normal and cancerous epithelial cells. Since glycoproteins and glycolipids are major components of surface membranes of mammalian cells, especially those of the intestinal mucosa, and glycoconjugates have been implicated in diverse cellular phenomena, our laboratory has been examining these 0-8412-0452-7/78/47-080-295$05.00/0 ©

1978 A m e r i c a n C h e m i c a l Society


296

GLYCOPROTEINS AND


components i n normal and cancerous c o l o n i c mucosal c e l l s . Three experimental models are being used f o r our i n v e s t i g a t i o n : 1) human cancerous and normal c o l o n i c t i s s u e s obtained a t surgery, 2) human c o l o n i c cancer c e l l s maintained i n monolayer c u l t u r e , and 3) experimental c o l o n i c cancer i n rodents induced by a chemi c a l carcinogen, 1,2-dimethylhydrazine. Some o f the approaches t h a t we are u s i n g i n the study of membrane glycoconjugates i n normal and cancerous c o l o n i c c e l l s i n c l u d e : 1) the i s o l a t i o n and chemical and immunochemical chara c t e r i z a t i o n of membrane glycoconjugates, 2) the i d e n t i f i c a t i o n and c h a r a c t e r i z a t i o n of both membrane and cytoplasmic glycoconjugates u s i n g f l u o r e s c e i n - or r a d i o - l a b e l e d l e c t i n s , 3) e x t e r n a l c e l l s u r f a c e r a d i o " l a b e l i n g w i t h g a l a c t o s e oxidase and iodium b o r o t r i t i d e or w i t h l a c t o p e r o x i d a s e - c a t a l y z e d i o d i n a t i o n , and 4) a study of the metabolism and turnover of membrane g l y c o c o n j u gates u s i n g i n v i v o and i n v i t r o i n c o r p o r a t i o n of r a d i o - l a b e l e d p r e c u r s o r s . In the presen p r i m a r i l y of our data o t i e s of human c o l o n i c tumors and the i s o l a t i o n and c h a r a c t e r i z a t i o n of s u r f a c e membrane g l y c o p r o t e i n s . Studies i n Human C o l o n i c Adenocarcinomatous T i s s u e s . Many a n t i g e n i c a c t i v i t i e s have been a s c r i b e d t o g l y c o p r o t e i n s and g l y c o l i p i d s . For example, g a s t r o i n t e s t i n a l mucins and i n t e s t i n a l mucosal c e l l surface membrane glycoconjugates have been reported to possess blood group a c t i v i t i e s . Immunofluorescence s t u d i e s on t i s s u e s i n d i c a t e d t h a t ABH blood group a c t i v i t y was low or absent i n primary g a s t r o i n t e s t i n a l tumors as w e l l as i n other e p i t h e l i a l tumors (60 . The l o s s of these a n t i g e n i c a c t i v i t i e s may be due t o e i t h e r decreased synt h e s i s or i n c r e a s e d degradation of these antigens i n tumor t i s sues. To determine i f d e l e t i o n of blood group a c t i v i t y occurs i n c o l o n i c cancer t i s s u e s and t o c l a r i f y the metabolic b a s i s f o r such a l t e r a t i o n s , the l e v e l s of blood group antigens were measured i n cancerous and adjacent normal c o l o n i c mucosa u s i n g hemagglutination i n h i b i t i o n methods (7^) . The r e s u l t s are shown i n Table I . The data r e p o r t e d here are f o r t i s s u e s taken from blood group A p a t i e n t s . The l e v e l s of these antigens were determined by measuring the a b i l i t y of membrane g l y c o p e p t i d e s t o i n h i b i t the a g g l u t i n a t i o n of red blood c e l l s by v a r i o u s agglut i n i n s . These a g g l u t i n i n s b i n d the s p e c i f i c sugar s t r u c t u r e s i n d i c a t e d i n the parentheses. A negative s i g n i n d i c a t e s no det e c t a b l e a n t i g e n . The g r e a t e r the number of p l u s e s , the higher the l e v e l of a n t i g e n . In a l l p a t i e n t s examined, blood group A a c t i v i t y could not be detected i n the cancer t i s s u e but normal l e v e l s were present i n adjacent t i s s u e s . Two p a t i e n t s who had h i g h l e v e l s of L e a c t i v i t y i n normal mucosa had no d e t e c t a b l e Lewis A a c t i v i t y i n the cancer t i s s u e . The use o f R i c i n u s communis l e c t i n (RCA), which recognizes s p e c i f i c sugar s t r u c t u r e s a


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Disease

297

i n c l u d i n g the immediate p r e c u r s o r o f the H s t r u c t u r e , detected h i g h e r l e v e l s o f t e r m i n a l g a l a c t o s e r e s i d u e s i n the cancerous t i s sues. No c o n s i s t e n t change i n Con A-binding g l y c o p e p t i d e s was observed. Since a l t e r a t i o n s i n blood group a n t i g e n i c i t y and l e c t i n r e a c t i v i t y suggested changes i n carbohydrate composition, the membrane f r a c t i o n o f cancerous and adjacent t i s s u e s was subjected to chemical a n a l y s i s . We measured the amounts o f 3 n e u t r a l sugars, 2 aminosugars and s i a l i c a c i d i n normal and cancerous t i s s u e s . The r e s u l t s i n d i c a t e d t h a t i n general the g l y c o p r o t e i n f r a c t i o n o f the cancer t i s s u e s contained l e s s o f each o f the sugars than d i d the normal t i s s u e s (Figure 1 ) . Fucose, glucosamine, galactosamine and s i a l i c a c i d were reduced s i g n i f i c a n t l y i n the cancerous t i s s u e s , w h i l e galactose was reduced t o a l e s s e r e x t e n t . The mannose content was o n l y s l i g h t l y reduced Not o n l y was the t o t a was reduced t o a d i f f e r e n t e x t e n t . s s e v i d e n t whe the mola r a t i o s o f each sugar normalized t o mannose are compared (Figure 2). For example, N-acetylgalactosamine which had a molar r a t i o to mannose o f 1.5 i n normal t i s s u e was reduced t o 0 . 4 i n cancer t i s s u e s . This r e d u c t i o n may be d i r e c t l y r e l a t e d t o the l o s s i n blood group A a c t i v i t y s i n c e N-acetylgalactosamine i s a p a r t o f t h i s blood group determinant. To e s t a b l i s h whether the observed a l t e r a t i o n s i n antigen l e v e l s and i n carbohydrate content were due t o decreased s y n t h e s i s o r t o i n c r e a s e d degradation o f the carbohydrate moiety, we examined two groups o f enzymes: g l y c o s y l t r a n s f e r a s e s , i n v o l v e d i n t h e i r b i o s y n t h e s i s , and g l y c o s i d a s e s , which degrade them. The A, B o r 0 blood group o f an i n d i v i d u a l i s determined by the presence o r absence o f two s p e c i f i c g l y c o s y l t r a n s f e r a s e s which are products o f the A o r B gene. The r e l a t i o n s h i p between blood groups o f an i n d i v i d u a l and the presence o f these two g l y c o s y l t r a n s f erases i s shown i n Table I I . Only i n d i v i d u a l s o f blood type A or AB c o n t a i n a s p e c i f i c N - a c e t y l g a l a c t o s a m i n y l t r a n s f e r a s e i n t h e i r i n t e s t i n a l mucosa, w h i l e o n l y those o f blood types B and AB have a s p e c i f i c g a l a c t o s y l t r a n s f e r a s e . Subjects who are blood type 0 l a c k both o f these enzymes. The immediate p r e c u r s o r f o r the A and B determinants i s the 0(H) s t r u c t u r e , so t h a t i t can be assumed t h a t a l l i n d i v i d u a l s who s e c r e t e A. B or 0 blood group substances have the g l y c o s y l t r a n s f e r a s e s r e q u i r e d t o s y n t h e s i z e the p r e c u r s o r o l i g o s a c c h a r i d e . Four o t h e r g l y c o s y l t r a n s f e r a s e s shown i n Table I I are not dependent on the i n d i v i d u a l ' s blood type. F i g u r e 3 shows some o f the g l y c o s y l t r a n s f e r a s e a c t i v i t i e s assayed i n tumors and i n t i s s u e s taken from the h i s t o l o g i c a l l y normal adjacent mucosa. A l s o shown are the l e v e l s o f these glycos y l t r a n s f erases i n normal c o l o n i c t i s s u e s taken from p a t i e n t s w i t h diseases other than cancer ( d i v e r t i c u l i t i s o r v o l v u l u s o f the c o l o n ) . There were no s i g n i f i c a n t d i f f e r e n c e s between the two



298

Table I Hemagglutination I n h i b i t i o n by the Membrane Glycopeptides of the Normal and Cancerous Mucosa (rz) Dolichos ,_ A n t i - L e . (A) b i f lorus serum

a

_ Ricinus _ Con A (Le ) . (Gal) communis

x

Patients

NL

1 2 3 4 5 6

++* ++++ ++++ ++++ ++++ +++

CA -

/ T

a

x

x

(Man .Glc N)

NL

CA

NL

CA

NL

CA

++++ ++++

-

-

+ ++ ++

+++ ++ ++++

+++ ++++ ++++

++++

++++

+

++++

++

*-, no i n h i b i t i o n up t o 300 ug membrane p r o t e i n ; +, i n h i b i t e d by 200-300 ug; ++, i n h i b i t e d by 100-200 ug; +++ i n h i b i t e d by 50-100 ug; and ++++, i n h i b i t e d by 50 ug. A, blood group A; Lea, blood group Lewis A; G a l , galactose; Man, mannose; GlcN, N-acetylglucosamine. f

Z

i

60-

•

NORMAL

•

CANCER

o

3

40-

i

o z

20-

MAN

Figure 1.

GAL

FUC

i

GLC GAL NEUAC -NAC -NAC

Carbohydrate composition of the membrane fraction of normal and cancerous colonic mucosa


16.

KIM E T AL.

Malignant

and Inflammatory

Colon

Disease

299

100Q_

u B GAL

B GAL (9GLC «<MAN o 90)

/ m

v

Colon carcinoma . ^ \ (Tissue c u l t u r e ) ' 2-

53 - 100

66 ( 0 * .

15

Stages I I & I I I carcinoma

77 (50 - > 80)

72 (60 - 80)

3

Stage I carcinoma

53 (20 - 70)-

(50 - 66)

Anti-N

(A

5

ign

52^ (30 - 75)

B e n

Anti-M

sera

Human

TT

Breast glandular t i s s u e

Table I . Percent Absorption o f A n t i - B l o o d Group M, N, and Precursor A g g l u t i n i n s with Human Breast Gland Membrane-Cytoplasm P r e p a r a t i o n s , Colon Carcinomata and Controls

314

G L Y C O P R O T E I N S A N D GLYCOLIPIDS I N DISEASE

PROCESSES

and source o f the sera were unknown; three serum standards were included as c o n t r o l s i n a l l determinations. Specimens were decoded only a f t e r completion o f the t e s t s . B i o s y n t h e t i c Procedures. For enzymatic transformation o f Tn to T group B r e d c e l l s o f C l a . R i c , which are completely Tntransformed, were used (19 ,20). Sera o f blood group B and A]B persons f r e e d o f a n t i - T and -Tn a g g l u t i n i n s served as p-galactos y l t r a n s f e r a s e source, UDP-Gal as Gal donor, and ATP, M n C l and MgCl2 as a c t i v a t o r s . A n a l y t i c a l grade chemicals were used; UDPGal and ATP were from Sigma Chemicals. Tn r e d c e l l s incubated i n absence o f one or more o f the other reactants served as c o n t r o l s ; they d i d not a c q u i r e T s p e c i f i c i t y , nor d i d normal MN r e d blood c e l l s when incubated under experimental c o n d i t i o n s with transf e r a s e - c o n t a i n i n g serum. For b i o s y n t h e s i s o f N and M s p e c i f i c determinants T antigens prepared by d e s i a l a t i o each from i s o l a t e d huma were used as s u b s t r a t e s , and f o r M - a c t i v a t i o n o f N, three d i f f e r ent human r e d c e l l NN g l y c o p r o t e i n preparations i s o l a t e d under gentle c o n d i t i o n s (13) were employed. Sera from three to s i x d i f f e r e n t donors o f NN as w e l l as MM blood types served as s i a l y l t r a n s f e r a s e source and CMP-NAN as NAN donor. CMP-NAN was prepared i n Dr. H. Schachter's l a b o r a t o r y as d e s c r i b e d by others (21+ 22) and C-CMP-NAN was from New England Nuclear Corp. In " c o l d experiments, the r e a c t i o n mixtures c o n s i s t e d o f 5 mg (0.5% f i n a l cone.) o f antigen with 2.0 (jmoles CMP-NAN and 500 serum as t r a n s f e r a s e source. In "hot" experiments, a l l r e a c t a n t s were reduced to 12.5% o f those i n the " c o l d " experiment. Incubation and work up were as described p r e v i o u s l y (JL9,_20). C o n t r o l samples were run throughout e x a c t l y as the experiment proper; they c o n s i s t e d o f r e a c t i o n mixtures l a c k i n g i n e i t h e r substrate or CMP-NAN. Substrates incubated with t r a n s f e r a s e i n absence o f CMP-NAN showed no change i n s p e c i f i c i t y . In a l l instances r e covery o f substrate was < 507o a t the end o f the experiment. Controls without added s u b s t r a t e o c c a s i o n a l l y acquired t r a c e s o f activity. 2

14

11

11

In v i v o Measurement o f Delayed-Type " R e c a l l H y p e r s e n s i t i v i ty. T antigen was d i s s o l v e d a t 1% f i n a l c o n c e n t r a t i o n i n buff e r e d s a l i n e c o n t a i n i n g 0.25% phenol; c o n t r o l s were solvent alone and solvent c o n t a i n i n g 1% MN a n t i g e n . A l l reagents were s t e r i l e and free o f pyrogens and Au-antigen. These s o l u t i o n s (0.1 ml each) were i n j e c t e d i . d . i n t o the upper arm. P a t i e n t s with mastectomy were i n j e c t e d i n the c o n t r a l a t e r a l arm. Induration and erythema were measured c a . 24 and 48 h r l a t e r and scored as by H o l l i n s h e a d ejt a l . (23). Reactions, i f any, given by the cont r o l s were s u b t r a c t e d from those due to T antigen. In v i t r o Measurement o f Cell-Mediated Immune Response.


Cell-

17.

SPRINGER E T A L .

Cancer-Associated

Blood Group

MN Precursors

315

u l a r immunity towards T antigen was assessed by the leukocyte migr a t i o n i n h i b i t i o n (LMI) assay i n agarose p l a t e s e s s e n t i a l l y as by Clausen (15,24). As c o n t r o l , leukocytes o f an apparently healthy person were t e s t e d i n p a r a l l e l on the same p l a t e s with those o f the p a t i e n t f o r MI by T antigen (5 o r 10 yg T antigen per 10 u l ) i n each agarose p l a t e . Each t e s t was run i n t r i p l i c a t e , and a r i t h m e t i c averages o f the r e s u l t s were used. C o n t r o l t e s t s with MN i n s t e a d o f T antigen were performed under l i k e c o n d i t i o n s . For healthy i n d i v i d u a l s , the average migration index a t 5 and 10 ug o f T antigen was 1.00; a migration index o f 75% f o r N - a c t i v a t i o n o f T, from four to > 34% f o r M - a c t i v a t i o n o f T and from two to > 75% f o r M - a c t i v a t i o n o f N antigen depending on the human o r animal antiserum i n h i b i t e d . Incorporation of ^C-NAN i n t o T- and Nantigens during incubation with t r a n s f e r a s e - c o n t a i n i n g s e r a was i n keeping with the s e r o l o g i c a l a c t i v i t i e s of the products obtained. S e r o l o g i c a l r e s u l t s were s i m i l a r f o r M - a c t i v a t i o n o f the a-1 glycopeptide i s o l a t e d from NN e r y t h r o c y t e s . The b i o s y n t h e t i c experiments are summarized i n Figure 1. We were s t r u c k by the s t r u c t u r a l s i m i l a r i t y o f the N- and Ms p e c i f i c groups o f the human red c e l l g l y c o p r o t e i n s and glycoprot e i n I ( e p i g l y c a n i n ) (26) of the TA3 mouse mammary adenocarcinoma; we found that t h i s g l y c o p r o t e i n indeed possessed blood group Nl i k e s p e c i f i c i t y (27). TA3 g l y c o p r o t e i n I o f s t r a i n A mice i s carcinoma-associated and does not occur i n normal s t r a i n A mouse t i s sue (28). Thus, a l i n k was e s t a b l i s h e d f o r the f i r s t time between an animal carcinoma-associated antigen and a human blood group substance (N and precursor T ) . The human a n t i - T a g g l u t i n i n of Thomsen and F r i e d e n r e i c h r e a d i l y k i l l e d TA3-St carcinoma c e l l s


316

GLYCOPROTEINS

A N D GLYCOLIPIDS

I N DISEASE

cx-GalNAc-OSer/Thr —

Tn

Figure 1. Biosynthetic pathway of the M- and Nspecific immunodeterminant structures of human blood group M and N glycoproteins


PROCESSES

17.

SPRINGER E T A L .

Cancer-Associated

Blood

Group

MN Precursors

317

i n the presence o f guinea p i g complement (28). We therefore turned our a t t e n t i o n from murine mammary glands and the TA3 carcinoma to human breast t i s s u e and i t s malignancies. Human breast gland c e l l membrane preparations were used to absorb human a n t i s e r a . The r e s u l t s of some o f these s t u d i e s are l i s t e d i n Table I . M- and N- s p e c i f i c s t r u c t u r e s are present i n both benign and malignant human breast glands, T- and Tn- s p e c i f i c i t i e s occurred i n a l l of the human b r e a s t (primary) and colon carcinomata t e s t e d but not i n corresponding preparations from benign o r normal b r e a s t t i s s u e s . T a c t i v i t y was low i n one i n v a s i v e Stage I I I b r e a s t carcinoma. A l l but one gland membrane p r e p a r a t i o n s p e c i f i c a l l y absorbed anti-M and anti-N a n t i b o d i e s i n agreement with the r e p r e s e n t a t i o n o f these antigens on the p a t i e n t ' s r e d cells. There was one exception where N but n o t M was expressed on the p a t i e n t ' s carcinoma c e l l membranes, while both antigens were present on her red c e l l s . We have t e s t e d metastase gery f o r primary d u c t a t i e n t s . A l l contained T- and the two t e s t e d f o r Tn contained a l s o T n - s p e c i f i c s t r u c t u r e s . This promising observation i n d i c a t e s that T- and Tn- s p e c i f i c i t i e s are not e l i m i n a t e d by e i t h e r somatic mutation or modulation and thus remain a c c e s s i b l e to p o t e n t i a l immunodiagnosis and immunotherapy. The T - s p e c i f i c i t y on adenocarcinomata prompted our determinat i o n of a n t i - T t i t e r scores o f human s e r a , which o r d i n a r i l y range from 22 to 24. They were s e v e r e l y depressed i n a h i g h l y s i g n i f i cant number o f a l a r g e p o p u l a t i o n o f breast carcinoma p a t i e n t s as compared to 270 p a t i e n t s with benign breast d i s o r d e r s and 470 cont r o l s a l l together (Table I I ) . S i m i l a r r e s u l t s were obtained i n p a t i e n t s with lung and g a s t r o i n t e s t i n a l (G.I.) carcinomata (15; to be p u b l i s h e d ) . Of the j u s t described p a t i e n t s with breast disease, 32 with mastectomy f o r carcinoma and 32 with breast biopsy who s u f f e r e d from benign disease were r e b l e d once w i t h i n 14 months of surgery. Of the 32 breast carcinoma p a t i e n t s r e b l e d , 21 (65.6%) showed an i n c r e a s e of > 25 to 90% i n a n t i - T t i t e r , while only one o f 32 (3.1%) benign breast disease p a t i e n t s and none o f 11 p a t i e n t s w i t h major operations f o r non-malignant d i s o r d e r s showed a s i g n i f i c a n t i n c r e a s e i n a n t i - T . This d i f f e r e n c e between the carcinoma pat i e n t s and the c o n t r o l s was s i g n i f i c a n t (JD < 0.001). In some p a t i e n t s , b l e d a t h i r d time, c l i n i c a l recurrence o f the carcinoma was a s s o c i a t e d with renewed lowering o f serum a n t i - T l e v e l s (to be p u b l i s h e d ) . Table I I I summarizes the f i n d i n g s on delayed-type s k i n hypers e n s i t i v i t y (DTH) observed on i . d . i n j e c t i o n of T antigen, and those o f the leukocyte migration i n h i b i t i o n assay (LMI). I t can be c l e a r l y seen that the immune response towards T antigen of breast carcinoma p a t i e n t s extends from humoral to c e l l - m e d i a t e d r e a c t i v i t y and i s demonstrable i n v i v o as w e l l as i n v i t r o . F o r t y four breast carcinoma p a t i e n t s were s k i n t e s t e d (Table I I I ) , 36 o f


In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978. 3.62

A l l noncarcinomatous persons studied (B) C

17 /470

8.30

8.15

14^/270

£

39 /470

22^/270

61/189

32.28-

40/189

No. o f persons

No. of persons

12 or
: that d i f f e r e n c e between a n t i - T scores of breast carcinoma p a t i e n t s and c o n t r o l group (A) as w e l l as (B) i s due to chance i s < 0.001 throughout (X t e s t ) . £ Two of the 14 p a t i e n t s have s i n c e developed carcinoma.

5.19

Benign breast disease (A)

21.16^

%

10 or
3 weeks a f t e r surgery or chemotherapy and > 12 months a f t e r r a d i a t i o n therapy were s t u d i e d , because of the known immunosuppression due to these i n t e r v e n t i o n s (46). Individuals of Chinese and Japanese descent not i n c l u d e d . — DTH p o s i t i v e = > 4.5 mm i n d u r a t i o n a f t e r 24-48 hr or erythema at 48 hr > 25 mm (average of 2 diameters measured p e r p e n d i c u l a r l y ) (23). — Leukocyte migration i n h i b i t i o n on agarose p l a t e ; 10 (jbg T antigen/1.5 x 10 leukocytes; not t o x i c up to > 45 jig. — Negative was the only i n f i l t r a t i n g l o b u l a r carcinoma. — The two negative persons had i n s i t u l o b u l a r carcinoma. — 2/38 p o s i t i v e . I n t r a d u c t a l papilloma was the most ominous s t r u c t u r e i n both. £ Vietnam war veteran with typhoid, c h o l e r a and tetanus t o x o i d v a c c i n a t i o n s .

"Healthy"

11/84

8/27

14-/16

I 2-/38

17/31

13/13

II

Benign

10/21

8^/9

7/14

Persons p o s i t i v e / P e r s o n s

III

tested

6/6

Persons positive—/Persons

LMI

IV

Disease stage

DTH

Table I I I . Delayed-Type R e c a l l Skin R e a c t i v i t y upon i . d . I n j e c t i o n of T and i n v i t r o Cell-Mediated Immune Response to T Antigen i n P a t i e n t s with Breast Carcinoma, Benign Breast Disease and i n Apparently Healthy Controls-^

GLYCOPROTEINS AND

320

GLYCOLIPIDS IN

DISEASE

PROCESSES

these s u f f e r e d from carcinoma of d u c t a l o r i g i n , 2 were noni n v a s i v e . The remaining 8 p a t i e n t s had l o b u l a r carcinoma. A l l p a t i e n t s with d u c t a l carcinoma gave a p o s i t i v e DTH r e a c t i o n regardless of Stage and invasiveness (15,29). F i v e with l o b u l a r carcinomata a l s o gave a p o s i t i v e r e a c t i o n but one of the p a t i e n t s with i n v a s i v e and two with non-invasive l o b u l a r tumors d i d not r e a c t (29). In c o n t r a s t , none of the 23 healthy i n d i v i d u a l s t e s t ed had any delayed h y p e r s e n s i t i v i t y r e a c t i o n toward T antigen, and 36 of the 38 p a t i e n t s diagnosed h i s t o l o g i c a l l y to have f i b r o adenoma-fibrocystic disease had a negative r e a c t i o n . The r i g h t hand s i d e of Table I I I shows that i n LMI 34 (51.5%) of the 66 p a t i e n t s with breast carcinoma Stages I I , I I I and IV had a p o s i t i v e r e a c t i o n , while of 27 p a t i e n t s with Stage I disease, eight (29.6%) were p o s i t i v e . Seven of the e i g h t p a t i e n t s with l o b u l a r carcinoma r e f e r r e d to above were t e s t e d i n LMI. The leukocytes of only two of these p a t i e n t s , both with i n f i l t r a t i n g tumor showed migration i n h i b i t i o n Of 84 p a t i e n t s wit p o s i t i v e i n LMI with T antigen. No e f f e c t of T antigen was found on the p e r i p h e r a l leukocytes of a l a r g e number of presumably healthy people. LMI was p o s i t i v e i n only one of 95 healthy i n d i v i d u a l s , a Vietnam war veteran v a c c i n a t e d repeatedly with S.typhi, V. choJLerae and Tetanus t o x o i d a l l of which possess T - s p e c i f i c i t y (see Table IV). T antigen up to 50 yg d i d not a f f e c t v i a b i l i t y (trypan blue) or migration of the white c e l l s of healthy persons, MN antigen at the same high concentration had no e f f e c t on migrat i o n of leukocytes of any of the p a t i e n t s and c o n t r o l s t e s t e d . Discussion In t h i s communication we report that we have reversed the degradative steps l e a d i n g from blood group M - s p e c i f i c s t r u c t u r e s to i t s precursors and synthesized T - s p e c i f i c groups (19,20) on Tn erythrocytes as w e l l as N- and M- s p e c i f i c groups on i s o l a t e d T antigens. The peptide core of T antigen i n M- d i f f e r s i n 2 amino acids from that i n N- i n d i v i d u a l s and M a n t i g e n i c determinants possess more carbohydrate (30,31,32,33,34). Since g l y c o s y l t r a n s ferases g e n e r a l l y recognize only l i m i t e d areas (one to two monosaccharides) u n d e r l y i n g the acceptor molecule, s i a l y l t r a n s f e r a s e s from MM and MN donors produced N and M a c t i v a t i o n , whereas those from NN donors produced only N regardless of the MN type o r i g i n of T antigen. I s o l a t e d N antigen could be transformed to M only with s e r a from MM and MN donors. The g e n e t i c a l sequence of these immunological s p e c i f i c i t i e s i s t h e r e f o r e : Tn-*- T* N->- M (M could conceivably a r i s e d i r e c t l y from T). These s t r u c t u r e s have a l s o been found on l i p i d i c c a r r i e r s , i n c l u d i n g those from a carcinoma (35). The discovery that human adenocarcinoma but not healthy human t i s s u e s and benign s t r u c t u r e s possess T- and Tn- s p e c i f i c i t i e s i n r e a c t i v e , unmasked form e s t a b l i s h e s that these precursors are



P a s t e u r e l l a pseudotuberculosis— Pneumococcus XIV p o l y s a c c h a r i d e — BCG, T i c e s t r a i n (Univ. of Illinois).^ T u b e r c u l i n , PPD ( T u b e r s o l ) - * Influenza v i r u s : A / V i c t o r i a + B/Hong Kong (Parke-Davis )— Pseudomonas—*^

VACCINES:

Aerobacter aerogenes K l e b s i e l l a pneumoniae 3/3 Proteus s t r a i n s

LPS:

BACTERIA:

Absent

(LPS) and

— Non-dialyzable p a r t . SL A c t i v e with some but not a l l human a n t i - T . — Donated by Dr. M. H e i d e l berger. ^ Donated by Dr. R. G. Crispen. — A p o s s i b l e c o n t r i b u t i o n to a c t i v i t i e s by growth^medium has not been excluded. -= From Connaught L a b o r a t o r i e s . Donated by Dr. J . U. Gutterman. — Donated by Wellcome L a b o r a t o r i e s , - i From L e d e r l e , Orimune t r i v a l e n t .

Salmonella t y p h i (Wyeth, E. L i l l y ) Corynebacterium parvum (Coparvax)—>|l C l o s t r i d i u m t e t a n i t o x o i d (Wyeth)—*— V i b r i o cholerae (Wyeth)—>— Poliomyelitis v i r u s ^ ' i

4/8 E. c o l i 6/9 Salmonella s t r a i n s S. typhimurium S. abortus equi S. t y p h i S. minnesota 2/3 S h i g e l l a s t r a i n s S e r r a t i a marcescens Bacterium t u l a r e n s i s 2/3 Chromobacterium violaceum

LPS:

VACCINES:

9/9 E s c h e r i c h i a c o l i 3/3 E. f r e u n d i i Salmonella poona 1/3 Pseudomonas s t r a i n s

T Specificity

T - S p e c i f i c Substances i n Microbes, t h e i r Lipopolysaccharides M i c r o b i a l Vaccines^

Present

IV.

BACTERIA:

Table

322

GLYCOPROTEINS

A N D GLYCOLIPIDS I N DISEASE

PROCESSES

c h a r a c t e r i s t i c o f adenocarcinomata o f human b r e a s t , G.I. t r a c t and probably lung. The occurrence o f f u l l y r e a c t i v e T- and Tna n t i g e n i c s p e c i f i c i t i e s i n these cancers i s probably due to i n complete b i o s y n t h e s i s o f normal c e l l - s u r f a c e components, as i n d i cated by the presence of a l l these s p e c i f i c i t i e s except M, i n the breast carcinoma o f one p a t i e n t whose r e d c e l l s c a r r i e d M and N antigens as w e l l as by the extensive absorption (70%) of anti-N by b r e a s t carcinoma membranes from one p a t i e n t o f e r y t h r o c y t e group MM. T- and Tn- s p e c i f i c a n t i g e n i c determinants have r e c e n t l y been demonstrated d i r e c t l y with immunofluorescent l e c t i n s i n a l l carcinomatous b r e a s t glands t e s t e d but not i n n o n - c a r c i nomatous glandular areas o f the same s e c t i o n s (36; Dr. Cr. McNeil, p e r s o n a l communication). O r d i n a r i l y humans have no anti-N o r anti-M a n t i b o d i e s s i n c e a l l possess b l o o d group N, M, o r NM, and i n d i v i d u a l s homozygous f o r M uniformly have some N as w e l l (1,37). Healthy mammals do not form a n t i b o d i e s agains with t h e i r immune machinery are not ubiquitous as are A and B s p e c i f i c i t i e s (3,7,38). On the other hand, T- and Tn- s p e c i f i c s t r u c t u r e s are always masked i n healthy humans, but are widespread among i n t e s t i n a l and a i r b o r n e b a c t e r i a (see below); consequently a n t i b o d i e s against these s t r u c t u r e s are found i n a l l humans (16,39). Levels o f these a n t i bodies are q u i t e constant under o r d i n a r y circumstances (29,39). Anti-T was s e v e r e l y depressed i n p a t i e n t s with carcinoma o f the b r e a s t , lung and G.I. t r a c t much more f r e q u e n t l y when compared to populations o f comparable age and socio-economic s t a t u s who had e i t h e r benign disease o r were apparently h e a l t h y . Of a l l i n d i v i duals with s e v e r e l y depressed a n t i - T , > 80% s u f f e r e d from c a r c i noma. This depression was not due to IgG, IgM, o r IgA decrease. S i m i l a r observations on a n t i - T of p a t i e n t s with b r e a s t cancer have r e c e n t l y been reported by others (40). Anti-T antibody may be depressed because of i t s i n t e r a c t i o n with T - s p e c i f i c substances e i t h e r attached to cancer c e l l - s u r f a c e and/or r e l e a s e d from t h e c e l l - s u r f a c e i n t o the c i r c u l a t i o n . This hypothesis i s supported by the o b s e r v a t i o n of r e l e a s e o f T - s p e c i f i c antigens from human mammary carcinoma-derived t i s s u e c u l t u r e c e l l s i n t o the c u l t u r e f l u i d (41). We d i d not f i n d depressed a n t i - T i n p a t i e n t s with sarcoma, melanoma o r with b r a i n tumors t e s t e d so f a r , n o r Ts p e c i f i c s t r u c t u r e s on these tumors. As mentioned above, there i s evidence that humoral a n t i - T as w e l l as a n t i - T n r e s u l t l a r g e l y from immunization by the host's own i n t e s t i n a l f l o r a (7,14,38,42,43,44,45). We s t u d i e d numerous, predominantly gram-negative b a c t e r i a and/or t h e i r l i p o p o l y s a c c h a r i d e s , and some v a c c i n e s . Table IV shows that many microbes indeed possess T - s p e c i f i c s t r u c t u r e s as determined by hemagglut i n a t i o n i n h i b i t i o n and by absorption assays. Three of the 12 a c t i v e E s c h e r i c h i a c o l i and one o f the two a c t i v e Chromobacterium violaceum s t r a i n s , Salmonella milwaukee and JS. abortus equi i n h i b i t e d human a n t i - T only when t e s t e d a f t e r b o i l i n g . It is


17.

SPRINGER E T A L .

Cancer-Associated

Blood

Group

MN Precursors

323

noteworthy that Corynebacterium parvum v a c c i n e , i n wide use to s t i m u l a t e " u n s p e c i f i c " r e s i s t a n c e against cancer, possessed s i g n i f i c a n t T s p e c i f i c i t y , while the one commercial BCG t e s t e d and the Pseudomonas v a c c i n e (also used as " u n s p e c i f i c " s t i m u l a n t against cancer) and PPD t u b e r c u l i n had no T a c t i v i t y . Tn a c t i v i t y o f b a c t e r i a l p r e p a r a t i o n s was a l s o found i n 25 of 30 Enterobacteariaceae ( i n c l u d i n g S.typhi). Bacterium t u l a r e n s i s , and i n JC. parvum, typhoid, tetanus and p o l i o m y e l i t i s v a c c i n e s . Tn a c t i v i t y was not demonstrable i n BCG, PPD t u b e r c u l i n , three Pseudomonas s t r a i n s and some Enterobacteriaceae. The discovery o f T- and Tn- s p e c i f i c immunologically r e a c t i v e determinants i n the c e l l membranes of breast and colon adenocarcinomata but not i n healthy t i s s u e s e s t a b l i s h e s f o r the f i r s t time a p h y s i c a l l y and chemically w e l l - d e f i n e d immunospecific s t r u c t u r e c h a r a c t e r i s t i c a l l y a s s o c i a t e d with human carcinoma. These precursor s p e c i f i c i t i e d i r e a c t i v for be due to incomplete b i o s y n t h e s i normal c e l l - s u r f a c e components gen a l s o occurs i n other malignancies and o c c a s i o n a l l y i n some permanently benign human tumors. The T antigen q u i t e l i k e l y can be considered a paradigm f o r other p r e c u r s o r antigens, e.g. Tn, and a l s o f o r the Forssman antigens and r e l a t e d blood group antigens, with s i m i l a r f u n c t i o n s i n malignant processes. T antigen i s a v a i l a b l e i n u n l i m i t e d q u a n t i t y , i s not contaminated with HL-A- and Au- antigens, and i s o b t a i n a b l e pyrogen-free. I t can be r e a d i l y prepared by removal of NAN from i s o l a t e d human r e d c e l l blood group NM antigens. T antigen and the a n t i - T a n t i b o d i e s which are present i n a l l humans (conceivably coupled with r a d i o a c t i v e i s o t o p e s ) may be u s e f u l i n d i a g n o s i s , prognosis, and p o s s i b l y therapy and immunoprophylaxis of some adenocarcinomata. Acknowledgments G. F. S. i s J u l i a S. Michels I n v e s t i g a t o r i n S u r g i c a l Oncology. This work was supported by the N a t i o n a l I n s t i t u t e s o f Health grants CA 19083 and CA 22540. We thank surgeons and patho l o g i s t s of Evanston and St. F r a n c i s H o s p i t a l s , Evanston, IL and the B e r k s h i r e Medical Center, P i t t s f i e l d , MA f o r f r e s h t i s s u e s . We are g r a t e f u l t o Mrs. H. Tegtmeyer, Mrs. T. Mo, Mr. M. Coleman, Mrs. V. Scanlon, Mrs. E. M i t c h e l l and Mrs. J . K e l l o g g f o r e x c e l lent assistance.



324 Literature Cited

1. Landsteiner, K.; Levine, P. J. Exp. Med. (1928), 47, 757-775. 2. Springer, G. F.; Ansell, N. J. Proc. Natl Acad. Sci. USA (1958), 44, 182-189. 3. Springer, G. F. Naturwissenschaften (1970), 57, 162-171. 4. Kosjakov, P. N.; Tribulev, G. P. J. Immunol. (1939), 37, 283-295. 5. Boorman, K. E.; Dodd, B. E. J. Pathol. Bacteriol. (1943), 55, 329-339. 6. Stalder, K.; Springer, G. F. Fed. Proc. (1960), 19, 70. 7. Springer, G. F.; Horton, R. E. J. Clin. Invest. (1969), 48, 1280-1291. 8. Davies, D. A. L. Immunology (1966), 11, 115-125. 9. Mann, D. L.; Rogentine, G. N., Jr.; Fahey, J. L.; Nathenson, S. G. Nature (1968), 217, 1180-1181. 10. Tom, B. H.; Rutzky C. I.; Kahan, B. D 11. Springer, G. F.; Desai, P. R.Carbohydr.Res. (1975), 40, 183-192. 12. Springer, G. F.; Desai, P. R.; Scanlon, E. F. Cancer (1976), 37, 169-176. 13. Springer, G. F.; Nagai, Y.; Tegtmeyer, H. Biochemistry (1966), 5, 3254-3272. 14. Springer, G. F.; Desai, P. R.; Banatwala, I. J. Natl. Cancer Inst. (1975), 54, 335-339. 15. Springer, G. F.; Desai, P. R.; Murthy, M. S.; Scanlon, E. F. J. Surg. Oncol. (1978), in press. 16. Moreau, R.; Dausset, J.; Bernard, J.; Moullec, J . Bull. Soc. Méd. Hôp. (1957), 73, 569-587. 17. Sturgeon, P.; McQuiston, D. T.; Taswell, H. F.; Allan, C. J. Vox Sang. (1973), 25, 481-497. 18. Stratton, F.; Renton, P. H. "Practical Blood Grouping", p. 34, Blackwell, Oxford, 1958. 19. Springer, G. F.; Desai, P. R.; Schachter, H.; Narasimhan, S. Naturwissenschaften (1976), 63, 488-489. 20. Desai, P. R.; Springer, G. F. Proc. 4th Int. Symp. Glycoconjugates (1977), in press. 21. Comb, D. G.; Roseman, S. J. Biol. Chem. (1960), 235, 25292537. 22. Kean, E. L. J. Biol. Chem. (1970), 245, 2301-2308. 23. Hollinshead, A. C.; Stewart, T. H. M.; Herberman, R. B. J. Natl. Cancer Inst. (1974), 52, 327-338. 24. Clausen, J. E. Acta Allergol. (Kbh.) (1971), 26, 56-80. 25. Springer, G. F.; Desai, P. R. Transplant. Proc. (1977), 9, 1105-1111. 26. Codington, J. F.; Sanford, B. H.; Jeanloz, R. W. Biochemistry (1972), 11, 2559-2564. 27. Springer, G. F.; Codington, J. F.; Jeanloz, R. W. J . Natl. Cancer Inst. (1972), 49, 1469-1470.


17.

28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46.

SPRINGER ET AL.

Cancer-Associated Blood Group MN Precursors

Springer, G. F.; Desai, P. R. Ann. Clin. Lab. Sci. (1974), 4, 294-298. Springer, G. F.; Desai, P. R.; Murthy, M. S.; Scanlon, E. F. Naturwissenschaften (1978), in press. Springer, G. F.; Yang, H. J.; Huang, I.-Y. Naturwissenschaften (1977), 64, 393. Springer, G. F.; Yang, H. J. Immunochemistry (1977), 14, 497-502. Yang, H. J.; Springer, G. F. Proc. 4th Int. Symp. Glycoconjugates (1977), in press. Waśniowska, K.; Drzeniek, Z.; Lisowska, E. Biochem. Biophys. Res. Commun. (1977), 76, 385-390. Friedensen, B.; Yang, H. J.; Springer, G. F., in preparation. Cantrell, J. L.; Springer, G. F.; Desai, P. R., in preparation. Fischer, K.; Poschmann Wochenschr. (1977), , Springer, G. F.; Tegtmeyer, H.; Huprikar, S. V. Vox Sang. (1972), 22, 325-343. Hoskins, L. C.; Boulding, E. T. J. Clin. Invest. (1976), 57, 74-82. Friedenreich, V. "The Thomsen Hemagglutination Phenomenon" Levin and Munksgaard, Copenhagen, 1930. Luner, S. J.; Wile, A. G.; Sparks, F. C. Proc. 68th Annu. Meet. Am. Assoc. Cancer Res. (1977), Abstract 375. Anglin, J. H.; Lerner, M. P.; Nordquist, R. E. Nature (1977), 269, 254-255. Springer, G. F.; Desai, P. R. Annu. Meet. Am. Soc. Microbiol. (1977), Abstracts, E42, p. 88. Springer, G. F.; Desai, P. R.; Murthy, S. M.; Scanlon, E. F. Proc. 4th Int. Symp. Glycoconjugates (1977), in press. Boccardi, V.; Attinà, D.; Girelli, G. Vox Sang. (1974), 27, 268-272. Springer, G. F.; Horton, R. E.; Forbes, M. J . Exp. Med. (1959), 110, 221-244. Daban, A.; Schneider, M.; Calle, R. Bull, du Cancer (1975), 62, 21-28.

RECEIVED

March 30, 1978.


18 Alpha-fetoprotein as an Indicator of Early Events in Chemical Carcinogenesis STEWART SELL Department of Pathology, M-012, University of California, San Diego, La Jolla, CA 92093 Alphafetoprotein (AFP) concentration in fetal sera, y sera, has attracted increasing attention among oncologists and developmental biologists. AFP was discovered by the great Soviet scientist, Garri Israelevich Abelev, in 1963, when he noted the presence of a specific serum protein in neonatal mice and in mice bearing transplantable hepatomas (1). Several other laboratories soon were able to confirm this observation in other species, including man (2, 3, 4, 5, 6). However, further advances in understanding the biologic and diagnostic significance of AFP were hindered by lack of a sensitive accurate technique for measuring AFP. Accurate radioimmunoassays for AFP were established in the early 1970's (7, 8, 9, 10). This was followed by development of specific immunoassays for AFP synthesis by labeled amino acid incorporation and immunofluorescent labeling techniques. Using these assays, AFP production and cellular localization both in vivo and in vitro under a variety of experimental systems has been examined (11, 12). These experimental studies led to understandings which have been expanded clinically in regard to the use of AFP in the diagnosis of congenital anomalies, the differentiation of benign from malignant liver disease, and the determination of the effects of therapy on AFP-producing tumors, as well as to insights into the significance of AFP in normal development and of the sequence of events culminating in development of hepato-cellular carcinomas after exposure of rats to chemical hepato-carcinogens. Properties of AFP AFP is a single protein chain with physical chemical properties similar to albumin (Table I). In native configuration AFP is immunochemically distinct from albumin, but cross inhibition of antibody binding between AFP and albumin is observed after unfolding of the molecules by disulfide reduction and carboxyaminylmethylation (13). In addition, preliminary amino acid 0-8412-0452-7/78/47-080-326$05.00/0 © 1978 American Chemical Society In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

18.

SELL

Alpha-fetoprotein

as an Indicator

in

Carcinogenesis

327

sequencing i n d i c a t e s a 45% homology (14). AFP of murine species has a high estrogen b i n d i n g a f f i n i t y (10~^M) , whereas the AFP of other species does not (15, 16). TABLE I PROPERTIES OF RAT AFP AND ALBUMIN AFP Molecular Weight 70,000 I s o e l e c t r i c Point 4.9 CHO Content 4% Serum Concentration (ug/ml) Adult .03 Fetal 8,000 Estrogen Binding -9 Ka 10 pm/mg P r o t e i n 50 Sequence Homology Cross R e a c t i v i t y 0 Native + Unfolded*

ALBUMIN 60,000 5.6 None 60,000 100 -5 10 0.2

* D i s u l f i d e r e d u c t i o n plus carboxamidomethylation. A s e r i e s of systematic experimental s t u d i e s on the nature and s i g n i f i c a n c e of a l p h a f e t o p r o t e i n (AFP) i n normal development, during r e s t i t u t i v e h e p a t o c e l l u l a r p r o l i f e r a t i o n , during tumor growth and therapy, and a f t e r exposure to hepatocarcinogens has been c a r r i e d out i n our l a b o r a t o r y . A summary of the serum conc e n t r a t i o n s of AFP under v a r i o u s c o n d i t i o n s i s given i n Chart 1. Normal Development A l p h a f e t o p r o t e i n i s synthesized by the f e t a l l i v e r and y o l k sac (18, 19), crosses the p l a c e n t a i n t o the maternal c i r c u l a t i o n and i s r a p i d l y c a t a b o l i z e d by the mother (20). There i s a r e c i p r o c a l r e l a t i o n s h i p between the serum c o n c e n t r a t i o n of AFP and albumin. E a r l y i n development AFP concentrations are high and albumin concentrations low (4, 5, 21). When the serum concent r a t i o n s of AFP f a l l during neonatal development, the concentrat i o n of albumin r i s e s . The d e l i n e a t i o n of the feto-maternal synthesis and d i s t r i b u t i o n i n the r a t has e s t a b l i s h e d a base f o r understanding e l e v a t i o n s of AFP concentrations i n aminotic f l u i d or maternal serum a s s o c i a t e d with c o n g e n i t a l anomalies i n the human (Table II) (22, 25).


328

GLYCOPROTEINS A N D

GLYCOLIPIDS IN

DISEASE

PROCESSES

TABLE I I INCREASED AMNIOTIC FLUID AFP IN ABNORMAL PREGNANCY ABNORMALITY MECHANISM F e t a l Death Release of F e t a l AFP i n t o AMF Neural Tube Defects Transfer of AFP from F e t a l CSF Esophageal A t r e s i a Reduced Turnover, Lack of Swallowing Congenital Nephrosis Loss of F e t a l AFP from Renal Defect M u l t i p l e Pregnancy Increased Production Rh I n c o m p a t i b i l i t y ?Increased Production A l p h a f e t o p r o t e i n i s synthesized by the l i v e r of the newborn r a t u n t i l four weeks of age where there i s an abrupt c e s s a t i o n of s y n t h e s i s (11). This s t r i k i n g c e s s a t i o n of AFP s y n t h e s i s i s a s s o c i a t e d with the termination of neonatal l i v e r c e l l p r o l i f e r a t i o n and change i n the ploid. There i s a c l o s e r e l a t i o n s h i p between the f e t a l t i s s u e s that synthesize AFP and tumors i n a d u l t s that produce AFP. Tissue s i t e s of s y n t h e s i s of AFP, measured by i n c o r p o r a t i o n of r a d i o l a b e l e d aminoacids, were determined using d i f f e r e n t f e t a l t i s s u e s (19). The f e t a l l i v e r and the y o l k sac elements are the f e t a l t i s s u e s which produce the highest amounts of AFP; i n a d u l t s l i v e r and teratocarcinoma (yolk sac) f e t a l tumors produce AFP with the highest frequency (26, 27, 28). The f e t a l GI t r a c t (stomach, small i n t e s t i n e and pancreas) a l s o produces AFP although at much lower amounts than l i v e r or y o l k sac; human tumors of the GI t r a c t may produce AFP but the frequence of AFP production by tumors of these t i s s u e s i s much l e s s than the production by hepatomas or teratocarcinomas (29). Small amounts of AFP are produced by f e t a l lung and a small number of bronchiogenic carcinomas produce (21, 29). F i n a l l y , although not produced d e t e c t a b l y by f e t a l mesenchymal t i s s u e s , i t i s p o s s i b l e that mesenchymal tumors may a l s o produce AFP (30). These r e s u l t s support the concept of d i f f e r e n t l e v e l s of expression of developmental products by tumors (31, 32). Such exposures a l s o a p p l i e s to e c t o p i c hormone product i o n and carcinoembryonic antigen by tumors (Table I I I ) .


18.

SELL

Alpha-fetoprotein

as an Indicator

in Carcinogenesis

329

TABLE I I I LEVELS OF EXPRESSION OF SOME ONCQDEVELQPMENTAL GENE PRODUCTS BY TUMORS AFP HORMONE CEA FIRST LEVEL (e.g. ACTH) F e t a l or Adult Pituitary Colonic Hepatoma, Tissue Which Adenoma Teratocarcinoma Carcinoma Normally With Yolk Sac Produce I t Elements SECOND LEVEL C l o s e l y Related Embryologically (Same C e l l Line) THIRD LEVEL More D i s t a n t l y Related (Same C e l l Line) FOURTH LEVEL Different Cell Line

Pancreas, GI CA

Pancreas, Gastric Liver CA

Medullary CA of Thyroid

Breast CA

of Lung

Sarcoma, Lymphoma

Pulmonary CA

Pulmonar

?Sarcoma Lymphoma

The c o n t r o l of AFP s y n t h e s i s by f e t a l hepatocytes has been determined by q u a n t i t a t i v e assay of AFP and albumin synthesis i n a f e t a l hepatocytes c u l t u r e system developed by Hyam L e f f e r t of the Salk I n s t i t u t e (18). The data i n d i c a t e that albumin i s synthesized throughout the c e l l c y c l e , whereas AFP i s l a r g e l y synthesized p r i o r to S and r e l e a s e d p r i o r to M (19). Factors such as hormones, which may a f f e c t AFP s y n t h e s i s , appear to do so through a l t e r a t i o n s i n the growth s t a t e of f e t a l hepatocytes. When f e t a l hepatocytes are not i n an a c t i v e growth s t a t e , product i o n o f AFP i s not d e t e c t a b l e . Hepatotoxic

L i v e r Injury

The l i v e r of adult r a t s subjected to 70% p a r t i a l hepatectomy (16) or f o l l o w i n g exposure to chemical hepatotoxic agents such as C C l ^ or galactosamine (35, 36) w i l l synthesize AFP during the r e s t i t u t i v e phase of l i v e r regeneration. The serum concentrat i o n s of AFP become elevated s h o r t l y a f t e r p r o l i f e r a t i o n i s observed and d e c l i n e to normal s h o r t l y a f t e r p r o l i f e r a t i o n stops (16). This e f f e c t i s r e p r o d u c i b l e i n v i t r o as adult hepatocytes c u l t u r e s w i l l re-express f e t a l phenotype during the a c t i v e growth phase but r e - e s t a b l i s h adult phenotype when becoming conf l u e n t (37). The absence of an e l e v a t i o n i n serum AFP f o l l o w i n g l i v e r i n j u r y i s a poor prognostic s i g n , as a l l r a t s not demons t r a t i n g an AFP e l e v a t i o n d i e d . In humans, AFP concentrations may be used i n a s i m i l a r manner to determine the extent of l i v e r


330

GLYCOPROTEINS

A N D GLYCOLIPIDS IN

DISEASE

PROCESSES

damage f o l l o w i n g l i v e r n e c r o s i s or h e p a t i t i s and to determine the occurrence of r e s t i t u t i v e p r o l i f e r a t i o n (38,39). By immunof l u o r e s c e n c e , AFP may be i d e n t i f i e d i n hepatocytes undergoing p r o l i f e r a t i o n (40). A s p e c i f i c f u n c t i o n f o r AFP has not been c o n v i n c i n g l y demonstrated. Some of the p o s t u l a t e d f u n c t i o n s of AFP are l i s t e d i n Table IV. Several of these deserve f u r t h e r comment. Those f u n c t i o n s r e l a t e d to the high b i n d i n g c a p a c i t y of AFP f o r estrogen seem not to be g e n e r a l l y a p p l i c a b l e s i n c e only the AFP of r a t and mouse have t h i s property. The p o s s i b i l i t y of AFP p r o t e c t i n g the fetus from maternal immune r e j e c t i o n i s an a t t r a c t i v e one, but c o n f l i c t i n g experimental data have been obtained (41, 42, 43, 44). I t i s c l e a r that AFP does c a r r y out some of the osmotic and c a r r i e r f u n c t i o n s of albumin I n the f e t u s s i n c e AFP i s the major serum p r o t e i n during a c o n s i d e r a b l e time i n the f e t u s (Table I ) .

SOME PROPOSED FUNCTION OBSERVATION S t r u c t u r e Homology with Albumin Inverse Serum Concentration to ALB Immunosuppressive In V i t r o Binds Estrogen Produced During Hepatocyte Proliferation Produced During L i v e r Lobule Formation

POSSIBLE FUNCTION F e t a l Albumin - C a r r i e r and

Osmotic

Blocks Maternal R e j e c t i o n of Fetus P r o t e c t s Fetus From Maternal Estrogen Growth C o n t r o l Developmental Signal

T i s s u e Organizing

The c o r r e l a t i o n of AFP production with normal neonatal l i v e r development and with r e s t i t u t i v e l i v e r c e l l p r o l i f e r a t i o n has suggested that the f u n c t i o n of AFP may be to serve as a t i s s u e o r g a n i z a t i o n s i g n a l f o r proper alignment of d i f f e r e n t c e l l types i n the l i v e r (31). In both neonatal development and r e s o t r a t i o n of the a d u l t l i v e r a f t e r p a r t i a l hepatectomy, not only must the number of hepatocytes be r a p i d l y i n c r e a s e d , but a l s o these c e l l s must assume the proper alignment with s i n u s o i d a l c e l l s , b i l e c a n n i c u l i and v e s s e l s . The r o l e of AFP as an o r g a n i z a t i o n s i g n a l i s supported by analogy to other "oncodevelopmental" markers such as the T locus markers of the mouse and the T c e l l antigens of lymphocytes, one being r e s p o n s i b l e f o r c e l l - c e l l r e c o g n i t i o n during normal development of the e a r l y embryo, the other b e l i e v e d to be r e q u i r e d f o r proper c e l l - c e l l i n t e r a c t i o n s i n immune responses. I t i s a t t r a c t i v e to assume other "oncodevelopmental products such as AFP might f u n c t i o n i n a s i m i l a r manner (31). 11


18.

SELL

Alpha-fetoprotein

as an Indicator

in

Carcinogenesis

331

Tumor Growth The e l e v a t i o n s of serum concentrations of AFP f o l l o w i n g t r a n s p l a n t a t i o n of a v a r i e t y of hepatomas i n syngeneic r a t s has been analysed (45, 46, 47). Not a l l t r a n s p l a n t a b l e hepatomas produce elevated serum c o n c e n t r a t i o n s of AFP. The ]evel of product i o n of AFP i s r e l a t e d to m u l t i p l e f a c t o r s i n c l u d i n g degree of aneuploidy, growth r a t e and degree of h i s t o l o g i c d i f f e r e n t i a t i o n . Upon t r a n s p l a n t a t i o n of tumors that produce AFP an exponential continuous e l e v a t i o n of serum c o n c e n t r a t i o n s of AFP occurs (48) (see Chart 1). This i s i n c o n t r a s t to the t r a n s i e n t , u s u a l l y much l o w e r , e l e v a t i o n s found f o l l o w i n g l i v e r c e l l n e c r o s i s and proliferation. Thus, t h i s model suggests that s e r i a l determinat i o n s of AFP i n p a t i e n t s with undiagnosed hepatomas may be u s e f u l i n d i f f e r e n t i a t i n g c i r r h o s i s or h e p a t i t i s (with l i v e r c e l l p r o l i f e r a t i o n ) from hepatom (49 31) The serum c o n c e n t r a t i o may be used to f o l l o w the e f f e c t s of therapy (48, 50, 51). In a model system, s u r g i c a l removal of an AFP producing tumor r e s u l t s i n an immediate d e c l i n e of serum AFP concentrations. A prolonged r e t u r n to normal i s found i n long-term s u r v i v o r s ; r e - e l e v a t i o n a s s o c i a t e d wtih development of pulmonary or lymph node metastases. R a d i a t i o n to the lung s h o r t l y f o l l o w i n g tumor removal r e s u l t s i n a s i g n i f i c a n t i n c r e a s e i n the number of long-term s u r v i v o r s (51). Again, t h i s animal model p o i n t s out the p o t e n t i a l u s e f u l n e s s of AFP i n c l i n i c a l d i a g n o s i s and prognosis (31). Tumor Development For over twenty years the i n d u c t i o n of r a t h e p a t o c e l l u l a r carcinomas has been used as a model f o r chemical c a r c i n o g e n e s i s (52, 53, 54). In t h i s system a sequence of morphologic a l t e r a t i o n s i s observed during exposure of r a t s culminating i n the appearance of h e p a t o c e l l u l a r carcinomas a f t e r 16-20 weeks. The sequence of morphologic changes proceeds from the appearance of small f o c i of a few hepatocytes with a l t e r e d s t a i n i n g c h a r a c t e r i s t i c s through l a r g e r microscopic f o c i of c e l l s which impinge upon the surrounding parenchyrra and g r o s s l y v i s i b l e n e o p l a s t i c nodules to frank h e p a t o c e l l u l a r carcinomas (55, 56, 57, 58). The hypot h e s i s that t h i s sequence i s r e l a t e d and that the nodules r e p r e sent pre-malignant but r e v e r s i b l e l e s i o n s i s supported by the presence of a l t e r e d h e p a t i c enzyme expression i n the n e o p l a s t i c nodules that i s a l s o found i n h e p a t o c e l l u l a r carcinomas (59, 60). Data obtained from the a p p l i c a t i o n of AFP measurements and c e l l u l a r l o c a l i z a t i o n as an e a r l y i n d i c a t o r of events i n chemical carcinogenesis may r e s u l t i n s i g n i f i c a n t changes i n the current concept of the sequence of events l e a d i n g to development of hepatoc e l l u l a r carcinomas. This use of AFP has been made i n c o l l a b o r a t i o n with F.F. Becker, E. Smuckler, B. Lombardi and H. Shinozuka. Exposure of r a t s to hepatocarcinogens such as e t h i o n i n e (61),


332

GLYCOPROTEINS A N D

GLYCOLIPIDS IN

DISEASE

PROCESSES

acetylaminofluorene (62) and 3-methyl-4-dimethylaminoazobenzene (63) r e s u l t s i n e l e v a t i o n of the serum concentrations of AFP w i t h i n a few hours or days even though (?premalignant) morphologic changes may not be observed u n t i l a f t e r 6-8 weeks of exposure and frank hepatomas do not develop u n t i l 16-20 weeks a f t e r the i n i t i a l exposure (64). D i s c o n t i n u a t i o n of exposure to the carcinogen a f t e r AFP e l e v a t i o n s have occurred does not cause a c e s s a t i o n of AFP production. For instance, serum concentrations of AFP remain elevated f o r 12-18 weeks a f t e r one 3-week pulse of a non-carcinogenic dose of 2-AAF,and four such pulses are r e q u i r e d to produce tumors (62). Therefore, the k i n e t i c s of AFP f o l l o w i n g administ r a t i o n of non-hepatotoxic doses of hepatocarcinogens i s q u i t e d i f f e r e n t than the k i n e t i c s a f t e r a d m i n i s t r a t i o n of agents which induce l i v e r c e l l n e c r o s i s . On the other hand, continued feeding of 2-AAF does not maintain the serum AFP e l e v a t i o n as the serum AFP c o n c e n t r a t i o n of r a t s fed a continuous d i e t of 2-AAF w i l l f a l l back to j u s t about norma observed when exposure i i s that the serum c o n c e n t r a t i o n of AFP i s a c t u a l l y f a l l i n g at the time of maximal development of n e o p l a s t i c nodules (65) (Chart 2). The serum AFP r i s e s sharply with the appearance of an AFP producing h e p a t o c e l l u l a r carcinoma at 16-20 weeks. About two-thirds of the h e p a t o c e l l u l a r carcinomas that are produced by 2-AAF exposure a c t u a l l y produce AFP (65). The i d e n t i f i c a t i o n of the c e l l s producing AFP a f t e r exposure to chemical hepatocarcinogens has been the subject of much unrewarding study, but immunofluorescent s t u d i e s r e c e n t l y c a r r i e d out have c l a r i f i e d the s i t u a t i o n . Several groups reported that some of the " o v a l " or t r a n s i t i o n a l c e l l s which appear s e v e r a l weeks a f t e r exposure to diaminoazobenzene (DAB) contained AFP (66-71). However, DAB i s a potent n e c r o t i z i n g agent f o r l i v e r c e l l s so that the r e l a t i o n s h i p between o v a l c e l l s and carcinogenesis i s d i f f i c u l t to i n t e r p r e t . On the other hand, O k i t a et a l . reported that not o v a l c e l l s but some of the n o e p l a s t i c nodules that appear a f t e r exposure to e t h i o n i n e c o n t a i n AFP (72). They use t h i s evidence to support Farber's concept of a sequence of metab o l i c and morphologic changes l i n k i n g the n e o p l a s t i c nodule to the hepatoma as a premalignant l e s i o n . Other data does not support t h i s concept. As mentioned above, there i s a reverse c o r r e l a t i o n between serum AFP concentrations and the appearance of n e o p l a s t i c nodules i n r a t s fed FAA (65). In a d d i t i o n , the l i v e r s of r a t s t r e a t e d with e t h i o n i n e or DEN and fed a c h o l i n e d e f i c i e n t d i e t d i s p l a y a marked o v a l c e l l p r o l i f e r a t i o n that occurs w i t h i n 2-3 weeks i n the absence of d e t e c t a b l e l i v e r c e l l n e c r o s i s . This "oval c e l l " p r o l i f e r a t i o n i s a s s o c i a t e d with serum AFP concentrat i o n s two logs higher than that found with e t h i o n i n e or DEN alone (60 ug/ml vs. 0.6-1.0 ug/ml). Immunofluorescent l a b e l i n g r e v e a l s that many of these o v a l c e l l s c o n t a i n albumin and approximately 3% a l s o c o n t a i n AFP (27^, and unpublished o b s e r v a t i o n s ) . Those elements r e c o g n i z a b l e as b i l e ducts i n the p r o l i f e r a t i v e l e s i o n s are negative f o r AFP and albumin. Double immunofluorescent


SELL

Mpha-fetoprotein

as an Indicator

SERUM ALPHAFETOPROTEIN

in

Carcinogenesis

CONCENTRATIONS

IN

RATS TRANSPLANTED

PREGNANCY

2IOI2345

333

HEPATOMA 7777

012

2 3 4 5 6 7 8 9 10II 12 131415 16 17 1819 20 WEEKS

Chart 1. Serum alphafetoprotein trations of rats during normal gestation, after partial hepatectomy and chemically induced liver injury, after exposure to chemical carcinogens, and during growth of an AFP producing transplantable hepatoma are presented (from Ref. 17).

Chart 2. Lack of relationship between serum AFP concentrations and development of neoplastic nodule after FAA feeding of AC I rats. Following feeding of 0.06% FAA the serum concentrations of AFP become elevated with the first week of feeding, attain a maximum after six weeks, and then fall during the third and fourth feeding cycles when neoplastic nodule formation is most evident (see Ref. 65).



Fk [ure 1. Edge of neoplastic nodule (left margin) and adjacent liver with marked increase in "oval cells" The oval cells are the smaller cells which alternate with larger hepatocytes. H & E X245. Figure 2. Same section as Figure 1 labeled for AFP by immunofluorescence. Note AFP containing oval cells. X245. Figures 3 and 4. Oval cells containing AFP. X245.


Figure 5. Area of "atypical" hyperplasia of liver. Note gland-like collection of atypical hepatocytes. H & E X230. The rectangle outlines the area shown in Figures 7 and 8. Figure 6. AFP immunofluorescence staining of area of "atypical" hyperplasia. X90. Figure 7 (H & E) and 8 (AFP immunofluorescence). Area shown within Figure 5. AFP containing cells are in gland-like area of "atypical" hyperplasia. X-230.

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GLYCOLIPIDS IN

DISEASE

PROCESSES

s t a i n i n g f o r albumin and AFP r e v e a l s that the AFP c e l l s are a l s o p o s i t i v e f o r albumin. E a r l y f o c i of c e l l u l a r a l t e r a t i o n and small n e o p l a s t i c nodules are AFP negative although many of the c e l l s i n these h e p a t o c e l l u l a r l e s i o n s contain albumin. Extensive examinat i o n of the l i v e r s of c h o l i n e supplemented r a t s r e c e i v i n g 1 i n j e c t i o n of DEN (75 mg/kg) r e v e a l s that s t r o n g l y AFP p o s i t i v e " o v a l " c e l s l (approximately 1 per 5,000 l i v e r c e l l s ) may be i d e n t i f i e d as s i n g l e c e l l s i n the p o r t a l zones only one week a f t e r i n j e c t i o n , at a time when l i t t l e or no morphologic change i s recognizable. This f i n d i n g has stimulated a more extensive a n a l y s i s of the r e l a t i o n s h i p of AFP production to the development of h e p a t o c e l l u l a r carcinomas a f t e r exposure of r a t s to chemical carcinogens (74). P r e l i m i n a r y observations show that the n e o p l a s t i c nodules that develQp a f t e r feeding 2-FAA to F i s h e r male r a t s f o r f o u r , 2-week on 1-week o f f , c y c l e s do not contain c e l l s with AFP although many of the c e l l s of these nodules are p o s i t i v e f o r a l b u min. At the time of t h i v a r y i n g i n s i z e from eigh been examined, and none of these contained c e l l s with AFP. Neop l a s t i c nodules are recognizable as d i s c r e t e well-demarcated c o l l e c t i o n s of l i v e r c e l l s s i m i l a r to normal hepatocytes without normal s i n u s o i d (74). In zones of these l i v e r s where there are not i d e n t i f i a b l e n e o p l a s t i c nodules the s i n u s o i d s contain a d i f f u s e increase i n " o v a l " c e l l s . Approximately 3-5% of these o v a l c e l l s are p o s i t i v e f o r AFP. In a d d i t i o n , some zones of " a t y p i c a l h y p e r p l a s i a " of hepatocytes a l s o contain c e l l s with AFP. Representative immunofluorescent s t a i n i n g patterns are shown i n Figures 1-8. Since none of the " n e o p l a s t i c nodules" c o n t a i n AFP but many of the tumors that a r i s e from carcinogen exposure produce AFP (65), the r e l a t i o n s h i p between n e o p l a s t i c nodules and h e p a t o c e l l u l a r carcinoma must be re-evaluated (75). The dogma among workers i n chemical carcinogenesis has been that h e p a t o c e l l u l a r carcinomas a r i s e from n e o p l a s t i c nodules. On the b a s i s of the above observations as w e l l as the p r e l i m i n a r y observ a t i o n s made i n c o l l a b o r a t i o n with Dr. L e f f e r t , that c e l l s from n e o p l a s t i c nodules do not p r o l i f e r a t e as w e l l i n v i t r o as normal hepatocytes, i t i s proposed that h e p a t o c e l l u l a r carcinomas do not a r i s e from n e o p l a s t i c nodules, but from oval c e l l s which are not part of the nodules. The production of AFP by these c e l l s a f t e r exposure to chemical carcinogens most l i k e l y represents inapprop r i a t e synthesis. Chemical carcinogens may s e l e c t a very l i m i t e d c e l l population (?stem c e l l s ) to produce AFP and t h i s s i g n a l may be misread by the normally c o n t r o l l i n g environment or by the developing stem c e l l s so that the stem c e l l s p r o l i f e r a t e and d i f f e r e n t i a t e r a t h e r than form normal l i v e r l o b u l e s , and eventua l l y progress to h e p a t o c e l l u l a r carcinomas (Figure 9). In c o n c l u s i o n , by c o r r e l a t i o n and analogy, i t i s hypothesized that the f u n c t i o n of AFP i s to serve as a s i g n a l f o r normal t i s s u e ( l i v e r ) o r g a n i z a t i o n (31). The c o r r e l a t i o n i s that AFP i s produced by f e t a l l i v e r and by the adult l i v e r f o l l o w i n g p a r t i a l


18.

SELL

Alpha-fetoprotein

as an Indicator

STIMULATION

OF AFP

RESTITUTIVE PROLIFERATION

in

Carcinogenesis

ADULT LIVER

CHEMICAL CARCINOGENESIS

PARTIAL HEPATECTOMY GALACTOSAMINE CCI 4

OVAL CELL PROLIFERATION

PORTAL

ATYPICAL DIFFERENTIATION HEPATOCELLULAR CARCINOMA

Figure 9. Representation of hypothesized targets for chemical toxins and chemical carcinogens in relationship to target cell and resulting AFP synthesis (for explanation, see text)


337

338

GLYCOPROTEINS


PROCESSES

hepatectomy or c h e m i c a l l y induced n e c r o s i s . In these s i t u a t i o n s , the developing or growing l i v e r must a l i g n a number of c e l l types i n c l u d i n g hepatocytes, s i n u s o i d s , b i l e c a n n i c u l i as w e l l as blood and lymphatic v e s s e l s . The analogy i s with other markers shared by f e t a l t i s s u e s and tumors i n a d u l t s (oncodevelopmental gene products) that have been demonstrated to play a r o l e i n c e l l - c e l l i n t e r a c t i o n s r e q u i r e d f o r normal development and t i s s u e o r g a n i zation. I t i s p o s t u l a t e d that chemical carcinogens a c t on a small number of u n d i f f e r e n t i a t e d h e p a t o c e l l u l a r stem c e l l s (?oval c e l l s ) causing the i n a p p r o p r i a t e s y n t h e s i s of AFP. Under the c o n t i n u i n g i n f l u e n c e of the carcinogen, these c e l l s develop i n t o h e p a t o c e l l u l a r carcinomas. T h i s occurs because e i t h e r the progeny of the stem c e l l s a r e unable to respond to environmental s i g n a l s or the appropriate environmental c o n t r o l s are not a v a i l a b l e .

Abstract Systematic quantitative sutdies and immunofluorescent c e l l u lar localization of alphafetoprotein (AFP) in experimental systems has provided a basis for understanding and applying AFP quantitation to the diagnosis of congenital anomalies, to the differentiation of benign from malignant l i v e r disease and to the determination of the effects of therapy upon AFP producing tumors. In addition, these studies have contributed to new insights into the significance of AFP in normal development and of the sequence of events in induction of hepatocellular carcinoma by chemical carcinogens. An hypothesis has been formulated that AFP functions during fetal development and in the adult during l i v e r undergoing regeneration as a marker for organization of growing l i v e r tissue into normal lobules. Production of AFP during exposrue to chemical hepatocarcinogens represents an inappropriate signal. Carcinogens induce a small number of immature hepatocytes (?stem cells) to proliferate. Under inappropriate signals for organization, some of these stem c e l l s , perhaps only one or two per l i v e r , eventually produce hepatocellular carcinomas. The previously accepted sequential evolution of hepatocellular carcinomas from foci of altered hepatocytes through neoplastic nodules to malignant tumors is not supported by the results of ongoing experiments. Neoplastic nodules may represent a non-cancerous response of differentiated hepatocytes to carcinogen exposure.


18. SELL

339 Alpha-fetoprotein as an Indicator in Carcinogenesis

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341 Alpha-fetoprotein as an Indicator in Carcinogenesis

60.

Pitot, H.C., Barsness, L., Goldsworthy, T., Kitagawa, T. Nature (1978) 271:456. 61. Smuckler, E.A., Koplitz, M., Sell, S. Cancer Res. (1976) 36:4558. 62. Becker, F.F., Sell, S. Cancer Res. (1974) 34:2489. 63. Watabe, H. Cancer Res. (1971) 31:1192. 64. Teebor, G.W., Becker, G.W. Cancer Res. (1971) 31:1. 65. Becker, F.F., Sell, S. Submitted. 66. Bannikov, G.A., Gel'Shtein, V.I., Chipysheva, T.A. Vestn. Akad. Med. Nank. SSSR (1977) 3:39. 67. Dempo, K.N., Chisaka, N., Yoshida, Y., Kaneko, A., Onoe, T. Cancer Res. (1975) 35:1282. 68. Fujita, S., Ishizuka, H., Kamimura, N., Kaneda, H., Ariga, K. Ann. N.Y. Acad. Sci. (1975) 259:217. 69. Onda, H. Gann (1976) 67:253. 70. Onoe, T., Kaneko, A. Dempo K. Ogawa K., Minase T Ann. N.Y. Acad. Sci 71. Tchipysheva, T.A. J. Cancer (1977) 20:388. 72. Okita, K., Gruenstein, M., Klaiber, M., Farber, E. Cancer Res. (1974) 34:2758. 73. Shinozuka, H., Lombardi, B., Sell, S., Iammarino, R.M. Cancer Res. in press. 74. Squire, R.A., Levitt, M.H. Cancer Res. (1975) 35:3214. 75. Sell, S. submitted. RECEIVED

December 29, 1977.


19 Carcinoembryonic Antigen—A Marker of Human Colonic Cancer CHARLES W. TODD and JOHN E. SHIVELY Division of Immunology, City of Hope National Medical Center, Duarte, CA 91010

The current literatur and quantitative change nancy. Despite the potential that these antigens offer for the control of cancer through detection, therapy, or prevention by immunization, in most cases the antigens are poorly defined and practical applications are lacking. One antigen which has achieved considerable application in the diagnosis and monitoring of cancer therapy is the carcinoembryonic antigen (CEA). Our study of the chemical nature of this complex molecule, or perhaps more accurately stated molecular complex, was undertaken with the goal of improving its clinical utility in the management of cancer. The initial reports by Gold and Freedman (1,2) suggested that CEA was synthesized by the rudimentary digestive system in the human fetus, that synthesis ceased prior to birth, but that it resumed with the onset of colonic adenocarcinoma in the adult. This sequence lead them to name the substance the carcinoembryonic antigen. Their development of a radioimmunoassay for CEA (3) and the potential importance of such a tumor marker stimulated studies of the diagnostic value of CEA assays in a variety of pathological conditions, both benign and malignant. The l i t erature on these clinical studies has been recently reviewed (4). These studies demonstrated that CEA-reactivity was not universally elevated in adenocarcinoma of the digestive system, that it was often elevated in other malignancies notably of breast, bladder, and lung, and that moderate elevations are often present in nonmalignant inflammatory diseases. Moreover, normal sera showed variable low levels of CEA-reactivity. Clearly the situation was more complex than originally visualized. Since radioimmunoassays are not completely specific (5), more precise chemical knowledge of the substances being detected was necessary. Most of the studies done on CEA have employed material isolated from hepatic metastases of colonic adenocarcinoma using essentially the method originally described by Krupey et al. (6), although the final block electrophoresis step is usually omitted. The CEA employed in most of our studies was obtained by homogeni0-8412-0452-7/78/47-080-342$05.00/0 © 1978 American Chemical Society In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

19.

TODD A N D SHIVELY

Carcinoembryonic

343

Antigen

z a t i o n of l i v e r metastases o f colon adenocarcinoma i n water, a d d i t i o n o f an equal volume o f 2M p e r c h l o r i c a c i d , c e n t r i f u g a t i o n , d i a l y s i s o f the supernatant, c o n c e n t r a t i o n , chromatography on Sepharose 4B, and rechromatography on Sephadex G200 ( 7 ) . The CEA a c t i v i t y during these i s o l a t i o n s was followed by a t r i p l e isotope double antibody radioimmunoassay (8.), which has been r e c e n t l y improved by the s u b s t i t u t i o n o f C o f o r N a as a v o l ume marker f o r the supernatant (9). CEA prepared i n t h i s way when examined by e l e c t r o n microscopy c o n s i s t e d o f twisted rod or c r u l l e r shaped p a r t i c l e s with dimensions o f 9 x 40 nm (10). Further p u r i f i c a t i o n o f CEA by a f f i n i t y chromatography on concanavalin A Sepharose removed a small amount (15%) of contaminants, which were not r e t a i n e d by the a f f i n i t y column. This m a t e r i a l was found to c o n t a i n mucopolysaccharides as i d e n t i f i e d by c e l l u l o s e acetate s t r i p e l e c t r o phoresis, depolymerization with bovine t e s t i c u l a r hyaluronidase and i d e n t i f i c a t i o n o f g l u c u r o n i d e r i v a t i v e o f i t s methy spectrometry (12). E a r l y s t u d i e s by Krupey et al. (13) revealed CEA to be a g l y c o p r o t e i n with a molecular weight about 180,000, comprising about 60% carbohydrate and H0% p r o t e i n . This high percentage o f carbohydrate may e x p l a i n the f a c t that the molecular dimensions as v i s u a l i z e d by e l e c t r o n microscopy i n d i c a t e a molecular volume about 16 times g r e a t e r than that c a l c u l a t e d f o r a c l o s e l y packed molecule o f 180,000 molecular weight. Presumably the frequency and s i z e o f the carbohydrate groupings i n t e r f e r e with compact f o l d i n g o f the chain. There appears to be only one polypeptide chain present i n CEA. Consistent with t h i s view i s the f i n d i n g that r e d u c t i o n and a l k y l a t i o n o f the d i s u l f i d e bonds p r i o r to e l e c t r o n microscopic examination o f CEA leads to the appearance of t h r e a d - l i k e s t r u c t u r e s . The maximum extended length observed, 220 nm, approaches that expected f o r a p r o t e i n o f the r e q u i s i t e s i z e a l l o w i n g 0.364 nm extension per amino a c i d r e s i d u e (14). C h a r a c t e r i z a t i o n o f CEA by i s o e l e c t r i c f o c u s i n g (15), i o n exchange chromatography (15,16) and a f f i n i t y chromatography on concanavalin A Sepharose ^11>17>_18) demonstrated considerable heterogeneity, as might be expected f o r a h i g h l y g l y c o s y l a t e d p r o t e i n . Indeed, v a r i a t i o n i s seen i n the r e l a t i v e amounts o f the carbohydrate components of CEA i s o l a t e s from d i f f e r e n t tumors (19). T y p i c a l r e s u l t s from a CEA p r e p a r a t i o n p u r i f i e d by a f f i n i t y chromatography on concanavalin A Sepharose (11_) are presented i n Table I . The carbohydrate compositions were determined by the method of Clamp (20) f o r n e u t r a l sugars, by the method o f Warren (21) f o r s i a l i c a c i d , and by the method o f L i u and Chang (22) with the amino a c i d a n a l y z e r f o r the amino sugars. The s i a l i c a c i d has been i d e n t i f i e d as N-acetylneuraminic a c i d (23)• These analyses r e q u i r e d samples o f approximately 250 yg. More r e c e n t l y we have developed a method approximately 2 orders o f magnitude more s e n s i t i v e employing methanolysis and t r i f l u o r o a c e t y l a t i o n to generate methyl N , 0 - t r i f l u o r o a c e t y l g l y c o s i d e s , which a r e separated by gas chromatography on 7m x 0.5 mm packed 5 7

2 2


344

GLYCOPROTEINS A N D GLYCOLIPIDS IN

DISEASE

PROCESSES

c a p i l l a r y columns and q u a n t i t a t e d by e l e c t r o n capture d e t e c t i o n (24). In c o n t r a s t to the v a r i a b i l i t y seen f o r the carbohydrate composition, the amino a c i d composition o f v a r i o u s CEA i s o l a t e s remains q u i t e constant (25). A t y p i c a l a n a l y s i s i s presented i n Table 1. Using the r e s u l t s i n Table 1 some deductions regarding the s t r u c t u r e o f CEA can be made. The t o t a l number o f amino a c i d residues i s 829. A s i n g l e polypeptide chain with 829 amino a c i d residues with a length of 0.364 nm per residue (14) would have a length of 302 nm, i f f u l l y extended. This compares f a v o r a b l y with the 220 maximum observed by e l e c t r o n microscopy c o n s i d e r i n g the approximations i n v o l v e d . The low content of N - a c e t y l galactosamine (1.5? of the sugar r e s i d u e s ) suggests that the o l i gosaccharide u n i t s are not bound to the p r o t e i n backbone through N-acetylgalactosamine l i n k e d to s e r i n e or threonine The f a i l ure to accomplish 3 - e l i m i n a t i o n a t i v e l y , i f the attachmen to asparagine, the t o t a l a s p a r t i c acid/asparagine content l i m i t s the number o f o l i g o s a c c h a r i d e u n i t s to 128 with an average s i z e of 4 r e s i d u e s . Subsequently we s h a l l see evidence that there are about h a l f as many u n i t s with an average of twice t h i s s i z e . The amount of N-acetyglucosamine i n d i c a t e s some o f these residues must be d i s t a l from the attachment s i t e s i n c e the r a t i o o f Nacetylglucosamine to a s p a r t i c acid/asparagine i s 1.5. The sum of s e r i n e (91 r e s i d u e s ) and threonine (74 r e s i d u e s ) i s 165. Thus there are ample residues to e s t a b l i s h the asp,x,ser/thr sequences i n the p r o t e i n chain, b e l i e v e d to code f o r N-acetylglucosamineasparagine attachment (26). More p r e c i s e information on the s t r u c t u r e o f the o l i g o saccharide u n i t s was obtained by methylation a n a l y s i s (27) employing gas chromatography and mass spectrometry as depicted i n Figure 1. Samples were methylated by the method of Hakomori (28_) i n which the m e t h y l s u l f i n y l anion (29) was used to generate the polysaccharide alkoxide before the a d d i t i o n o f methyl i o d i d e . A c e t o l y s i s , h y d r o l y s i s , r e d u c t i o n , and a c e t y l a t i o n o f the permethylated p o l y s a c c h a r i d e s were performed using the procedures described by S t e l l n e r et al. (30). I d e n t i f i c a t i o n o f the p a r t i a l l y methylated a l d i t o l acetates was performed by the method of Bjorndal et al. (31) f o r the n e u t r a l sugar d e r i v a t i v e s and by the method of S t e l l n e r et al. (30) f o r the amino sugar d e r i v a tives. The products are i d e n t i f i e d by t h e i r r e t e n t i o n times i n the gas chromatograph and t h e i r fragments as produced i n the mass spectrometer. There are c e r t a i n p r e f e r r e d cleavage p o i n t s that occur on e l e c t r o n impact i n the mass spectrometer. The order of the preference o f these cleavages i s i n d i c a t e d by the numbers 1, 2, and 3 (Figure 1). A d e t a i l e d procedure has been published (32). The r e s u l t s of these s t u d i e s (27) on 4 CEA samples are presented i n Table 2 together with the r e s u l t s on a sample analyzed by Hammarstrom et al. (33). I t i s apparent that considerable


TODD A N D SHIVELY

Carcinoembryonic

Antigen

TABLE 1 COMPOSITION OF CEA

Moles per 180,000 MW

Components

Sugars N-Acetylgalactosamine

188.2

N-Ace tylglucosamine N-Acetylneuraminic

7.5

acid

12.1

Fucose

102.4

Galactose

110.9

Mannose Amino Acids Alanine

49.3

Arginine

20.1

A s p a r t i c acid/Asparagine

127.8

Cysteine

15.8

Glutamic acid/glutamine

84.6

Glycine

47.7

Histidine

20.1

Isoleucine

31.3

Leucine

67.8

Lysine

20.3

Methionine

Trace

Phenylalanine

18.9

Proline

58.6

Serine

91.2

Threonine

74.0

Tryptophan

11.9

Tyrosine

36.1

Valine

53.5

Carbohydrate

49.9?

Protein

50.1?

These v a l v e s were c a l c u l a t e d from the data f o r F r a c t i o n V, Table 2, Reference 11.


346

GLYCOPROTEINS

A N D GLYCOLIPIDS IN

DISEASE

PROCESSES

v a r i a t i o n e x i s t s among these samples, but that a b a s i c p a t t e r n i s being followed. As expected from patterns p r e v i o u s l y observed i n mammalian g l y c o p r o t e i n s (34_), a l l the fucose i s t e r m i n a l . S i m i l a r l y a l l the N-acetylneuraminic a c i d i s t e r m i n a l . This f o l l o w s not from the methylation a n a l y s i s , but from the observ a t i o n that removal of the N-acetylneuraminic a c i d with neuraminidase exposed to a t t a c k by periodate a d d i t i o n a l moles of galactose e q u i v a l e n t to the moles o f N-acetylneuraminic a c i d removed (23). This observation together with the n e g l i g i b l e amounts of branching galactose observed by methylation a n a l y s i s (Table 2) a l s o f i x e s the p o i n t of attachment of the N - a c e t y l neuraminic a c i d at the 3 - p o s i t i o n of galactose (23). Threefourths of the mannose r e s i d u e s i n a l l the CEA samples were l i n k e d to 3 other sugar residues (branching mannose). Except f o r the small amounts of 3,4- and 2,3-linked g a l a c t o s e , the only other branching sugar i s N-acetylglucosamine of which 32-41? i s present a t branching p o i n t s The data i n Table 2 ber of o l i g o s a c c h a r i d e u n i t s l i n k e d to the p r o t e i n chain and t h e i r average s i z e (35). The number of o l i g o s a c c h a r i d e u n i t s equals the sum of the nonreducing terminal residues minus the sum o f the branching r e s i d u e s . From t h i s the average s i z e can be obtained by d i v i d i n g the t o t a l number of residues by the number of o l i g o s a c c h a r i d e u n i t s . Thus the numbers of o l i g o s a c c h a r i d e u n i t s f o r the CEA samples i n Table 2 are 51, 77, 124, 64, and 69 r e s p e c t i v e l y . On t h i s b a s i s the comparable o l i g o s a c c h a r i d e u n i t s contain 9, 7, 4, 8, and 6 monosaccharide residues each. Considering the cumulative e r r o r s and the expected i n h e r ent v a r i a t i o n , the data are i n reasonably good accord. The p i c ture that emerges i s many small o l i g o s a c c h a r i d e u n i t s attached to about 1 out o f 10 of the amino a c i d residues u t i l i z i n g about 2/3 of the a s p a r t i c acid/asparagine r e s i d u e s . The high carbohydrate content of CEA suggested the p o s s i b i l i t y that the a n t i g e n i c s i t e as detected i n the radioimmunoassay might r e s i d e there. Although the p r e c i s e nature of t h i s s i t e i s s t i l l undefined and may vary depending on the a n t i s e r a used to define i t , the cumulative weight of evidence i n d i c a t e s that the p r o t e i n p o r t i o n of the molecule plays the major, i f not e x c l u s i v e , r o l e i n determining the s i t e . This does not exclude the p o s s i b i l i t y that many CEA a n t i s e r a contain a n t i b o d i e s to carbohydrate. Under the c o n d i t i o n s of the usual radioimmunoassay only a r a t h e r narrow p o p u l a t i o n of high a f f i n i t y a n t i b o d i e s p l a y s a s i g n i f i cant r o l e (4). A consequence of t h i s s e l e c t i o n i s that the antiserum used may contain l a r g e populations of a n t i b o d i e s p l a y i n g an i n s i g n i f i c a n t r o l e i n the immunoassay t i t r a t i o n , but quite capable of r e a c t i n g with the antigen under the more concentrated c o n d i t i o n s p r e v a i l i n g i n immunodiffusion t e s t s . For t h i s reason i t i s dangerous to assume that phenomena observed by various immunodiffusion techniques are n e c e s s a r i l y involved i n immunoassay t i t r a t i o n s ( 4 _ ) .



residues

Terminal 4 3,4

Terminal 2 6 2,4 2,6 3,6

103 43 61 5 11 5 125 9 9 Trace 20 16 23 77 Trace 92 63 155

Terminal Terminal 3 2 6 3,4 and 2,3

a

x

CEA

23

linkage

Terminal

Glyoosidio

103 72 22 18 2 2 116 14 7 Trace 18 18 25 83 16 94 76 186

11

2

CEA

of h

95 63 18 9 4 4 98 13 7 Trace 13 18 22 73 20 110 83 213

13

CEA

CEA

96 4 14 Trace 18 23 18 77 5 83 41 129

110 36 11 36 13

18

h

CEA-14

A l l tumors were l i v e r

104 58 45 18 11 2 134 9 9 Trace 22 20 11 71 32 88 58 178

34

Z

CEA

5

8-10 g

T h e s u b s c r i p t s 1 to 4 i n d i c a t e CEA p u r i f i e d from d i f f e r e n t tumors. metastases that had o r i g i n a t e d i n the colon. ^Data from Hammarstrom et al. (33).

a

Total

Total N-Acetylglucosamine

Total Mannose

N-Acetylneuraminic acid Fucose Galactose

Carbohydrate

Moles/1.

STRUCTURAL UNITS OF THE POLYSACCHARIDE PORTION OF CEA

TABLE 2

348

GLYCOPROTEINS

A N D GLYCOLIPIDS

IN

DISEASE

PROCESSES

The evidence supporting the r o l e of the p r o t e i n p o r t i o n i n determining the a n t i g e n i c s i t e may be summarized as f o l l o w s . S e r i a l p e r i o d a t e o x i d a t i o n , Smith degradation (36), of CEA demonstrated p e r s i s t e n c e of a n t i g e n i c a c t i v i t y even a f t e r removal o f 90? of the carbohydrate ( 2 3 , 3 3 , 3 7 ) . A n t i s e r a to CEA can detect as l i t t l e as l O ' * M o f CEA. Such high b i n d i n g a f f i n i t i e s are c h a r a c t e r i s t i c a l l y achieved a g a i n s t p r o t e i n a n t i gens but not a g a i n s t carbohydrates (38). These high b i n d i n g a f f i n i t i e s can not be explained by b i v a l e n t antibody b i n d i n g to adjacent s i t e s on the CEA molecule, s i n c e e q u i v a l e n t concentrat i o n s of b i n d i n g s i t e s of monovalent Fab' fragments of CEA a n t i bodies were e q u a l l y e f f e c t i v e as the i n t a c t antibody i n the CEA radioimmunoassay (39_). D i l u t e a l k a l i treatment of CEA destroys i t s a n t i g e n i c a c t i v i t y , but does not a f f e c t i t s l e c t i n b i n d i n g ability (33). I t i s w e l l known that p r o t e i n s t r u c t u r e i s r a p i d l y degraded by a l k a l i , whereas the carbohydrate l i n k a g e s o f asparagine l i n k e d g l y c o p r o t e i n Westwood et al. (40) hav o f CEA destroys i t s a n t i g e n i c a c t i v i t y , producing N-terminal p r o l i n e peptides with i n t a c t carbohydrate chains except f o r the l o s s of s i a l i c a c i d and fucose. I t had p r e v i o u s l y been shown that d e s t r u c t i o n of s i a l i c a c i d and fucose by periodate o x i d a t i o n does not a l t e r the a n t i g e n i c a c t i v i t y (23). Reduction and a l k y l a t i o n o f the c y s t i n e d i s u l f i d e bonds i n CEA destroys i t s a n t i g e n i c a c t i v i t y (22,33,41,42). C l e a r l y the secondary and t e r t i a r y s t r u c t u r e s o f CEA are c r i t i c a l i n maintaining i t s a n t i genic nature. 1

The sequence o f the f i r s t 24 amino a c i d s at the N-terminus of CEA was r e a d i l y determined by standard automated Edman techniques. T h i s sequence i s presented i n Figure 2 (43). I t has been confirmed s e v e r a l times (44) and extended an a d d i t i o n a l 6 r e s i d u e s (45), although the choice between glutamic a c i d and glutamine at c e r t a i n r e s i d u e s has not been uniform (46). A d d i t i o n a l sequencing encountered t e c h n i c a l problems a r i s i n g from the high degree of g l y c o s y l a t i o n . These d i f f i c u l t i e s began with the r e s i s t a n c e to s p e c i f i c cleavage, presumably due to s t e r i c hinderance by the carbohydrate groups. Since the carbohydrate s u b s t i t u e n t s are h i g h l y heterogeneous i n degree of s u b s t i t u t i o n among a p o p u l a t i o n o f CEA molecules, s p e c i f i c cleavage methods d i d not g i v e uniform or s t o i c h i o m e t r i c y i e l d s of expected p e p t i d e s . Once peptides were obtained, t h e i r p u r i f i c a t i o n was rendered d i f f i c u l t by these heterogeneous carbohydrate substituents. Glycopeptides have broad, i l l d e f i n e d peaks i n separations based on c o n v e n t i o n a l s i z i n g techniques. Edman degradations o f g l y c o p e p t i d e s f r e q u e n t l y come to an abrupt h a l t when an amino a c i d to which carbohydrate i s attached i s encountered. The f i r s t of these d i f f i c u l t i e s was solved by the use of the detergent T r i t o n X100 (0.25?), which rendered CEA suscept i b l e to t r y p s i n cleavage (47). Although CEA c o n t a i n s s u f f i c i e n t a r g i n i n e and l y s i n e r e s i d u e s to produce about 40 t r y p t i c p e p t i d e s ,


TODD AND SHIVELY

Carcinoembryonic

Antigen

,0H

v

0-R

R-0

R-0

OCH,

HO

0-R

OCH,

OCH. 0

CH OH

CH OCCH

2

I

HCOCH3

HCOCH,

© (D (D

CH OCH

CH 0CH

3

3

I 0 HCOCCH,

HCOH

HCOCCH,

HCOH

I

I CH OCH 2

CH OCH 2

3

3

Cancer Research Figure 1. Methylation analysis. Numbers indicate preferred order of bond rupture under electron bombardment in the mass spectrometer.

CARCINOEMBRYONIC ANTIGEN A m i n o - T e r m i n a l Residues 5 10 Leu Thr He Glu Ser Thr Pro Phe A s n 15 Vol Ala Glu Gly Lys Glu Vol L e u Leu Leu vol His Asn L e u Lys

f at the N-terminus of CEA 2

S

e

q

u

e

n

c

e

0

a

m

i

n

0

a c i d s

—[AAAAAAAA)— ORIGINAL GENE

I —[AAAAAAAA]

|AAAAAAAA|—

DUPLICATED GENES ^ —fvvwvw] [wwwv^j— DIVERGENT GENES

t

Figure 3. Proposed genetic evolui f th protein chains of CEA and related antigens on

or

e


350


PROCESSES

only 10 were i s o l a t e d . These were f r a c t i o n a t e d by a combinat i o n o f anion and c a t i o n exchange r e s i n s using high pressure l i q u i d chromatography. In some cases these peptides could be sequenced through 20 Edman degradation c y c l e s . D e t a i l s are presented elsewhere (47). C u r r e n t l y methods of d e g l y c o s y l a t i n g CEA are being evaluated with the o b j e c t i v e o f s i m p l i f y i n g the sequencing problem. I t has long been recognized that radioimmunoassay o f serum from normal i n d i v i d u a l s f o r CEA always showed some v a r i a b l e low l e v e l o f CEA r e a c t i v i t y . The demonstration i n normal t i s s u e s o f m a t e r i a l c r o s s r e a c t i v e with but d i s t i n g u i s h a b l e from CEA appeared to e x p l a i n the observed normal l e v e l s . These molecules included NCA (48), NGP (49), CCEA-2 (50), CEX (51), and 6 p r o t e i n (52). These molecules a l l have lower molecular weights than does CEA. Subsequently other groups (53-55) reported the presence i n normal serum and normal t i s s u e e x t r a c t s o f m a t e r i a l with CEA-like a c t i v i t y and s i m i l a r t umns. Unfortunately th mal t i s s u e was so low that s u f f i c i e n t m a t e r i a l could not be obtained f o r chemical c h a r a c t e r i z a t i o n . In 1974 Go et al. (56) measured the s e c r e t i o n i n various p o r t i o n s o f the g a s t r o i n t e s t i n a l t r a c t o f m a t e r i a l that i n h i b i t e d i n the assay f o r CEA. The colon secreted the l a r g e s t q u a n t i t y o f the m a t e r i a l . I t e l u t e d from a Sephadex G-200 column i n the same p o s i t i o n as CEA. The presence o f these q u a n t i t i e s of m a t e r i a l with CEA-like a c t i v i t y i n the colon lavages o f healthy i n d i v i d u a l s made i t p o s s i b l e to i s o l a t e s u f f i c i e n t m a t e r i a l f o r chemical characteri z a t i o n and d i r e c t comparison with CEA (57). The m a t e r i a l with CEA-like a c t i v i t y from c o l o n i c lavages was p u r i f i e d by g e l f i l t r a t i o n on Sepharose 6B and Sephadex G200 followed by a f f i n i t y chromatography on concanavalin A l i n k e d to Sepharose. The p u r i f i e d m a t e r i a l migrated i n polyacrylamide-sodium dodecyl s u l f a t e e l e c t r o p h o r e s i s as a s i n g l e d i f f u s e band with m o b i l i t y i d e n t i c a l to that o f tumor CEA. I t possessed the same s p e c i f i c a c t i v i t y as CEA i n radioimmunoassay. The carbohydrate and amino a c i d compositions were s i m i l a r to those o f CEA. Moreover, methylat i o n a n a l y s i s demonstrated that the monosaccharide linkages were s i m i l a r t o those i n CEA (57), and i t s N-terminal amino a c i d sequence was homologous to CEA (j>8). Thus by a l l c r i t e r i a a p p l i e d i t was i n d i s t i n g u i s h a b l e from CEA. I t thus seems l i k e l y that CEA i s a normal t i s s u e product. This r a i s e s the question of whether the elevated serum l e v e l s observed i n malignancy r e s u l t from increased c e l l u l a r s y n t h e s i s or by r e d i r e c t i o n to the blood stream o f m a t e r i a l normally e l i m i n a t e d i n the g a s t r o i n t e s tinal tract. During the i s o l a t i o n o f CEA from l i v e r metastases o f c o l o n i c adenocarcinoma, i t was observed that a lower molecular weight component possessing CEA a c t i v i t y i n the radioimmunoassay was present i n the eluate from the Sepharose 4B column. This materi a l , which we have designated TEX, has now been i s o l a t e d and puri f i e d (59). Like CEA, TEX i s a g l y c o p r o t e i n . I t binds to E


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concanavalin A Sepharose from which i t can be e l u t e d by d i s p l a c e ment with methyl a-D-mannoside. By Sephadex G200 g e l f i l t r a t i o n and by sodium dodecyl s u l f a t e polyacrylamide g e l e l e c t r o p h o r e s i s i t s molecular weight was shown to be about 110,000 d a l t o n s . TEX contained 35% carbohydrate, c o n s i d e r a b l y l e s s than that present i n CEA. Linkage s t u d i e s of the carbohydrate by methylat i o n a n a l y s i s revealed that TEX contained s u b s t a n t i a l l y l e s s t e r minal g a l a c t o s e , as w e l l as l e s s 4-linked i n t r a c h a i n and 3,4branched N-acetylglucosamine than CEA. A s i n g l e treatment with periodate destroyed a l l of the s i a l i c a c i d and fucose and 25% of the N-acetylglucosamine as with CEA, but i n a d d i t i o n twice as much mannose and g a l a c t o s e are destroyed as compared with CEA (59). When the s i z e of the p r o t e i n component i s c a l c u l a t e d from the molecular weight and percent p r o t e i n , i t i s found that t h i s s i z e i s n e a r l y i d e n t i c a l to that of CEA. S i m i l a r l y the amino a c i d composition o f TEX i s n e a r l y i d e n t i c a l to that o f CEA, except f o r the presence methionine i n TEX, but no 24 N-terminal amino a c i d s of TEX d i f f e r s from CEA only i n the presence o f a l a n i n e , at p o s i t i o n 21 i n l i e u of v a l i n e i n CEA. The combined evidence, both immunological and chemical, i n d i cates that TEX i s c l o s e l y r e l a t e d to CEA, but d i f f e r s i n i t s mode and degree o f g l y c o s y l a t i o n and has at l e a s t one and probably more amino a c i d a l t e r a t i o n s i n i t s polypeptide chain (59). Since TEX, which was i s o l a t e d from a tumor, may be s i m i l a r to NCA and r e l a t e d m a t e r i a l s i s o l a t e d from normal t i s s u e , a preparation o f NCA was obtained from normal spleen t i s s u e by an immunochemical method u t i l i z i n g i n s o l u b i l i z e d a n t i b o d i e s to CEA and NCA (60). The molecular weight of the NCA obtained was found to be about 100,000 d a l t o n s . The carbohydrate composition was 30%. Unfortunately the m a t e r i a l a v a i l a b l e was i n s u f f i c i e n t f o r methylation a n a l y s i s and periodate o x i d a t i o n s t u d i e s . The sample possessed n e a r l y the same amino a c i d a n a l y s i s as CEA, but l i k e TEX contained a small but demonstrable amount of methionine. The sequence o f the f i r s t 26 N-terminal amino a c i d s was i d e n t i c a l to that o f CEA, except a t p o s i t i o n 21, where as i n TEX a l a nine i s present i n s t e a d of the v a l i n e o f CEA (60). The immunological data show that NCA and TEX are i d e n t i c a l i f tested with e i t h e r goat anti-NCA or r a b b i t anti-TEX, but TEX c r o s s r e a c t s with CEA when tested with monkey anti-CEA (59), an antiserum which was p r e v i o u s l y shown not to c r o s s r e a c t with spleen NCA (6l_). An a l t e r n a t i v e i n t e r p r e t a t i o n i s that TEX cont a i n s t r a c e amounts o f CEA]_ , r e c e n t l y described as i d e n t i c a l to CEA i n a l l respects except, perhaps, i n degree of g l y c o s y l a t i o n (62). Whatever may be the i n t e r r e l a t i o n s h i p s o f the v a r i o u s a n t i gens c r o s s r e a c t i v e with CEA (^-52,62), i t appears that from the standpoint o f primary s t r u c t u r e , i.e. sequence, of the p r o t e i n component, CEA, NCA, and TEX are c l o s e l y r e l a t e d . NCA and TEX may or may not d i f f e r from one another i n t h i s respect, but they both d i f f e r from CEA. We b e l i e v e that they a r i s e from d i s t i n c t 0W



352

but related genes which have a common evolutionary origin in a single gene. This primoridal gene (Figure Z) has duplicated, or replicated to more than 2 genes, which have subsequently undergone divergent evolution. A more profound understanding of the interaction of these genes and their gene products in benign and malignant disease will hopefully lead to improved clinical utility of this system. ACKNOWLEDGMENTS This review was supported in part by National Cancer Institute grants CA 16434 and CA 19163 from the National Large Bowel Cancer Program. REFERENCES 1. 2.

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Slayter, H.S. and Coligan, J.E.: Electron microscopy and physical characterization of the carcinoembryonic antigen. Biochemistry, 14: 2323-2330, 1975. Slayter, H.S. and Coligan, J.E.: Characterization of carcinoembryonic antigen fractionated by concanavalin A chromatography. Cancer Res., 36: 1696-1704, 1976. Pritchard, D.G. and Todd, C.W.: Purification of carcinoembryonic antigen by removal of contaminating polysaccharides. Cancer Res., 36: 4699-4701, 1976. Krupey, J . , Gold, P. and Freedman, S.O.: Physicochemical studies of the carcinoembryonic antigens of the human digestive system. J. Exp. Med., 128: 387-398, 1968. Pauling, L., Corey, R.B. and Branson, H.R.: The structure of proteins: Two hydrogen-bonded helical configurations of the polypeptide chain. Proc. Nat. Acad. Sci. USA, 37: 205-240, 1951 Coligan, J.E., Henkart Heterogeneity o carcinoembryoni antigen chemistry, 10: 591-599, 1973. Eveleigh, J.W.: Heterogeneity of carcinoembryonic antigen. Cancer Res., 34: 2122-2124, 1974. Chism, S.E., Bell, P.M. and Warner, N.L.: Heterogeneity of CEA and 'CEA-like' preparations determined by Farr assays for lectin binding. J. Immunol. Meth., 13: 83-89, 1976. Harvey, S.R. and Chu, T.M.: Demonstration of two molecular variants of carcinoembryonic antigen by concanavalin A Sepharose affinity chromatography. Cancer Res., 35: 3001-3008, 1975. Egan, M.L., Coligan, J.E., Morris, J.E., Schnute, W.C., Jr. and Todd, C.W.: Physical characterization and structural studies of the carcinoembryonic antigen. Cancer Res., 36: 3482-3485, 1976. Clamp, J.R., Bhatti, T. and Chambers, R.E.: The examination of carbohydrate in glycoproteins by gas-liquid chromatography. In: Glycoproteins. A. Gottschalk, (ed.), Elsevier Publishing Co., 5 (part A): 300-321, 1972. Warren, L.: The thiobarbituric acid assay of sialic acids. J. Biol. Chem., 234: 1971-1975, 1959. Liu, T.-Y. and Chang, Y.H.: Hydrolysis of proteins with ptoluenesulfonic acid. J. Biol. Chem., 246: 2842-2848, 1971. Coligan, J.E. and Todd, C.W,: Structural studies on carcinoembryonic antigen: Periodate oxidation. Biochemistry, 14: 805-810, 1975. Wrann, M.M. and Todd, C.W.: Sensitive determination of sugars utilizing packed capillary columns and electron capture detection. J. Chromatog. 147: 309-316, 1978.


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Coligan, J.E., Egan, M.L., Guyer, R.L., Schnute, W.C., Jr. and Todd, C.W.: Structural studies on the carcinoembryonic antigen. Ann. N.Y. Acad. Sci., 259: 355-365, 1975. 26. Spiro, R.G.: Glycoproteins. Ann. Rev. Biochem., 39: 599638, 1970. 27. Coligan, J.E., Pritchard, D.G., Schnute, W.C., Jr. and Todd, C.W.: Methylation analysis of the carbohydrate portion of carcinoembryonic antigen. Cancer Res., 36: 1915-1917, 1976. 28. Hakomori, S.: A rapid permethylation of glycolipid and polysaccharide catalyzed by methylsulfinyl carbanion in dimethylsulfoxide. J. Biochem. Tokyo, 55: 205-208, 1964. 29. Sandford, P.A. and Conrad, H.E.: The structure of the Aerobacter aerogenes A3(S1) polysaccharide. I. A reexamination using improved procedures for methylation analysis. Biochemistry, 5: 1508-1517 1971 30. Stellner, K., Saito aminosugar linkage Biochem. Biophys., 155: 464-472, 1973. 31. Björndal, H., Hellerqvist, C.G., Lindberg, B. and Svensson, S.: Gas-liquid chromatography and mass spectrometry in methylation analysis of polysaccharides. Angew. Chem. Intern. Ed. Engl., 9: 610-619, 1970. 32. Pritchard, D.G., Todd, C.W. and Egan, M.L.: Chemistry of carcinoembryonic antigen. Methods in Cancer Research, 14: 55-85, 1977. 33. Hammarström, S., Engvall, E., Johansson, B.G., Svensson, S., Sundblad, G. and Goldstein, I.J.: Nature of the tumor-associated determinant(s) of carcinoembryonic antigen. Proc. Nat. Acad. Sci. USA, 72: 1528-1532, 1975. 34. Heath, E.C.: Complex polysaccharides. Ann. Rev. Biochem., 40: 29-56, 1971. 35. Shively, J.E. and Todd, C.W.: Carcinoembryonic antigen. Scand. J. Immunol., in press. 36. Smith, F. and Unrau, A.M.: On the presence of l-->6 linkages in laminarin. Chem. Ind. London, 881, 1959. 37. Bessel, E.M., Thomas, P. and Westwood, J.H.: Multiple Smith degradations of carcinoembryonic antigen (CEA) and of asialo CEA. Carbohyd. Res., 45: 257-268, 1975. 38. Eisen, H.N., pp 376-377 in Davis, B.D., Dulbecco, R., Eisen, H.N., Ginsberg, H.S. and Wood, W.B., Microbiology, Harper and Row, New York, 1968. 39. Morris, J.E., Egan, M.L. and Todd, C.W.: The binding of carcinoembryonic antigen by antibody and its fragments. Cancer Res., 35: 1804-1808, 1975. 40. Westwood, J.H., Bessel, E.M., Bukhari, M.A., Thomas, P. and Walker, J.M.: Studies on the structure of the carcinoembryonic antigen. I. Some deductions on the basis of chemical degradations. Immunochemistry, 11: 811-818, 1974.


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41. Westwood, J.H. and Thomas, P.: Studies on the structure and immunologic activity of carcinoembryonic antigen -the role of disulfide bonds. Br. J. Cancer, 32: 708-719, 1975. 42. Westwood, J.H., Thomas, P. and Foster, A.B.: The treatment of carcinoembryonic antigen with sodium metaperiodate. Biochem. Soc. Trans., 2: 1250-1251, 1974. 43. Terry, W.D., Henkart, P.A., Coligan, J.E. and Todd, C.W.: Structural studies of the major glycoprotein in a preparation with carcinoembryonic antigen activity. J. Exp. Med., 136: 200-204, 1972. 44. Terry, W.D., Henkart, P.A., Das, S., Coligan, J.E. and Todd, C.W.: Characterization of human carcinoembryonic antigens. Proceed, of Miles Symposium, pp 241-247, 1973. 45. Chu, T.M., Bhargava, A.K. and Harvey, S.R.; Structure studies of the glycoproteins associated with carcinoembryonic antige 46. Coligan, J.E., Egan and Todd, C.W.: Structural studies on the carcinoembryonic antigen. Ann. N.Y. Acad. Sci., 259: 355-365, 1975. 47. Shively, J.E., Kessler, M. and Todd, C.W.: The antigenic determinants of carcinoembryonic antigen: The formation, isolation, composition, and N-terminal sequences of its tryptic peptides. Cancer Res., submitted for publication. 48. von Kleist, S., Chavanel, G. and Burtin, P.: Identification of an antigen from normal human tissue that crossreacts with the carcinoembryonic antigen. Proc. Nat. Acad. Sci. USA, 69: 2492-2494, 1972. 49. Mach, J.-P. and Pusztaszeri, G.: Carcinoembryonic antigen (CEA): Demonstration of a partial identity between CEA and a normal glycoprotein. Immunochemistry, 9: 1031-1034, 1972. 50. Turberville, C., Darcy, D.A., Laurence, D.J.R., Jones, E.W. and Neville, A.M.: Studies on carcinoembryonic antigen (CEA) and a related glycoprotein, CCEA-2. Preparation and chemical characterisation. Immunochemistry 10: 841843, 1973. 51. Darcy, D.A., Turberville, C. and James, R.: Immunological study of carcinoembryonic antigen (CEA) and a related glycoprotein. Br. J. Cancer, 28: 147-160, 1973. 52. Ørjasaeter, H.: Study of substances related to carcinoembryonic antigens, CEA-NCA and association with α-antichymotrypsin. Acta Path. Microbiol. Scand., 84: 235-244, 1976. 53. Chu, T.M., Reynoso, G. and Hansen, H.J.: Demonstration of carcinoembryonic antigen in normal human plasma. Nature, 238: 152-153, 1972. 54. Kupchik, H.Z. and Zamcheck, N.: Carcinoembryonic antigen(s) in liver disease. Gastroenterology, 63: 95-101, 1972. 1


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Pusztaszeri, G. and Mach, J . : Carcinoembryonic antigen (CEA) in nondigestive cancerous and normal tissues. Immunoohemistry, 10: 197-204, 1973. Go, V.L.W., Ammon, H.V., Holtermuller, K.H., Krag, E. and Phillips, S.F.: Quantification of carcinoembryonic antigen-like activities in normal human gastrointestinal secretions. Cancer, 36: 2346-2350, 1975. Egan, M.L., Pritchard, D.G., Todd, C.W. and Go, V.L.W.: Isolation and immunochemical and chemical characterization of carcinoembryonic antigen-like substances in colon lavages of healthy individuals. Cancer Res., 37: 2638-2643, 1977. Shively, J.E., Todd, C.W., Go, V.L.W. and Egan, M.L.: Amino terminal sequence of a carcinoembryonic antigen-like glycoprotein isolated from the colonic lavages of healthy individuals. Canoer Res., in press. Kessler, M.J., Shively, J.E., Pritchard, D.G. and Todd, C.W.: Isolation, immunologi studies of a tumo onic antigen. Canoer Res., in press. Engvall, E., Shively, J.E. and Wrann, W.: Isolation and characterization of the normal crossreacting antigen (NCA). Homology of its N-terminal amino acid sequence with that of carcinoembryonic antigen (CEA). Proc. Nat. Acad. Sci. USA, in press. Ruoslahti, E., Engvall, E., Vuento, M. and Wigzell, H.: Monkey antisera with increased specificity to carcinoembryonic antigen (CEA). Int. J. Canoer, 17: 358-361, 1976. Hammarström, S., Svenberg, T., Hedin, A. and Sundblad, G.: Antigens related to CEA. Scand. J. Immunology, in press.

RECEIVED

December 29, 1977.


20 Status of Blood Group Carbohydrate Chains in Human Tumors WILLIAM W. YOUNG, JR. and SEN-ITIROH HAKOMORI Biochemical Oncology, Fred Hutchinson Cancer Research Center, and Departments of Pathobiology and Microbiology/Immunology, University of Washington,1124Columbia, Seattle, WA 98104 1. Introduction Clear changes of glycolipi files in cell surface membranes associated with oncogenic transformation have been described during the past ten years. Possibly the first clear documentation for this topic was the decrease of hematoside and increase of lactosylceramide in polyoma transformed BHK cells as described in 1968 (1). A number of studies carried out by different investigators agreed that two kinds of changes in carbohydrate chains may occur during oncogenic transformation (see for review ref. 2). One is the block of synthesis certain glycolipids resulting in the associated accumulation of precursor structures and the second is induction of synthesis of a unique glycolipid for the transformed cells which most probably depends on activation of a new glycosyltransferase. Since in vitro cellular systems are only models for in vivo tumors, it is important to observe the changes of membrane glycolipids in tumors, particularly in human cancer. Among the great number of carbohydrate chains in human cells and tissues, the most well-defined carbohydrate chains, in terms of their chemistry, enzymology and genetics, are the blood group determinants. For this reason, our major effort has been directed to characterizing the changes of blood group determinants in human cancer as compared to comparable normal tissue; however, it should be noted that such comparisons may not be ideal as the precursor cell of human cancer may represent only a small population of cells present in normal tissue. However, for practical purposes such as diagnosis and therapy of human tumor using a specific cell surface marker, a gross difference of carbohydrate determinants observable between tumor and normal tissue may offer useful information. The first clear change of blood group determinants in human cancer was described in the laboratory of the late Dr. Masamune, who was a good friend of the late Dr. Ward Pigman to whom this Symposium is dedicated. Oh-uti in 1949, while working in Masamune's laboratory, described the deletion of blood group activity in "blood group polysaccharide" prepared from human gastric cancer (3). A number of subsequent studies were made in 0-8412-0452-7/78/47-080-357$05.00/0 © 1978 American Chemical Society In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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Masamune s l a b o r a t o r y comparing the immunological and chemical d i f f e r e n c e s o f b l o o d group g l y c o p r o t e i n s from tumors and normal mucosa (^_,J5). Independently, Davidsohn and h i s co-workers ( 6 , T _ ) , Kay and Wallace ( 8 J , Prendergast (9.), and Dabelsteen and F u l l i n g ( 1 0 ) , have s t u d i e d the s t a t u s o f b l o o d group A and B i n human tumors as compared t o normal t i s s u e . Although a few d i s c r e p e n c i e s were d e s c r i b e d , they e s s e n t i a l l y agreed that blood group A or B determinants are d e l e t e d i n v a r i o u s human tumors and t h a t the degree o f d e l e t i o n c o u l d be c o r r e l a t e d with the degree o f m a l i g nancy (6_,T_). Most o f these s t u d i e s were made b e f o r e the chemical s t r u c t u r e s o f A and B determinants were e s t a b l i s h e d . Since the s t r u c t u r a l r e l a t i o n s h i p between A, B, H, L e , Le^, I and i d e t e r minants was e s t a b l i s h e d l a t e i n the 1960 s, s t u d i e s have focused on the s t a t u s o f the p r e c u r s o r s o f ABH determinants. As seen i n Table I , accumulation o f p r e c u r s o r carbohydrate chains i s q u i t e obvious f o r some human cancers i f not a l l o f the cases. A s i m i l a r p r e c u r s o vivo transformed c e l l s an p r e c u r s o r s c o u l d be u s e f u l s u r f a c e markers o f tumor c e l l s (13). Another category o f changes i n blood group determinants that has been observed i s t h e appearance o f new b l o o d group determinants i n some human tumor t i s s u e . Examples i n c l u d e the p o s s i b l e s y n t h e s i s o f P and P]_ antigens i n the g a s t r i c tumor o f a p a t i e n t belonging t o the extremely r a r e b l o o d group p ( l ^ i ' l ^ ) and induct i o n o f A - l i k e antigen i n tumors o f host blood group 0 ( 1 6 , 1 7 ) • The A - l i k e antigen appearing i n b l o o d group 0 or B could have been the r e s u l t o f the new s y n t h e s i s o f Forssman antigen i n tumors ( l 8 , 19), These items w i l l be d i s c u s s e d e x t e n s i v e l y i n subsequent sections. a

?

2 . Accumulation o f Precursor Carbohydrates o f Blood Group Determinants Since I - , and i-determinants have been assigned as p r e cursors o f b l o o d group ABH determinants ( 2 6 ) , we have s t u d i e d the i / l r e a c t i v i t y o f w a t e r - s o l u b l e blood group g l y c o p r o t e i n s e x t r a c t e d from g a s t r o i n t e s t i n a l tumors and normal mucosal t i s s u e . The g l y c o p r o t e i n f r a c t i o n was e x t r a c t e d according t o the procedure d e s c r i b e d by Masamune et al (5) and by the p e r c h l o r i c a c i d e x t r a c t i o n method d e s c r i b e d by Krupey et al {2J). As shown i n Table I I , the p e r c h l o r i c a c i d e x t r a c t a b l e f r a c t i o n o f tumor t i s s u e showed s i g n i f i c a n t i - a c t i v i t y i n c o n t r a s t t o the normal mucosa i n h cases, whereas, 3 cases showed higher i - a c t i v i t y i n normal mucosa, and one case d i d not show any s i g n i f i c a n t d i f f e r ence (Watanabe, Hakomori and Warner, unpublished d a t a ) . Recently, the hexasaccharide s t r u c t u r e , G a i e i + 4 G l c N A c $ l + 3 G a l B l + 4 G l c N A c 3 l - * 3 Gal3l->-i+Glc was found t o be a determinant f o r i - s p e c i f i c i t i e s ( 2 8 ) . Such a s t r u c t u r e may appear o r i n c r e a s e i n some human cancer. In order t o study the s t a t u s and d i s t r i b u t i o n o f blood group c a r r i e r carbohydrate chains i n normal and cancer t i s s u e s , the r e a c t i v i t y o f g l y c o l i p i d s d i r e c t e d a g a i n s t three s t r u c t u r e s , as shown i n F i g u r e 1 , were compared u s i n g t h e i r r e s p e c t i v e a n t i bodies (2h). The complement f i x i n g r e a c t i v i t i e s o f g l y c o l i p i d s


20.

YOUNG A N DHAKOMORI

Blood

Group

Carbohydrate

Table I. Status o f Blood Group Determinants 1.

D e l e t i o n o f AB-determinants G a s t r o i n t e s t i n a l tumors

359

i n Human Cancer.

(Oh-uti 1 9 ^ 9 ; Masamune et al 1 9 5 2 ; 1 9 5 8 ; Davidsohn et al 1 9 6 6 ; I s e k i et al 1 9 6 2 ; S t e l l n e r et al 1 9 7 3 )

C e r v i c a l cancer

(Davidsohn et al 1 9 6 9 )

Bladder cancer

(Kay and Wallace 1 9 6 1 )

Oral

(Prendergast 1 9 6 8 ; Dabelsteen and F u l l i n g 1971)

carcinoma

2 . Precursor accumulation A s s o c i a t i o n o f CEA with I (Ma) antigen

(Simmons and Perlman 1 9 7 3 )

Presence o f I (Step antigen

(Feiz

Presence o f i (Dench) antigen

(Watanabe and Hakomori unpublished)

Accumulation o f l a c t o N-triosylceramide (GlcNAcCTH) 3.

Chains

Induction o f "incompatible" determinants P i , P, P antigen i n tumor o f p - i n d i v i d u a l

1975)

(Watanabe and Hakomori 1 9 7 7 ; Karlsson 1 9 7 6 )

k

(Levine et al 1 9 5 1 )

A - l i k e antigen i n tumors of 0 - , B - i n d i v i d u a l s (Hakomori et al 1 9 6 7 ; Hakkinen 1971) k. Heterophile F-antigen as a p o s s i b l e new i s o a n t i g e n i n human and the presence o f F-antigen i n tumors o f F"-tissues

(Hakomori, Wang and Young 1 9 7 7 ; Makita 1 9 7 7 )


360

GLYCOPROTEINS A N D GLYCOLIPIDS I N DISEASE PROCESSES

Table I I . The i - a c t i v i t y o f the p e r c h l o r i c - a c i d - e x t r a c t able f r a c t i o n o f normal mucosa and tumor t i s s u e

Dench i a c t i v i t y i n Case

Blood group

MIM Tn V. His G. Gutt. A. Dalz. C. Walt. M. Hutch. Anonym.

0 A. ND 0, 0, 0, ND ND

Normal mucosa

MN Le N, L e L e , MN a

a

Tumor tissue

>50 >50

3 6

>50 6 3 >50

6 25 12 >50

A c t i v i t y i s expressed by doses o f g l y c o p r o t e i n which showed obvious hemagglutination i n h i b i t i o n i n yg. The t e s t system contained a n t i - i (Dench) serum (3 hemagglutination doses) and 1% cord e r y t h r o c y t e s K. Watanabe, S. Hakomori and G.A. Warner, unpublished data).

L-a-Fuc 1—26-Gal 1—4 B-GlcNAc1 B-Gal 1—4 B-GlcNAc 1—3B-Gal 1— 4B-Glc—Ceramide L-a-Fuc1—2B-Gal 1 - 4 B - G l c N A d '

(structure 4)_ (structure 3)

(structure 2 ) . ^structure 1)_ Journal of Experimental Medicine

Figure 1.

Structure of H glycolipid and its degradation products to which antibodies were directed 3


20.

YOUNG A N D HAKOMORI

Blood

Group

Carbohydrate

Chains

361

e x t r a c t e d from normal and colon mucosa with a n t i b o d i e s d i r e c t e d against s t r u c t u r e s 1, 2 and h are shown i n Table I I I . The average r e a c t i v i t y with a n t i - s t r u c t u r e h a n t i s e r a was 1 t o l 6 0 f o r normal mucosal t i s s u e and 1 t o 750 f o r cancer t i s s u e . While the antibody d i r e c t e d against s t r u c t u r e h showed t h i s remarkable d i f f e r e n t i a l r e a c t i v i t y between normal and tumor g l y c o l i p i d s , the antibodies d i r e c t e d against s t r u c t u r e 1 and 2 showed s i m i l a r r e a c t i v i t y towards normal and tumor g l y c o l i p i d s . The g l y c o l i p i d s of s e v e r a l cases o f colon carcinoma were compared with those o f normal mucosal t i s s u e by t h i n - l a y e r chromatography (TLC). An i n t e n s i f i e d spot corresponding t o s t r u c t u r e h (GlcNAcBl^GalBl-Û Glc31-^1ceramide) was observed i n the g l y c o l i p i d f r a c t i o n o f colon carcinoma as compared t o normal mucosa. Such a remarkable accumulation o f t h i s s t r u c t u r e i n some human cancers can be regarded as a precursor accumulation. Recently, K a r l s s o n b r i e f l y mentioned the accumulation o f the same g l y c o l i p i d i n human melanoma t i s s u e (25). Sinc f o r a l l blood group ABH t i o n o f t h i s g l y c o l i p i d can be c o r r e l a t e d t o a p r e c u r s o r accumulation due t o a blocked synthesis o f these antigens. 3. Induction o f "incompatible" determinants and Forssman g l y c o lipid. The appearance o f incompatible blood group determinants o f "both the P and ABH systems have been reported (see Table I ) . Because o f the well-known c r o s s - r e a c t i o n s o f the A and Forssman antigens, there was t h e p o s s i b i l i t y t h a t the apparent "neo-A" r e a c t i v i t y t h a t was detected i n tumors o f type 0 p a t i e n t s (l6,17) could have been caused by the presence o f Forssman antigen. The heterogenetic Forssman antigen (29) i s a glycosphingol i p i d whose s t r u c t u r e has been i d e n t i f i e d as GalNAcal-*3GalNAc31"*3 Galal->UGalBl->UGlc-*ceramide (30,31). In the e a r l y l i t e r a t u r e , reviewed by Buchbinder i n 1935 1*32), the various animal species were c a t e g o r i z e d as being e i t h e r Forssman-positive or - negative. It i s now c l e a r t h a t a l l Forssman-positive animal species possess a g l y c o l i p i d with a carbohydrate s t r u c t u r e i d e n t i c a l t o that d e s c r i b e d above (33-36). In a d d i t i o n the immunodeterminant o f the Forssman antigen, namely a t e r m i n a l d i s a c c h a r i d e GalNAcal*3Gal NAc31-*-R, has been found i n other " c a r r i e r " s t r u c t u r e s as w e l l , a l l of which are Forssman a c t i v e : ( l ) a ceramide t e t r a s a c c h a r i d e o f hamster f i b r o b l a s t s , GalNAcal+3GalNAc3l+3Galal+UGal3l+ceramide (37); a p o l y s a c c h a r i d e o f Streptococcus type c (3

v


364

G L Y C O P R O T E I N S A N D GLYCOLIPIDS IN

DISEASE

PROCESSES

l i p i d s can be d i s s o l v e d f i r s t i n hot MeOH or EtOH followed byb u f f e r , which appears to r e s u l t i n maximum antigen exposure as well. An a d d i t i o n a l problem of g l y c o l i p i d antigens i s t h e i r apparent "masking" when i n the presence of a l a r g e amount of other g l y c o l i p i d s . Forssman r e a c t i v i t y could not be detected i n the t o t a l n e u t r a l g l y c o l i p i d f r a c t i o n s of the s u r g i c a l cases ment i o n e d above but became q u i t e strong a f t e r p u r i f i c a t i o n o f the ceramide pentasaccharide f r a c t i o n by Iatrobeads chromatography (19). A s i m i l a r s i t u a t i o n occurred with the H - a c t i v i t y o f Hg l y c o l i p i d (U3) and I i a c t i v i t y of I i - a c t i v e g l y c o l i p i d s (unpublished o b s e r v a t i o n ) . The immunochemical methods most f r e q u e n t l y used f o r g l y c o l i p i d a n a l y s i s i n c l u d e the f o l l o w i n g . Double d i f f u s i o n i n agar g e l i s the simplest but l e a s t s e n s i t i v e method and o c c a s i o n a l l y produces spurious p r e c i p i t i n bands. I n h i b i t i o n of hemagglutinat i o n i s simple, and o f can detect about 1 nanogra able e f f o r t . Release o f trapped markers from g l y c o l i p i d - l i p o somes by antibody-complement (hk) has a s e n s i t i v i t y l i m i t o f about 50 nanograms. Recently we have developed a radioimmunoassay (RIA) f o r d e t e c t i n g g l y c o l i p i d antigens. I n i t i a l attempts t o use r a d i o l a b e l e d g l y c o l i p i d s were u n s u c c e s s f u l because of t r a n s f e r of l a b e l between "hot" and " c o l d " liposomes. The present assay u t i l i z e s a g l y c o l i p i d - p o l y m e r synthesized as shown i n F i g . 2 . F i r s t , the o l e f i n i c double bond i n the ceramide moiety o f the g l y c o l i p i d i s converted to a c a r b o x y l i c a c i d by a recent improvement (U5) of our e a r l i e r procedure (W>). Then the g l y c o l i p i d i s coupled t o a p o l y a c r y l i c hydrazide (PAH) polymer (|+7). Radiol a b e l s can be attached to both the g l y c o l i p i d and PAH moieties as shown i n F i g . 3 . I t should be noted i n passing that such a "polyg l y c o l i p i d " should prove u s e f u l not only f o r d e t e c t i o n o f g l y c o l i p i d antigens as described below but a l s o f o r q u a n t i t a t i o n of a n t i - g l y c o l i p i d s e r a and as a polymeric antigen i n immunization protocols. The RIA procedure i s summarized i n F i g . h. "Cold" g l y c o l i p i d antigen i n the form o f l i p i d v e s i c l e s i s preincubated with d i l u t e d r a b b i t anti-Forssman immunoglobulin. Then the l a b e l e d Forssman-PAH complex i s added and allowed to b i n d t o the remaining a v a i l a b l e a n t i b o d i e s . A d d i t i o n of i n t a c t , f i x e d Staphylococcus aureus, which has " P r o t e i n A" b i n d i n g s i t e s f o r immunog l o b u l i n ( U 8 ) , and c e n t r i f u g a t i o n r e s u l t s i n p r e c i p i t a t i o n of any antigen-antibody complexes. The supernatant can then be counted to determine the amount of i n h i b i t i o n of p r e c i p i t a t i o n o f l a b e l e d antigen by the unlabeled competitor. Figure 5 demonstrates t h a t t h i s assay can r e a d i l y detect 1 nanogram of Forssman, while i n h i b i t i o n by r e l a t e d s t r u c t u r e s (globoside and blood group A g l y c o l i p i d s ) i s n e g l i g i b l e . Figure 6 compares the r e a c t i v i t y of the p u r i f i e d ceramide


20.

YOUNG AND HAKOMORI

Blood

Group

Carbohydrate

Chains

Table IV. Summary o f Forssman s t a t u s o f Taiwan tumor

Blood type

F"" cases

F

cases

cases

A

h

1

B 0

10 6

1 k

Total

20

6

SYNTHESIS OF "POLY-GLYCOLIPIDS"

Forssman g l y c o l i p i d a c e t a t e

3 ( H-labelled)

KMnO^ , crown e t h e r

"Forssman g l y c o l i p i d

Polyacrylic

acid"

hydrazide

Carbodiimide

H

t

1 4

CH0

NaBH4

Forssman-polyacrylic Figure 2.

hydrazide

Synthesis of a "poly-glycolipid" man-polyacrylic hydrazide

Forss-


365

366


PROCESSES

R NH OH 0

0 ,

[ H]GalNAcal+3GalNAc8l+3Gal 3

i H,[ C]NHNHC-CH 14

I

Forssman p o l y a c r y l i c Figure 3.

Step 1.

hydrazide

Structure of Forssman-polyacrylic

hydrazide

Glycolipid/phospholipid/cholesterol vesicles Rabbit anti-Forssman Ig d i l u t e d 1:1000 Incubate 4 ° , 2 hr 14

Step 2.

Step 3.

Figure 4.

3 C/ H 1 F o r s s m a n - p o l y a c r y l i c hydrazide (10,000 cpm per assay tube) Incubate 4 ° , 30 min [

I n t a c t Staphylococcus aureus Incubate 4 ° , 10 min C e n t r i f u g e and count supernatant Protocol for radioimmunoassay of Forssman

glycolipid


20.

YOUNG AND HAKOMORI

Blood

Group

Carbohydrate

Chains

367

Figure 5. Inhibition of precipitation of [ C/ H]-labeled Forssman-PAH by unlabeled glycolipid antigens. Lipid vesicles of the indicated composition were incubated with anti-Forssman (10 yL, 1/1000 dilution) for 2 hr at 4°C in a total volume of 100 ixL. [ C/ H]-labeled Forssman-PAH (10,000 cpm, 100 pL) was added and incubation continued for 30 min, 4°C. S. aureus (7 mg, 700 fiL) was added and after 10 min at 4°C removed by centrifugation at 100 g for 10 min. 450 fiL of supernatant was counted, and the results are expressed as follows: 14

14

3

3

% Inhibition =

-

fl)

X

1

0

0

where A = supernatant cpm of control tube without anti-Forssman; B = supernatant cpm of control tube with anti-Forssman but no inhibitor; C = supernatant cpm of experimental tube. (X—X) Forssman/SM/CHOL; (O—O) Globoside/SM/ CHOL; and (A—A) A glycolipid/SM/CHOL. Mole ratio glycolipid/ SM/CHOL =1/250/200. h


In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978. Tissue dry

w e i g h t , ug

Figure 6. Forssman reactivity of purified ceramide pentasaccharide fractions of human gastrointestinal tissues as determined by radioimmunoassay. Purified ceramide pentasaccharide fractions from the normal colonic mucosa (X—X) and colon tumor tissue (O—O) from a Taiwan patient were tested for Forssman reactivity by radioimmunoassay as described in the legend to Figure 5.

100

20.

YOUNG AND HAKOMORI

Blood

Group

Carbohydrate

Chains

369

pentasaccharide f r a c t i o n from the normal mucosa and tumor t i s s u e o f one Taiwan case which by chemical a n a l y s i s had been found t o be F , as d e f i n e d above. +

k.

Concluding remarks In c o n c l u s i o n , examples have been presented i n which s t r i k i n g a l t e r a t i o n s o f b l o o d group determinants occur i n human tumor t i s s u e apparently due t o one o f two mechanisms: (a) accumulation o f p r e c u r s o r s t r u c t u r e s due t o a b l o c k i n s y n t h e s i s o f more comp l e x determinants such as A o r B; and (b) the appearance o f apparently new determinants due t o the p o s s i b l e a c t i v a t i o n o f a l l e l o m o r p h i c genes. Many examples o f the f i r s t p o s s i b i l i t y have been r e v e a l e d , not o n l y i n tumors but a l s o i n in vitro model systems as w e l l , and i s a r a t h e r w e l l - a c c e p t e d mechanism. In cont r a s t , the second process has o n l y l i m i t e d experimental evidence. In f a c t u n t i l r e c e n t l y the o n l y example was a s i n g l e case o f apparent e x p r e s s i o n o f p individual (l]+,15). Now man antigen i n F" i n d i v i d u a l s (19^) may r e s u l t from t h i s process as w e l l . Both processes may represent the e x p r e s s i o n o f o n c o - f e t a l antigens as both the " s t r u c t u r e U" ceramide t r i s a c c h a r i d e (2*0 and Forssman g l y c o l i p i d (K. Watanabe, unpublished o b s e r v a t i o n ) have been d e t e c t e d i n human f e t a l t i s s u e . In a d d i t i o n both processes generate tumor c e l l surface determinants which could serve as s p e c i f i c t a r g e t s f o r immunotherapy or f o r immunochemotherapy.

This study has been supported by N a t i o n a l Cancer I n s t i t u t e Grant CA 1 9 2 2 U , and p a r t i a l l y supported by a grant from the Cancer Research I n s t i t u t e , Inc. W.W.Y. i s r e c i p i e n t o f a f e l l o w s h i p from the Cancer Research I n s t i t u t e , Inc.


370


REFERENCES 1. Hakomori, S., and Murakami, W.T. (1968) Proc. Nat. Acad. Sci. U.S.A. 59, 254. 2. Hakomori, S. (1975) Bioch. Biophys. Acta 4l7, 55. 3. Oh-uti, K. (1949) Tohoku J. Exp. Med. 51, 297. 4. Masamune, H., Yosizawa, Z., Oh-uti, K., Matuda, Y., and Masukawa, A. (1952) Tohoku J. Exp. Med. 56, 37. 5. Masamune, H., Kawasaki, H., Abe, S., Oyama, K., and Yamaguchi, Y. (1958) Tohoku J. Exp. Med. 68, 8 l . 6. Davidsohn, I., Kovarik, S., and Lee, C.L. (l966) Arch. Path. 81, 381. 7. Davidsohn, I., Kovarik, S., and Ni, Y. (1969) Arch. Path. 87, 306. 8. Kay, H.E.H., and Wallace, B.H. (1961) J. Natl. Cancer Inst. 26, 1349. 9. Prendergast, R.C., Tot P.D. d Garguilo A.W (1968) J. Dent. Res. 47, 306 10. Dabelsteen, E . , and Fulling, J.H. (1971) Scand. J. Dent. Res. 79, 387. 11. Iseki, S., Furukawa, K., and Ishihara, K. (1962) Proc. Jap. Acad. Sci. 38, 556. 12. Simmons, D.A.R., and Perlman, P. (1973) Cancer Res. 33, 313. 13. Rosenfelder, G., Young, W.W., J r . , and Hakomori, S. (1977) Cancer Res. 37, 1333. Ik. Levine, P., Bobbitt, O.B., Waller, R.K., and Kuhmichel, A. (1951) Proc. Soc. Exp. Biol. Med. 77, 403. 15. Levine, P. (1976) Ann. N.Y. Acad. Sci. 277, 428. 16. Hakomori, S., Koscielak, J., Bloch, K.J., and Jeanloz, R.W. (1967) J. Immunol. 98, 31. 17. Häkkinen, I. (1970) J. Natl. Cancer Inst. 44, 1183. 18. Makita, A. (1977) United States-Japan Joint Seminar "Structural and Functional Significance of Membrane Glycolipids", 19. Hakomori, S., Wang, S.-M., and Young, W.W., Jr. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 3023. 20. Stellner, K., Hakomori, S., and Warner, G.A. (1973) Biochem. Biophys. Res. Commun. 55, 439. 21. Iseki, S., Furukawa, K., and Ishihara, K. (1962) Proc. Jap. Acad. Sci. 38, 556. 22. Cooper, A.G. (1974). Immunol. 113, 1246. 23. Feizi, T., Turberville, C., and Westwood, J.H. (1975) Lancet 1, 391. 24. Watanabe, K., and Hakomori, S. (1976) J. Exp. Med. 144, 644. 25. Karlsson, K.A. In Structure of Biological Membranes (1976), S. Abrahamsson and I. Pascher (eds.) 34th Nobel Symposium, New York, Plenum Publishing. 26. Feizi, T., Kabat, E.A., Vicar, G., Anderson, B., and Marsh, W.L. (1971) J. Immunol. 106, 1578. 27. Krupey, J., Wilson, T., Freedman, S.O., and Gold, P. (1972) Immunochemistry 9, 6l7.


20. YOUNG AND HAKOMORI

Blood Group Carbohydrate Chains 371

28. Niemann, H., Watanabe, K., Hakomori, S., Childs, R.A., and Feizi, T. submitted for publication. 29. Forssman, J. (1911) Biochem. Z. 37, 78. 30. Siddiqui, B., and Hakomori, S. (1971) J. Biol. Chem. 246, 5766. 31. Stellner, K., S a i t o , and Hakomori, S. (1973) Arch. Biochem. Biophys. 155, 464. 32. Buchbinder, L. (1935) Arch. Pathol. 19, 841. 33. Sung, S.J., Esselman, W.J., and Sweeley, C.C. (1973) J. Biol. Chem. 248, 6528. 34. Taketomi, T., Hara, A., Kawamura, N., and Hayashi, M. (1974) J. Biochem. (Tokyo) 75., 197. 35. Ziolkowski, C.H.J., Fraser, B.A., and Mallette, M.F. (1975) Immunochemistry 12, 297. 36. Karlsson, K.-A., Pascher, I., Pimlott, W., and Samuelsson, B.E. (1974) Biomed Mass Spectrometry 1 49. 37. Gahmberg, C.G., an 2438. 38. Coligan, J . E . , Fraser, B.A., and Kindt, T.J. (1977) J. Immunol. 118, 6. 39. Slomiany, A., Slomiany, B.L., and Annese, C. (1977) FEBS Letters 81, 157. 40. Slomiany, A., and Slomiany, B.L. (1977) Eur. J. Biochem. 76, 491. 41. Tonegawa, Y., and Hakomori, S. (1977) Biochem. Biophys. Res. Commun. 76, 9. 42. Kawanami, J. (1972) J. Biochem. (Tokyo) 72, 783. 43. Stellner, K., Watanabe, K., and Hakomori, S. (1973) Biochemistry 12, 656. 44. Six, H.R., Young, W.W., J r . , Uemura, K., and Kinsky, S.C. (1974) Biochemistry 13, 4050. 45. Young, W.W., Jr., Laine, R.A., and Hakomori, S. Meth. Enzymol. in press. 46. Laine, R.A., Yogeeswaren, G., and Hakomori, S. (1974) J. Biol. Chem. 249, 4460. 47. Wilchek, M., and Miron, T. (1974) Meth. Enzymol. 34, 72. 48. Kessler, S.W. (1976) J. Immunol. 117, 1482. 49. Rapport, M.M., and Graf, L. (l969) Prog. Allergy 13, 273. 50. Marcus, D.M., and Schwarting, G.A. (1976) Advances in Immunology 23, 203. RECEIVED

March 15, 1978.


21 Role of Glycoconjugates in Expression of the Transformed Phenotype S. STEINER, D. VIA, M. KLINGER, G. LARRIBA, and S. SRAMEK Department of Virology, Baylor College of Medicine, Houston, TX 77030 R. LAINE Department of Biochemistry, University of Kentucky, Lexington, KY 40506 Recent studies hav glycolipids as being o importanc y proper ties associated with oncogenic transformation, e.g., altered antigenicity, decreased cellular adhesiveness, and altered lectin agglutinability. Moreover, in human tumors the blood group isoantigens, which are fucosyl glycolipids, are decreased (Hakomori and Jeanloz, 1964), as are the glycosyl transferases involved in the synthesis of these fucosyl glycolipids (Stellner et a l . , 1973). In contrast, the level of a simple and novel fucolipid is enhanced (Watanabe et a l . , 1976). Changes in fucoproteins in human tumors (Simmons and Pearlman, 1973) and in trypsin-sensitive surface fucopeptides in a variety of transformed cells (e.g., Buck et al. 1970; Pietropaolo et a l . , 1977) have also been observed. Furthermore, alterations in the serum level of fucosyl transferase in patients with tumors has been reported (Bauer et a l . , 1977; Kessel et a l . , 1977). These observations and related ones which suggested possible differences in the metabolism of fucosylated components in cancer cells as compared to normal cells prompted studies in this laboratory of changes in fucose metabolism in virus-transformed cells as compared to normal cells. These studies initially focused on the small molecular weight, organic-extractable fucosylated components. Murine sarcoma virus-transformed rat (MSV-NRK) cells and their nontransformed counterparts (NRK) were grown in medium supplemented with either [ H]- or [ C] fucose. The cells were extracted either with CHCl-CHOH (2:1, v/v) or 60% ethanol (v/v), and the extract chromatographed on AG50, H+ form, followed by silicic acid thin-layer chromatography (TLC). The distribution of radioactivity on the thin-layer chromatogram is shown in Figure 1. The normal cells have two prominent peaks of radioactivity, i.e., component FL4 and FL3. In contrast, in the MSV-transformed rat cells there is a marked decrease in the incorporation of labeled fucose into FL4. This decreased labeling of FL4 is approximately five-fold and is evident when either [ C]- or [ H] fucose is used. Moreover, all normal mammalian cells thus far examined 3

14

3

3

14

3

0-8412-0452-7/78/47-080-378$06.50/0 © 1978 American Chemical Society In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

21.

STEINER E T A L .

Glycoconjugates

and the Transformed

Phenotype

379

(human, mouse, r a t , baboon, monkey and hamster) contain FL3 and FL4. In a d d i t i o n , many d i f f e r e n t transformed c e l l s , e . g . , mouse mammary carcinoma, simian v i r u s 40-transformed c e l l s , herpes simplex virus-transformed c e l l s , manifest a decrease i n l a b e l i n g o f FL4 ( S t e i n e r e t a l . , 1973, 1974). The decreased i n c o r p o r a t i o n o f label i n t o FL4 appears to be more pronounced i n h i g h l y tumorigeni e (as compared to weakly or nontumorigenic) simian v i r u s 40transformed c e l l s , herpes simplex virus-transformed c e l l s ( S t e i ner and S t e i n e r , 1975/76), o r mouse mammary carcinoma c e l l s (unpublished o b s e r v a t i o n s ) . Based on these o b s e r v a t i o n s , s t u d i e s were undertaken to examine, i n a temporal manner, the r e l a t i o n s h i p between decreased i n c o r p o r a t i o n of l a b e l e d fucose i n t o FL4 and a l t e r a t i o n s in the metabolism o f other glycoconjugates as a f u n c t i o n o f the expression o f the transformed c e l l phenotype. We have observed that growth o f MSV-NRK c e l l s i n medium supplemented with 2 mM sodium butyrate r e s u l t s i n c e l l u l a the MSV-NRK c e l l s resembl (Figur 2). Moreover, the process i s f u l l y r e v e r s i b l e upon s h i f t o f c e l l s to standard medium. There i s a l s o an e l a b o r a t i o n o f the c y t o s k e l e t a l elements, i . e . , microfilaments and m i c r o t u b u l e s , i n these treated c e l l s (Altenburg e t a l . , 1976). Along with the morphologi c a l changes, there i s a t h r e e - f o l d increase i n the l e v e l o f P H ] fucose incorporated i n t o FL4 (Table I ) . The increased l a b e l i n g i s observed i n MSV-NRK c e l l s grown i n butyrate-supplemented medium f o r short (1 passage) and long (30 passages) periods o f time. Furthermore, s h i f t o f b u t y r a t e - t r e a t e d MSV-NRK c e l l s to c o n t r o l medium r e s u l t e d i n r e v e r s a l to the l e v e l o f c o n t r o l c e l l s . We a l s o examined the e f f e c t s o f sodium butyrate on the gangl i o s i d e composition o f the MSV-NRK c e l l s . As i n d i c a t e d i n Table I I , growth o f MSV-NRK c e l l s i n sodium butyrate-supplemented medium r e s u l t s i n an increase i n both hematoside GM3 and i n the d i s i a l o g a n g l i o s i d e G D ^ . The increased l e v e l s o f hematoside and GD-i^ were observed w i t h i n a s i n g l e passage i n sodium b u t y r a t e supplemented medium, although the maximal l e v e l o f hematoside was not observed f o r several passages. Another butyrate-induced e f f e c t i s the marked increase i n GD]/\ i n normal (NRK) c e l l s (Table II). The elevated l e v e l o f GD-|/\ i n NRK c e l l s (which are already f l a t t e n e d ) was p a r a l l e l e d by a change i n the o r g a n i z a t i o n o f the microfilaments as examined by i n d i r e c t immunofluorescent microscopy with a n t i - a c t i n . The microfilaments s h i f t e d from a random d i s t r i b u t i o n to a p a r a l l e l d i s t r i b u t i o n ( V i a and S t e i n e r , unpublished data). An increase i n the average s i z e d i s t r i b u t i o n o f t r y p s i n - s e n sitive surface fucopeptides has been observed i n a wide range o f transformed c e l l s as compared to normal c e l l s . This increase i s evident i n the MSV-NRK c e l l s as compared to NRK c e l l s (Figure 3A). There i s l i t t l e detectable s h i f t i n the molecular weight d i s t r i b u t i o n o f these components i n the f i r s t - p a s s a g e sodium b u t y r a t e t r e a t e d MSV-NRK c e l l s . By passage 2 i n sodium butyrate-supplemen-



Figure 1. Aminoacyl fucosides of NRK and MSV-NRK cells, (top) NRK cells; (bottom,) MSV-NRK cells; 3 — FL3; 4 = FL4. Cells were grown in [ H]fucose-supplemented medium for 72 hr, harvested by scraping into 60% ethanol (v/v), and extracted thrice in 60% ethanol at 100°C. The pooled extracts were subjected to column chromatography on AG50 H and the bound fraction eluted with 0.5N NHfiH. The NHÔH eluant was dried and chromatographed on plates in CHCU-CHsOH-NHfiH by volume). The distribution was determined by scraping 0.5-cm fractions from origin to solvent front. Radioactivity was quantitated with a scintillation spectrometer.

PROCESSES

3

+

0

5 10 15 FRACTION NUMBER

Figure 2. Morphological changes induced by sodium butyrate. MSV-NRK cells were cultured in control medium, i.e., untreated, and in 2mM sodium butyrate-supplemented medium for 24, 48, 72, and 96 hr and for 16 passages.


In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978. 3342

0.3

0.9

0.9

0.3

1.6

FL4/FL3

[^H]fucose l a b e l i n g , h a r v e s t i n g and a n a l y s i s o f FL3a and FL4a, see Figure 1.

* Average o f two separate experiments done i n d u p l i c a t e .

For d e t a i l s of

MSV-NRK

999

3690

30

MSV-NRK

reversal, 3 passages no sodium butyrate

4334

3732

1

MSV-NRK 4167

3062

3276

FL3 cpm/ mg p r o t e i n *

5320

FL4 cpm/ mg p r o t e i n *

1006

No. o f passages i n sodium butyrate

Fucosides o f MSV-NRK C e l l s

0

line

E f f e c t o f Sodium Butyrate on the Aminoacyl

MSV-NRK

NRK

Cell

Table I .


382

PROCESSES

FRACTION NUMBER

FRACTION NUMBER

Figure 3. Sephadex G-50 fucopeptide profile of 2mM "butyratetreated MSV-NRK cells. Exponentially growing cells labeled with radioactive fucose for 48 hr were used in all experiments. Fucopeptides were prepared and analyzed by the general method described by Buck and co-workers (1970). (A) H-labeled (*) NRK cells vs. C-labeled (O) control MSV-NRK cells. (B) H-labeled (%) MSV-NRK cells passaged twice in butyrate medium vs. C-labeled (O) control MSV-NRK cells. 3

14

3

14


STEINER E T A L .

Glycoconjugates

and the Transformed

Phenotype

BD

FRACTION NUMBER

(C) H-labeled (•) MSV-NRK cells passaged six times in butyrate medium vs. C-labeled (O) control MSV-NRK cells. (D) H-labeled (%) MSV-NRK cells passaged 19 times in butyrate medium vs. C-labeled (O) control MSV-NRK cells. The phenol red dye marker eluted at fractions 136-146 in column A, fractions 123-133 in column B, fractions 122-132 in column C, and fractions 125-135 in column D. Radioactivity was not detected in fractions beyond those shown in the figure. Labeling experiments with reversed radioisotopic forms of fucose, i.e., H and C, gave comparable results. 3

14

3

14

3


14


19

MSV--NRK + SB

MSV--NRK + SB

3

7.4

|16.1|

lll.2|

7.8

16.0

16.8

GM 2

6.3

6.2

6.5

6.7

3.0

2.4

GM

2

trace

trace

trace

trace

ND

ND

GMj

Ganglioside nmoles NANAV10 mg c e l l 1A

1.4

|2.4|

/2T2l

1.2

15.81

ND

GD

protein 1B

0.3

0.2

0.3

0.4

ND

ND

G D

3

1

N-acetylneuraminic a c i d detected by r e s o r c i n o l reagent, r e l a t i v e amount o f NANA in each component determined by densitometric scanning. 2 ND = Not d e t e c t e d . L i p i d s were extracted twice in CHCI3-CH3OH ( 2 : 1 , v/v) followed by two e x t r a c t i o n s i n CHCI3-CH3OH ( 1 : 2 , v / v ) . The l i p i d e x t r a c t was d r i e d , resuspended i n CHCI3-CH3OH ( 2 : 1 , v/v) and p a r t i t i o n e d according to the method o f Folch and coworkers (1957). The Folch upper phase was d r i e d , resuspended in a small volume o f H2O and d i a l y z e d . The sample was c h r o matographed on s i l i c i c a c i d t h i n - l a y e r plates (Q5, Quantum I n d u s t r i e s , F a i r f i e l d , N . J . ) i n CHC13-CH30H-H20 (60:35:8, by v o l ) . The i n d i v i d u a l g a n g l i o s i d e s were i d e n t i f i e d by comparison with authentic standards.

MSV--NRK

0

MSV -NRK

reversal (3 passages no sodium butyrate)

1

NRK + SB

No. passages in sodium butyrate

0

line

E f f e c t o f Sodium Butyrate on the Ganglioside Composition o f Murine Sarcoma Virus-Transformed NRK C e l l s as Compared with Normal NRK C e l l s

NRK

Cell

Table I I .

21. STEINER ET AL.

Glycoconjugates and the Transformed Phenotype

ted medium, a small shift can be observed (Figure 3B). By passage 6 (Figure 3C) in sodium butyrate-supplemented medium, there is a marked increase in the molecular weight distribution of the trypsin-sensitive surface fucopeptides to a distribution comparable to that of normal cells (Figure 3A). Thus, the shift in the size distribution of the trypsin-sensitive surface fucopeptides in butyrate-treated MSV-NRK cells not only appears to follow cellular flattening but also follows the increased incorporation of Ph]fucose into FL4 and the increased levels of the gangliosides GM3 (hematoside) and GDia. In order to understand the biochemical basis for the reduced level of FL4 in transformed cells, it was necessary to know the structures of FL3 and FL4. The initial examination involved indirect analyses with radioisotopically labeled FL3 and FL4 from cultured cells. These analyses indicated that these compounds had a positive charge, could be extracted with chloroform-methanol and might possess a lon ever, because of the difficultie structure by indirect means, an isolation method for the purification of sufficient quantities for direct chemical characterization was developed (Figure 4). Four- to six-week old rats were injected intraperitoneal^ with [ C]fucose. The liver, brain, intestine and kidneys were removed and extracted thrice with 60% ethanol . [ H]FL3 and [3h]FL4, purified from cell culture, were added to the extract to monitor for the fractions of interest during the purification and to evaluate the final recovery of FL3 and FL4. The ethanol-soluble fraction was chromatographed on Sephadex G-25, followed by high-voltage electrophoresis and descending paper chromatography. This last step separates FL3 from FL4, which in turn were chromatographed on silicic acid thin-layer plates and were each observed to be separable into two components, i.e., FL3a and FL3b, FL4a and FL4b. The purity of each component was examined in several TLC systems. Gas-liquid chromatography and an amino acid analyzer were used to identify the constituents of each component. The compounds were subjected to HC1methanolysis, silylated and the carbohydrates analyzed by gasliquid (GC) chromatography (Figure 5). The GC results indicated the presence of fucose in the FL3 series (as illustrated in Figure 5C for FL3b) and both fucose and glucose in the FL4 series (as illustrated in Figure 5D for FL4a). No other carbohydrate residues were observed (Figure 5A) nor did we observe the presence of long chain base (Figure 5B) or fatty acid (Larriba et a l . , 1977). Since these compounds contain a positively charged moiety, the possibility that there is an amino acid constituent was considered. Each component was subjected to strong acid hydrolysis and analyzed with an amino acid analyzer (Table III). FL3a contained threonine; FL3b, serine; FL4a, threonine; and FL4b, serine. The molar ratios of the amino acid and carbohydrate are indicated in Table IV. As can be seen, FL3a contains fucose and threonine; FL3b, fucose and serine; FL4a, glucose, fucose and threonine; and 14

3


GLYCOPROTEINS

386

A N D GLYCOLIPIDS

I N DISEASE

PROCESSES

[ C ] f u c o s e - l a b e l e d rat tissue 14

[ H ] f u c o s e - l a b e l e d F L 3 and F L 4 f r o m cultured cells Extract Centrifugation

r

ellet

Supernatant fraction Sephadex G - 2 5 High voltage e l e c trophoresis, pH 2 . 0 (2 M acetic acid) i Descending paper chromatography (ethyl acetate-acetic a c i d - H 2 0 3:1:1, by vol) I

1

F L 3 (a and b)

F L 4 (a and b) Silicic acid T L C

(CHCI0-CH3OH-NH4OH,

40:80:25, by vol)

1

FL4a

FL4b

FL3a

FL3b

s l i c i c a c i d T L C ^ ( b u t a n o l - p y r i d i n e - H O } 6:4;3, by v o l / 0

I FL4a

I

FL4b

\

FL3a

P u r i t y examined i n a variety of T L C s y s t e m s methods: c h a r r i n g , o r c i n o l and n i n h y d r i n ) .

Figure 4.

1

FL3b (detection

Purification of aminoacyl fucosides from rat tissue


STEINER E T A L .

Glycoconjugates

RETENTION

TIME

and the Transformed

Phenotype

frnlnules)

Biochemical and Biophysical Research Communications

Figure 5.

Gas-liquid chromatography of FL3b and FL4a.

(A) Top profile: equimolar standard mixture of monosaccharides. (B) Second profile from top: lactosylceramide. (C) Third profile from top: FL3b. (D) Bottom profile: FL4a. Peaks correspond to the following compounds: 1, 2, 3, fucose; 4, arabinotol; 5, 6, 7, galactose; 8, 9, glucose; 10,11, mannitol; 12,13, N-acetylgalactosamine; 14, N-acetylglucosamine; 15, N-acetylneuraminic acid; and 16, dihydrosphingosine. Samples were methanolyzed, N-acetylated and silylated (Larriba et al, 1977). 1-2 fiL of each sample was then injected onto six-foot columns packed with 3% OV-1 on 80/100 Supelcoport in a Packard Becker model 419 dual column gas chromatograph withflameionization detector. N flow rate (effluent), 30 mL/min. Injector and detector temperatures, 270°C. Temperature program: temperature maintained at 160°C for 5 min, then raised 5°/min to 250°C, and maintained at 250°C for 15 min. Peak areas were integrated using a Spectra-Physics Autolab System I computing integrator. 2


388

G L Y C O P R O T E I N S A N D GLYCOLIPIDS I N DISEASE PROCESSES

Table I I I . Amino Acid A n a l y s i s o f Aminoacyl Fucosides

Compound-

1

After hydrolysis

Before hydrolysis

FL3a

21.9

43.0

FL3b

23.6

46.2

FL4a

16.0

42.9

FL4b

ND2

46.3

Serine-*

--

46.8

Threonine-*

—

43.3

Samples wer 110 C f o r 24 h r , concentrated by f l a s h e v a p o r a t i o n , resuspended i n a small volume (0.05-0.1 ml) o f 0.11 M sodium c i t r a t e , pH 3 . 2 , and chromatographed on a Beckman 121 amino a c i d analyzer using a s i n g l e column, p h y s i o l o g i c a l f l u i d program. ND = Not done. 3 The amino acids with c l o s e s t r e t e n t i o n times to s e r i n e and threonine are a s p a r t i c a c i d (40.6) and p r o l i n e ( 6 5 . 9 ) . 1

2


OH O- ^-D-glucopyranosyl-(l-**3)-0- cC-L-fucopyranosyl-L-threonine Figure 6.

Structure of O-p-D-glucopyranosylfl-*

3)-0-

«-1,-fucopyranosyl-L-ihreonine



137

53

9

FL3b

FL4a

FL4b

0

46

0

84

Threonine-*

1.1

8

1.4

1.2

1.2

0

0 0

1.1

0

1.0

0

1.0

1.0

0

1.0

0

Fuc / Glc / Thr / Ser

1.1

120

0

Serine^

!

3


1 For d e t a i l s o f a n a l y s i s o f carbohydrate and amino a c i d s , see Figure 5 and Table I I I , respectively. 2 The nmoles o f amino a c i d and carbohydrate represent the t o t a l p u r i f i e d material r e c overed from the r a t t i s s u e s . As judged by exogenously added FL3a and FL4a from c u l tured c e l l s , recovery o f FL3a from r a t t i s s u e was approximately 16.0% and recovery o f FL4a was approximately 10.0%. The amount o f threonine and s e r i n e i s not c o r r e c t e d f o r breakdown (^10%) due to strong a c i d h y d r o l y s i s .

11

56

0

GI ucose

91

Fucose

nmoles^

FL3a

Compound

Table IV. Molar Ratios o f Carbohydrate and Amino Acids o f FL3a, FL3b, FL4a and FL4b

390

GLYCOPROTEINS


PROCESSES

FL4b, g l u c o s e , fucose and s e r i n e ; a l l in an approximate r a t i o o f 1:1. Like FL4a, which i s the f u c o s e - l a b e l e d component designated as FL4 i n the e a r l i e r l a b e l i n g s t u d i e s , a u r i n a r y f u c o s i d e , which contains f u c o s e , glucose and t h r e o n i n e , has r e c e n t l y been desc r i b e d (Hallgren et a l . , 1975). This u r i n a r y f u c o s i d e has the s t r u c t u r e shown in Figure 6, i n c l u d i n g the novel feature o f an i n ternal fucose 0 - g l y c o s i d i c a l l y l i n k e d to amino a c i d . GC/mass spectrometry studies were undertaken to determine the carbohydrate sequence and linkage o f FL4a. Synthetic f u c o s i d e , o f the same s t r u c t u r e as the u r i n a r y f u c o s i d e , was most generously supplied by Dr. Svensson ( U n i v e r s i t y H o s p i t a l , Lund, Sweden; synt h e s i z e d by Drs. Garegg and Norberg, 1976) and was used as a s t a n dard. FL4a and the s y n t h e t i c fucoside were permethylated and subj e c t e d to GC/mass spectrometric a n a l y s e s . A GC/mass fragmentogram i n d i c a t e s that four fragments (m/e 393, 329, 219 and 187) are obtained from both permethylated s y n t h e t i c f u c o s i d e (Figure 7A) and permethylated FL4a fro and s y n t h e t i c fucoside ha Moreover, the fragments m/e 219 and m/e 187 are c o n s i s t e n t with the presence of terminal hexose and m/e 393 and m/e 329 are cons i s t e n t with a d i s a c c h a r i d e i n which 6-deoxyhexose (fucose) i s i n ternal and hexose i s t e r m i n a l . Neither component had an observable fragment, m/e 189, t y p i c a l of terminal f u c o s e . The s t r u c t u r e of the permethylated compounds was f u r t h e r examined f o l l o w i n g methanolysis and a c e t y l a t i o n by a n a l y s i s of the p a r t i a l l y methylated a l d i t o l a c e t a t e s . Each compound gave two peaks with major fragment m/e 262 and m/e 264, r e s p e c t i v e l y . As i n d i c a t e d in Table V I , the m/e 262 peak from d e r i v a t i z e d FL4a and s y n t h e t i c f u coside had the same r e t e n t i o n times. S i m i l a r l y , the peaks which gave a major fragment m/e 264 from d e r i v a t i z e d FL4a and s y n t h e t i c fucoside had the same r e t e n t i o n times. The mass spectra of the p a r t i a l l y methylated a l d i t o l acetates from FL4a and s y n t h e t i c f u coside were compared. The components from FL4a and the s y n t h e t i c f u c o s i d e with a major peak m/e 262 a l s o had fragments m/e 290, 118 and 131 (Figure 8 ) . These fragments are c o n s i s t e n t with an i n t e r n a l 6-deoxyhexose (Figure 9 ) . The mass spectra of the components from FL4a and s y n t h e t i c fucoside with a major fragment m/e 264 a l s o y i e l d e d fragments m/e 292, 118, 161 and 162, which i s c o n s i s t e n t with a terminal hexose (Figure 10). The combined GC, amino a c i d a n a l y s i s , and GC/mass spectrometry data i n d i c a t e that FL4a has a s i m i l a r s t r u c t u r e , i . e . , glucosyl (1—^3) f u c o s y l — • t h r e o n i n e , to that o f the u r i n a r y f u c o s i d e . The novel s t r u c t u r e of t h i s component, i . e . , with a fucose moiety which appears to be both i n t e r n a l and O - g l y c o s i d i c a l l y l i n k e d to amino a c i d , r a i s e s a question o f i t s b i o s y n t h e t i c o r i g i n . I f FL4a i s derived from f u c o p r o t e i n which has fucose 0 - g l y c o s i d i c a l l y l i n k e d to amino a c i d , then i t should be p o s s i b l e to r e l e a s e a d i s a c c h a r i d e which contains g l u c o s y l f u c i t o l by a l k a l i n e borohydride treatment o f that f u c o p r o t e i n ( s ) . An e t h a n o l - i n s o l u b l e f r a c t i o n of f u c o s e - l a b e l e d c e l l s was subjected to a l k a l i n e boro-


21.

STEINER E T A L .

Glycoconjugates

and the Transformed

Phenotype

391

m/e 393.2

219.1

187

i

i""""l 100 J

I""""'! I'l'l"'"""!'""""!" 200 300 400

|'i

Figure 7. Mass fragmentogram of permethylated synthetic glucosyl fucosyl threonine and FL4a. (A) Authentic glucosyl fucosyl threonine. (B) FL4a. FL4a and synthetic glucosyl fucosyl threonine were permethylated (Hakomori, 1964) and analyzed by GC/ mass spectrometry. A preliminary total mass scan was used to detect the major ions of interest. GC parameters: OV-101 column at 175°C, N carrier gas. Mass parameters: Finnagen chemical ionization; cell volts, 34.2; lens volts, —0031; EM volts, —2318; lost mass, —0640; first mass, +0099; emission mass, +00.59; collective mass, -\-00.02; ion voltage mass, +00.58; elect, energy, +0150; ion energy, +011.9, —014.6; extr. volts, +008.6. 2



392

PROCESSES

Table V. Gas-Liquid Chromatographic Retention Times o f Permethylated Compounds Compound

Time (min)

S y n t h e t i c 0-/?-D-glucopyranosyl(1—^3)-0-oC-L-fucopyranosyl FL4a

3.83 3.83

FL4a and s y n t h e t i c glucosyl fucosyl t h r e o nine were d e r i v a t i z e d and analyzed as described i n Figure 7. The permethylated compounds were detected by scanning f o r the fragments m/e 393.2, 329.1, 219.1, and 187 which may be derived as i n d i c a t e d below hexose - jfucose

threonine

219 4,

187

Table V I .

! !

393 i 329

Gas-Liquid Chromatography Retention Times o f P a r t i a l l y Methylated A l d i t o l Acetates

Parent compound

Fragment used f o r d e t e c t i o n (m/e)

Retention time (min)

FL4a Synthetic

glc-fuc-thr

262 262

3.92 3.92

FL4a Synthetic

glc-fuc-thr

264 264

4.42 4.42

P a r t i a l l y methylated a l d i t o l acetates o f FL4a and s y n t h e t i c g l u cosyl fucosyl threonine were prepared by a c i d i c methanolysis o f the methylated compound followed by treatment with t r i f l u o r o a c e t i c a c i d , reduction with sodium borodeuteride and a c e t y l a t i o n with acet i c anhydride. The p a r t i a l l y methylated a l d i t o l acetates were analyzed by GC/mass spectrometry. The major ion o f each p a r t i a l l y methylated a l d i t o l acetate was determined in a p r e l i m i n a r y mass scan and used to d e t e c t the r e s p e c t i v e p a r t i a l l y methylated a l d i t o l acetates. GC parameters: 0V-101 column at 120 C. Mass parameters: as in Figure 7.


21.

STEINER

ET AL.

Glycoconjugates

and the Transformed

Phenotype

393

262

100

r25

118 • 50

290

131 150

100

200

300

250

350

262

100

118 131 290 50

iiUJuiiuXk 100

150

I, ,1a j u , . i t . i U ,i I , , j,i,lu-i.iJi.11., m , » i 350 200 250 300

Figure 8. Total mass spectra of partially methylated alditol acetates obtained from glucosyl fucosyl threonine and from FL4a. (A) Total mass spectrum of partially methylated alditol acetate, with a major ion m / e 262 from glucosyl fucosyl threonine. (B) Total mass spectrum of partially methylated alditol acetate, with the same retention time as in (A), from FL4a. For experimental details see Table VI.



394

I

"(total ion) MH-59

"323 129^^31 (OCH )\

? 9 CH-OC-CH,

(CH3C cb) O)

3

3

C H - O - C H ^ _ _ 118 CH-0-CH CH-O-CH CH

-o-c?c

CH -0-CH 2

3

T e r m i n a l hexose ^^ l E&3l] Con A b i n d i n g t h a t i s s p e c i f i c a l l y d i s p l a c e d by m e t h y l a - D - m a n n o p y r a n o s i d e . This i s c o n f i r m e d f u r t h e r by t h e low amount o f n o n - s p e c i f i c b i n d i n g ( l e s s t h a n 15%) o f [ 5 | ] Con A i n t h e b i n d i n g a s s a y ( t a b l e I V ) . As d i s c u s s e d e a r l i e r , h e p a t o c y t e s u s p e n s i o n s o b t a i n e d by c o l l a g e n a s e p e r f u s i o n p o s s e s s e d 28% o f t h e c e l l s i n a g g r e g a t e o f 2-6 c e l l s . The amount o f [ 5 | ] Con A nbound t o p a p a i n - d i g e s t e d h e p a t o c y t e s i s n o t e n h a n c e d by an i n c r e a s e i n t h e number o f s i n g l e c e l l s i n the hepatocyte suspension f o l l o w i n g papain d i g e s t i o n s i n c e t h e a g g r e g a t e s i z e o f h e p a t o c y t e s o b t a i n e d by c o l l a n g e a s e p e r f u s i o n i s n o t a l t e r e d by p a p a i n d i g e s t i o n . These d a t a i n d i c a t e t h a t r a t h e p a t o c y t e s do n o t e x h i b i t i n c r e a s e d Con A b i n d i n g f o l l o w i n g p a p a i n d i g e s t i o n even t h o u g h t h e h e p a t o c y t e s become s i g n i f i c a n t l y more a g g l u t i n a b l e ( t a b l e l ) . T h u s , t h e s e d a t a do n o t s u p p o r t t h e h y p o t h e s i s o f i n c r e a s e d Con A - i n d u c e d a g g l u t i n a b i 1 i t y o f p a p a i n - d i g e s t e d h e p a t o c y t e s due t o e x p r e s s i o n of c r y p t i c l e c t i n b i n d i n g s i t e s f o l l o w i n g papain d i g e s t i o n . The unaltered l e c t i n binding o f the hepatocytes f o l l o w i n g papain d i g e s t i o n i s c o n s i s t e n t with the f i n d i n g that eel 1-surface, p a p a i n - l a b i l e g l y c o p e p t i d e s from e n z y m a t i c a l l y d i s p e r s e d hepat o c y t e s p o s s e s s no d e t e c t a b l e Con A r e c e p t o r a c t i v i t y , indica2

12

12



1.8 1.1 0.96 1.3

0.80 1.1

6

Specific binding Molecules Ka bound/cel1 Li ter/moles x 10" x 10" 1 3

a

D e t e r m i n e d by b i n d i n g a s s a y o f D a v i s e t a l b Data p l o t t e d a c c o r d i n g t o t h e method ofSTeck

1ntact Incubated c o n t r o l Papain-digested

Condi t i o n s or treatment

IV

(41).

0.13

0.13 0.16

8

8

N o n - s p e c i f i c binding* Molecules/cel1 x 10"

HEPATOCYTES

and W a l l a c h

BINDING OF CON A TO RAT

TABLE

5

o o w

>

2 S

r

8

O

>

3

n o

$

to

STARLING E T A L .

Normal

and Malignant

Rat Liver

Cells

Experimental Cell Research

Figure 3. Fer-Con A topographical distribution of rat hepatocytes. Electron micrograph of rat hepatocytes labeled with Fer-Con A as described in the Materials and Methods section. (A) Intact + Fer-Con A; (B) incubated control + Fer-Con A; (C) papain-digested hepatocytes + FerCon A; (D) intact hepatocytes + 0.17M methyl a-v-mannopyranoside + Fer-Con-A. X55fl00.


428


PROCESSES

Experimental Cell Research

Figure 4. Cell-surface morphology of rat hepatocytes. Electron micrograph of glutaraldehyde-fixed cells post-fixed with OsO^. Sections were stained with uranyl acetate and lead citrate. (A) Intact; (B) incubated control; (C) papain-digested hepatocytes. XH,800.


23.

STARLING E T A L .

Normal

and Malignant

Rat Liver

Cells

429

t i n g t h a t t h e s e g l y c o p e p t i d e s a r e n o t i n v o l v e d i n t h e Con A induced a g g l u t i n a t i o n o f the i n t a c t c e l l s ( t a b l e I I I ) . Con A r e c e p t o r t o p o g r a p h y . N i c o l s o n (39) o b s e r v e d t h a t F e r - C o n A r e c e p t o r s on t r a n s f o r m e d and t r y p s i n i z e d 3T3 c e l l s were i n a c l u s t e r e d t o p o g r a p h i c a l d i s t r i b u t i o n compared w i t h n o n - t r a n s f o r m e d 3T3 c e l l s . Based on t h i s o b s e r v a t i o n , N i c o l s o n p o s t u l a t e d t h a t t h e i n c r e a s e d a g g 1 u t i n a b i 1 i t y o f t r y p s i n i z e d n o n - t r a n s f o r m e d and t r a n s f o r m e d 3T3 c e l l s was due t o t h e c l u s t e r e d d i s t r i b u t i o n o f Con A r e c e p t o r s on t h e s u r f a c e o f t h e s e c e l l s w h i c h e n a b l e a g g l u t i n a t i o n t o o c c u r between c e l l - s u r f a c e a r e a s o f h i g h l e c t i n receptor density. The t o p o g r a p h i c a l d i s t r i b u t i o n s o f h e p a t o c y t e s l a b e l e d w i t h F e r - C o n A a r e shown i n F i g . 3. Altered distribution o f Con A r e c e p t o r s i t e s a f t e r p a p a i n d i g e s t i o n does n o t a p p e a r t o be r e s p o n s i b l e f o r t h e i n c r e a s e d a g g l u t i n a b i 1 i t y o f p a p a i n d i g e s t e d h e p a t o c y t e s s i n c e i n t a c t , i n c u b a t e d c o n t r o l , and p a p a i n digested hepatocytes a l Fer-Con A molecules ( f i g t o r s (5^§j7_) h a v e r e p o r t e d t h a t c l u s t e r e d d i s t r i b u t i o n s o f l e c t i n m o l e c u l e s may r e f l e c t t h e l a t e r a l m o b i l i t y o f t h e l e c t i n r e c e p t o r s r a t h e r than t h e i r i n h e r e n t t o p o g r a p h i c a l d i s t r i b u t i o n , these d a t a do n o t r u l e o u t p o s s i b l e d i f f e r e n c e s w h i c h may e x i s t i n t h e i n h e r e n t t o p o g r a p h i c a l d i s t r i b u t i o n s o f h e p a t o c y t e Con A r e c e p t o r s , a l t h o u g h s u c h d i f f e r e n c e s w o u l d n o t e x p l a i n t h e i n c r e a s e d Con A i n d u c e d a g g l u t i n a b i 1 i t y o f p a p a i n - d i g e s t e d h e p a t o c y t e s compared w i t h i n t a c t and i n c u b a t e d c o n t r o l h e p a t o c y t e s . C e l l - s u r f a c e morphology. A p l a u s i b l e e x p l a n a t i o n f o r the i n c r e a s e d Con A - i n d u c e d a g g 1 u t i n a b i 1 i t y o f p a p a i n d i g e s t e d h e p a t o c y t e s i s d e r i v e d f r o m t h e change i n s u r f a c e m o r p h o l o g y f r o m a v i l l o u s t o a smooth membrane as a r e s u l t o f p a p a i n d i g e s t i o n (fig. 4). T h i s membrane a l t e r a t i o n may a l l o w g r e a t e r a r e a s o f c o n t a c t between a g g l u t i n a t e d c e l l s t h e r e b y p e r m i t t i n g more e x tensive l e c t i n bridging to occur. T h i s m o r p h o l o g i c a l change w i l l also result i n a h i g h e r s u r f a c e d e n s i t y o f Con A bound r e c e p t o r s on p a p a i n - d i g e s t e d h e p a t o c y t e s as compared t o i n c u b a t e d c o n t r o l h e p a t o c y t e s a l t h o u g h t h i s i n c r e a s e d r e c e p t o r d e n s i t y may n o t account e n t i r e l y f o r the increased a g g l u t i n a b i 1 i t y o f papain d i g e s t e d h e p a t o c y t e s (3j0 • The a b o v e c o n c l u s i o n s a r e f u r t h e r s u p p o r t e d by t h e o b s e r v a t i o n t h a t h i g h Con A - a n d WGA-induced a g g l u t i n a t i o n o f human l y m p h o b l a s t s and normal human p e r i p h e r a l l y m p h o c y t e s was c o r r e l a t e d w i t h a smooth c e l l s u r f a c e w h e r e a s low Con A and WGA a g g l u t i n a b i 1 i t y was c o r r e l a t e d w i t h a v i l l o u s s u r f a c e morphology ( 4 0 ) . The a b o v e d a t a s u g g e s t t h a t t h e r e a r e s i g n i f i c a n t structural d i f f e r e n c e s i n t h e c e l l - s u r f a c e g l y c o p r o t e i n s o f normal and m a l i g n a n t r a t l i v e r c e l l s and t h a t v i a b l e h e p a t o c y t e s u s p e n s i o n s o f f e r a v a l u a b l e in v i v o system f o r s t u d y i n g c e l l - s u r f a c e p r o p e r t i e s a s s o c i a t e d w i t h normal c e l l s .


430


ACKNOWLEDGEMENTS The authors express appreciation to Dr. Max Schlamowitz and Dr. Kay Hillman for their assistance in the Con A iodination procedure. This work was supported by grants from NCI (CA-11710), the Robert A. Welch Foundation (G-354), the Paul and Mary Haas Foundation and the George and Mary Josephine Hamman Foundation. J.J.S. was recipient of a Rosalie B. Hite predoctoral fellowship in cancer research. Portions of the tables and figures from references 19 and 31 are reproduced by permission of Academic Press. Literature Ci ted 1. Emmelot, P. (1973) Eur. J. Cancer 9, 319. 2. Nicolson, G. L. (1974) Int. Rev. Cytol. 39, 89. 3. Burger, M. M. (1969) Proc. Natl. Acad. Sci. US 62, 944. 4. Burger, M. M. (1970) Permeability and Function of Biological Membranes (ed. L. Bolis et al.), p. 107. North-Holland, Amsterdam. 5. Nicolson, G. L. (1973 6. Nicolson, G. L. (1974 Cells (ed. B. Clarkson and R. Baserga), p. 251. Cold Spring Harbor, New York. 7. Rosenblith, J. A., Ukena, T. E., Yin, H. H., Berlin, R. D., and Karnovsky, M. J. (1973) Proc. Natl. Acad. Sci. US 70, 1625. 8. Rutishauser, U. and Sachs, L. (1975) J. Cell Biol. 66, 76. 9. Collard, J. G. and Temmink, J. H. M. (1975) J. Cell Sci. 19, 21. 10. Jansons, V. K., Sakamoto, C. K., and Burger, M. M. (1973). Biochim. Biophys. Acta 291, 136. 11. Walborg, E. F., Jr., Wray, V. P., Smith, D. F., and Neri, G. (1975) Immunological Aspects of Neoplasia, p. 123. Williams and Wilkins Co., Baltimore, Md. 12. Neri, G., Smith, D. F., Gilliam, E. B., and Walborg, E. F., Jr. (1974) Arch. Biochem. Biophys. 165, 323. 13. Smith, D. F., Walborg, E. F., Jr., and Chang, J. P. (1970) Cancer Res. 30, 2306. 14. Smith, D. F., Neri, G., and Walborg, E. F., Jr. (1973) Biochem. 12, 2111. 15. Becker, F. F. (1974) Proc. Natl. Acad. Sci. US 71, 4307. 16. Schreiber, G. and Schreiber, M. (1972) Sub-cell. Biochem. 2, 321. 17. Jacob, S. T. and Bhargava, P. M. (1962) Exp. Cell Res. 27, 453. 18. Bonney, R. J. (1974) In Vitro 10, 130. 19. Starling, J. J., Capetillo, S. C., Neri, G., and Walborg, E. F., Jr. (1977) Exp. Cell Res. 104, 177. 20. Phillips, H. J. (1973) Tissue Culture: Methods and Applications (ed. P. F. Kruse and M. K. Patterson) p. 406. Academic Press, New York. 21. Walborg, E. F., Jr. Lantz, R. S., and Wray, V. P. (1969) Cancer Res. 29, 2034.


23.

22. 23. 24. 25.

26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42.

STARLING ET AL.

431 Normal and Malignant Rat Liver Cells

Davis, E. M., Starling, J. J., and Walborg, E. F., Jr. (1976) Exp. Cell Res. 99, 37. Wray, V. P. and Walborg, E. F.,Jr. (1971) Cancer Res. 31, 2079. Hanks, J. H. and Wallace, R. E. (1949) Proc. Soc. Exp. Biol. Med. 71, 196. Leseney, A. M., Thomas, M. W., O'Neil, P. A., and Walborg, E. F., Jr. (1974) Méthodologie de la Structure et du Métabolisme des Glycoconjugues. Actes du Colloque Internationaux n 221 due Centre National de la Recherche Scientifique, vol. 2, p. 859. Editiones du CNRS, Paris. Aminoff, D. (1961) Biochem. J. 81, 384. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F. (1956) Anal. Chem. 28, 350. Gatt, R. and Berman, E. R. (1966) Anal. Biochem. 15, 167. Sonoda, S. and Schlamowitz Agrawal, B. B. L 96, 23C. Starling, J. J., Hixson, D. C., Davis, E. M., and Walborg, E. F., Jr. (1977) Exp. Cell Res. 104, 165. Davis, E. M., Tsay, D. D., Schlamowitz, M., and Walborg, E. F., Jr. (1977) Anal. Biochem. 80, 416. Seglen, P. 0. (1972) Exp. Cell Res. 74, 450. Warren, L. (1969) Current Topics in Developmental Biology (ed. A. A. Moscona and A. Monroy) vol. 4, p. 197. Academic Press, New York. Walborg, E. F., Jr., Davis, E. M., Gilliam, E. B., Smith, D. F., and Neri, G. (1975) Cellular Membranes and Tumor Cell Behavior, p. 337. Williams and Wilkins, Baltimore, Md. Cline, M. J. and Livingston, D. C. (1971) Nature New Biol. 232, 155. Ozanne, B. and Sambrook, J. (1971) Nature New Biol. 232, 156. Noonan, K. D. and Burger, M. M. (1973) J. Cell Biol. 59, 134. Nicolson, G. L. (1971) Nature New Biol. 233, 244. Temmink, J. H. M., Collard, J. G., Roosien, J. and van den Bosch, J. F. (1976) J. Cell Sci. 21, 563. Steck, T. L. and Wallach, D. F. H. (1965) Biochim. Biophys. Acta 97, 510. Montgomery, R. and Lee, Y. C. (1965) Biochem. 4, 566.

RECEIVED

March 30, 1978.


24 Membrane Glycoproteins of Rat Mammary Gland and Its Metastasizing and Nonmetastasizing Tumors KERMIT L. CARRAWAY, JOHN W. HUGGINS, ANNE P. SHERBLOM, ROBERT W. CHESNUT, ROBERT L. BUCK, SUSAN P. HOWARD, CHARLOTTE L. OWNBY, and CORALIE A. CAROTHERS CARRAWAY Department of Biochemistry, Oklahoma State University, Stillwater, OK 74074

Glycoproteins are ubiquitous components of animal cell surfaces (1) and are generally believed to play important roles in neoplastic diseases (2). Glycoprotein differences between nonmalignant and malignant cells have been demonstrated by such diverse techniques as lectin agglutination (3), glycopeptide analyses (4), gel electrophoresis (5) and enzymatic cell surface labeling (6). The origin of these differences and the roles of the glycoproteins in most cellular phenomena are still unknown. One intriguing possibility is that cell surface glycoproteins may be involved in the mechanism by which tumor cells escape destruction by the immune system of the host. Two very different hypotheses have been presented to explain the role of glycoproteins in this escape from immune surveillance. The first involves the shedding of antigens from the cell surface. These antigens combine with circulating antibodies to form complexes which block antibody-mediated destruction of the tumor cells (7). Kim et al. (8) have extended this hypothesis to suggest that glycoprotein shedding in mammary tumors is related to their metastatic potential. Using a series of mouse mammary tumors selected for variations in ability to metastasize, they found an inverse correlation between metastasis and three parameters: 5'-nucleotidase activity, cell surface glycocalyx staining and immunogenicity of the tumors in rabbits. It was suggested that the loss of the cell surface constituents was due to shedding, but the mechanism was not specified. A second means by which glycoproteins might be involved in "protection" of the tumor cells was suggested by studies on the TA3 mouse mammary tumor. Two sublines of this tumor which are different in their immunological properties have been isolated. The TA3-St subline grows only in the syngeneic host, but the TA3-Ha subline is able to cross histocompatibility barriers (9). This loss of strain specificity is accompanied by a greatly increased amount of a large sialoglycoprotein (10) named 0-8412-0452-7/78/47-080-432$05.00/0 © 1978 American Chemical Society In Glycoproteins and Glycolipids in Disease Processes; Walborg, E.; ACS Symposium Series; American Chemical Society: Washington, DC, 1978.

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e p i g l y c a n i n . I t was hypothesized that the g l y c o p r o t e i n i n the TA3-Ha s u b l i n e "covers" the h i s t o c o m p a t i b i l i t y antigens at the c e l l surface (11), preventing t h e i r expression and thereby prev e n t i n g the r e c o g n i t i o n of these c e l l s as f o r e i g n to the host. Our own work has been concerned with the c e l l surface g l y coproteins of r a t mammary tumors and the r e l a t i o n s h i p of these g l y c o p r o t e i n s to the p r o p e r t i e s of the tumor c e l l s . In t h i s p r e s e n t a t i o n we s h a l l focus on three questions. 1) Is there a necessary r e l a t i o n s h i p between the expression of c e l l surface g l y c o p r o t e i n s and metastasis of mammary tumors as suggested by Kim et a l . (8)? 2) I s there a r e l a t i o n s h i p between the "form" or environment of the tumor and the c e l l surface g l y c o p r o t e i n s i t expresses? 3) How are the c e l l s u r f a c e p r o p e r t i e s of tumors r e l a t e d to t h e i r b i o l o g i c a l a c t i v i t i e s as tumors? Comparison of membrane g l y c o p r o t e i n s from normal mammary gland, a nonmetastasizin (R3230 tumor (13762). To determine whethe y expression of c e l l surface g l y c o p r o t e i n between nonmetastasizing and m e t a s t a s i z i n g mammary tumors, we have examined the R3230 AC and 13762 r a t mammary adenocarcinomas, both grown i n the F i s c h e r 344 r a t . The R3230 AC i s a slow-growing tumor d e r i v e d from a spontaneous carcinoma (12). I t i s hormonally responsive and e x h i b i t s l i t t l e or no m e t a s t a t i c c a p a c i t y under the c o n d i t i o n s used i n our s t u d i e s . The 13762 r a t mammary adenocarcinoma i s a methylcholanthrene-induced s o l i d tumor (13) which i s hormonally responsive and metastasizes widely and h e a v i l y . For comparison of c e l l surface g l y c o p r o t e i n s i t i s necessary to p u r i f y plasma membranes from the t i s s u e samples. P u r i f i c a t i o n of plasma membrane and other s u b c e l l u l a r t i s s u e f r a c t i o n s i s d i f f i c u l t f o r s e v e r a l reasons (14). D i s r u p t i o n of the c e l l s i n the t i s s u e almost n e c e s s a r i l y fragments the plasma membrane. Fragmentation i s not uniform and may r e s u l t i n a heterogeneous d i s t r i b u t i o n of membrane fragments, both i n terms of s i z e and of p h y s i c a l and f u n c t i o n a l a t t r i b u t e s . This complicates the s e p a r a t i o n of p a r t i c u l a r morphological u n i t s because of the v a r i a t i o n of the s i z e s , d e n s i t i e s , compositions and other f a c t o r s upon which s e p a r a t i o n of fragments i s based. Identif i c a t i o n of morphological u n i t s i s a l s o complicated by fragment a t i o n , which destroys morphological c h a r a c t e r i s t i c s and reduces membranes t o sheets or v e s i c l e s . In a d d i t i o n to morphological c r i t e r i a membrane f u n c t i o n a l a c t i v i t i e s (enzymes, antigens) must be used f o r i d e n t i f i c a t i o n of s u b c e l l u l a r components. T h i s comb i n a t i o n of heterogeneity and l o s s of morphological i d e n t i t y makes the p r e p a r a t i o n of coherent, h i g h l y p u r i f i e d s u b c e l l u l a r components very d i f f i c u l t . Most of the work i n t h i s area has been performed on l i v e r . Plasma membranes have been p u r i f i e d by a number of methods, most of which tend to give membranes derived from the b i l e c a n a l i c u l a r regions (15). Mammary t i s s u e presents i t s own p a r t i c u l a r problems.


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Because of the presence of l a r g e amounts of connective t i s s u e the e l a s t i c i t y of the t i s s u e makes homogenization d i f f i c u l t . The procedure which g i v e s the best r e s u l t s f o r normal l a c t a t i n g mammary t i s s u e i n v o l v e s vigorous homogenization i n a S o r v a l l Omni-mixer and i s o l a t i o n of a microsomal f r a c t i o n by d i f f e r e n t i a l c e n t r i f u g a t i o n (5). Microsomes are f u r t h e r f r a c t i o n a t e d by f l o t a t i o n through a sucrose d e n s i t y gradient ( F i g . 1 ) . 5'-Nucleotidase i s used as a marker f o r p r e l i m i n a r y assessment of plasma membrane p u r i f i c a t i o n , s i n c e t h i s enzyme has been shown to be present predominantly i n the plasma membrane of a number of c e l l types (14), i n c l u d i n g mammary gland and mammary tumors (16, 17). S i g n i f i c a n t p u r i f i c a t i o n of 5'-nucleotidase (>25-fold) was achieved i n both of the l i g h t e r f r a c t i o n s ( F and F ) of the discontinuous g r a d i e n t , and there i s a p a r a l l e l p u r i f i c a t i o n of Na , K -ATPase (5), another plasma membrane marker. S u c c i n i c dehydrogenase (a m i t o c h o n d r i a l enzyme) i s markedly reduced, but plasmic r e t i c u l u m marke t o s y l t r a n s f e r a s e i s s i g n i f i c a n t l y concentrated. The l a s t observation suggests G o l g i apparatus contamination (16). Further p u r i f i c a t i o n i s achieved by t r e a t i n g F i and F f r a c t i o n s s e p a r a t e l y with d i g i t o n i n and s u b j e c t i n g these to r e f l o t a t i o n on a s i m i l a r discontinuous gradient c o n t a i n i n g d i g i t o n i n . D i g i t o n i n complexes with c h o l e s t e r o l and would be expected t o s h i f t the c h o l e s t e r o l - e n r i c h e d plasma membrane f r a c t i o n to a higher d e n s i t y (18). Cholesterol-poor f r a c t i o n s such as G o l g i fragments should be unchanged i n d e n s i t y . By t h i s procedure a p o r t i o n of the d i g i t o n i n - t r e a t e d F i s s h i f t e d to F x D F , which shows a 3 - f o l d increase i n n u c l e o t i d a s e a c t i v i t y and i s devoid of g a l a c t o s y l t r a n s f e r a s e and NADPH-cytochrome c reductase (19). C l e a r l y i t has the c h a r a c t e r i s t i c s expected of a h i g h l y p u r i f i e d plasma membrane f r a c t i o n . The F f r a c t i o n behaves d i f f e r e n t l y . Part of i t r e e q u i l i b r a t e s to the Fj. p o s i t i o n upon r e f l o t a t i o n . When t r e a t e d with d i g i t o n i n , part of F i s s h i f t e d to a higher d e n s i t y (F DF ). F D F i s enriched i n 5'-nucleotidase, c h o l e s t e r o l , s i a l i c a c i d and g a l a c t o s y l t r a n s f e r a s e , p r o p e r t i e s suggesting that t h i s s u b f r a c t i o n comes from a plasma membrane c o n t a i n i n g galactosyltransferase. These procedures have been used with s i m i l a r r e s u l t s to p u r i f y plasma membranes from the s o l i d tumors. As shown i n Table I, the 5'-nucleotidase of the m e t a s t a s i z i n g 13762 tumor i s not g r e a t l y diminished when compared t o the nonmetastasizing R3230 AC. S i a l i c a c i d analyses (20) of membrane f r a c t i o n s from these two tumors a l s o f a i l to show any s i g n i f i c a n t d e p l e t i o n i n the m e t a s t a s i z i n g tumor (Table I I ) . S i a l o g l y c o p r o t e i n s a r e analyzed by polyacrylamide g e l e l e c t r o p h o r e s i s i n sodium dodecyl s u l f a t e (SDS). Whereas normal mammary membranes show three major c l a s s e s of g l y c o p r o t e i n s by t h i s technique ( F i g . 2), membranes from the tumor c e l l s have a x

2

+

+

2

x

3

2

2

2

3

2

3


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Figure 1. Schematic for membrane preparations from normal lactating mammary tissue and rat mammary tumor tissue samples

1 F.

I

A

•

A

F.DF,

i V XL

1^.

A

FJDFJ

/ MIGRATION DISTANCE (cm)

Figure 2. Membrane glycoproteins of normal lactating rat mammary tissue. Membrane fractions were prepared as previously described (5,19) and analyzed by electrophoresis on poly aery lamide gels in the presence of SDS (5). Gels were stained by the periodate-Schiff procedure and scanned on a Gilford spectrophotometer with a linear transport device.



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TABLE I Nucleotidase A c t i v i t i e s of Mammary Tumor F r a c t i o n s Fraction

R3230 AC

13762

ymoles/hr•mg p r o t e i n Homogenate FiDF F DF 2

3

3

3.5

4.2

95

126

64

61

TABLE I I S i a l i c A c i d Content Homogenate

F

x

F

2

ug/mg p r o t e i n R3230 AC 13762

0.7 0.4

13 19

11 16

f o u r t h c l a s s of g l y c o p r o t e i n s ( F i g . 3) with an apparently lower molecular weight than the other three (5). G l y c o p r o t e i n patterns from the two tumors are very s i m i l a r , though both have patterns more complex than those of membranes from the normal gland. I t i s not p o s s i b l e to a s c e r t a i n a t present whether these s i m i l a r e l e c t r o p h o r e t i c c l a s s e s are the same molecular s p e c i e s , but i t should be f e a s i b l e to o b t a i n a t l e a s t a p a r t i a l answer to that question using immunological techniques. C l e a r l y the d e p l e t i o n of c e l l surface g l y c o p r o t e i n s i s not a u n i v e r s a l c h a r a c t e r i s t i c of plasma membranes i s o l a t e d from m e t a s t a s i z i n g mammary tumors. T h i s does not imply that these tumors do not shed t h e i r surface c o n s t i t u e n t s , nor does i t i n d i c a t e that shedding i s unimportant to metastasis. The conc e n t r a t i o n of any component i n the c e l l r e f l e c t s a balance between s y n t h e t i c and degradative pathways. Since these need to be i n v e s t i g a t e d more c a r e f u l l y , we have examined a s c i t e s c e l l s derived from the m e t a s t a t i c 13762 tumor, because these c e l l s are more e a s i l y manipulated. Glycoproteins of the a s c i t e s form of the 13762 mammary adeno care inoma. The s o l i d 13762 mammary adenocarcinoma has been converted i n t o t r a n s p l a n t a b l e , m e t a s t a t i c a s c i t e s forms MAT-A, MAT-B and


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MAT-C, which we obtained from the Mason Research Laboratory, Worchester, Mass. To serve as models f o r g l y c o p r o t e i n behavior i n the s o l i d tumor, these a s c i t e s tumors must meet two c r i t e r i a . 1) The tumor s u b l i n e s and t h e i r c e l l surface p r o p e r t i e s must be s t a b l e to i n v i v o passage over the p e r i o d of experimentation. 2) The g l y c o p r o t e i n s of the a s c i t e s tumor must be the same as those of the s o l i d tumor. The question of the s t a b i l i t y of the s u b l i n e s proved to be an i n t e r e s t i n g one. During passage of MAT-B and MAT-C s u b l i n e s i n v i v o at weekly i n t e r v a l s over about s i x months, we noted that both were converted to v a r i a n t forms. We s h a l l term these MAT-B1 and MAT-C1 and w i l l discuss t h e i r a l t e r e d c e l l surface p r o p e r t i e s i n a l a t e r s e c t i o n . We suspect that the MAT-A subl i n e a l s o undergoes changes with prolonged passage i n v i v o , but we do not have an appropriate marker to demonstrate these changes. We are maintaining the MAT-B1 and MAT-C1 tumors by t r a n s p l a n t a t i o n and as comparisons. To analyze c e l l surface g l y c o p r o t e i n s of the a s c i t e s tumors, membranes were prepared by a m o d i f i c a t i o n of the method of Warren j2t a l . (21), using z i n c to " s t a b i l i z e " the c e l l surface membranes. Polyacrylamide g e l e l e c t r o p h o r e s i s of SDSs o l u b i l i z e d membranes was used f o r a n a l y s i s of s i a l o g l y c o p r o t e i n s . A l l a s c i t e s tumor s u b l i n e s showed a major band which b a r e l y penetrated the g e l s , i l l u s t r a t e d i n F i g . 4 f o r the MAT-B1 and MAT-C1 s u b l i n e s . Although SDS e l e c t r o p h o r e s i s i s u n r e l i a b l e f o r molecular weight determinations on g l y c o p r o t e i n s , g e l f i l t r a t i o n experiments a l s o suggest that t h i s s i a l o g l y c o p r o t e i n i s a l a r g e molecule. This g l y c o p r o t e i n i s observed as the major c e l l surface component when i n t a c t c e l l s are l a b e l e d with l a c t o peroxidase and I or with periodate and H-borohydride. These r e s u l t s i n d i c a t e that g l y c o p r o t e i n s i n the s o l i d tumor d i f f e r from those i n a s c i t e s l i n e s derived from i t . However, i t should be noted that the membrane preparations f o r s o l i d and a s c i t e s tumors are d i f f e r e n t . Perhaps the more s t r i n g e n t c o n d i t i o n s of c e l l d i s r u p t i o n used i n s o l i d tumor membrane p r e p a r a t i o n cause breakdown of a l a r g e g l y c o p r o t e i n to give m u l t i p l e smaller g l y c o p r o t e i n s . Two types of experiments argue against t h i s p o s s i b i l i t y . F i r s t , attempts to demonstrate a l a r g e g l y c o p r o t e i n i n the s o l i d 13762 tumor by p e r i o d a t e borohydride l a b e l i n g of the s o l u b i l i z e d t i s s u e followed by SDS e l e c t r o p h o r e s i s were negative. Second, milk f a t globule membranes contain the same three major s i a l o g l y c o p r o t e i n c l a s s e s as the normal mammary gland and s o l i d tumors. Since the milk f a t globule has not been subjected to mechanical d i s r u p t i o n , i t i s u n l i k e l y that the s i m i l a r c l a s s e s of g l y c o p r o t e i n s of normal mammary and the s o l i d tumors are products of the c e l l d i s r u p t i o n . What i s the o r i g i n of t h i s d i f f e r e n c e i n g l y c o p r o t e i n s between s o l i d and a s c i t e s forms of the tumor? Probably i t i s a response to the a l t e r e d environment of the a s c i t e s c e l l s , but 1 2 5

3


438


Mat - Bi IM-

MIGRATION DISTANCE (cm)

Figure 4. Membrane glycoproteins of the rat mammary adenocarcinoma MAT-B1 and MAT-CI sublines. Membranes were prepared essentially as described previously (17) for MAT-A cells. Electrophoresis and staining were performed as indicated in Figure 2. About 50% more protein was applied to the gel for the MAT-B1 sample.


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the mechanism of change i s u n c l e a r . One p o s s i b i l i t y i s an a l t e r e d gene expression r e s u l t i n g i n the synthesis of d i f f e r e n t p r o t e i n s . A l t e r n a t i v e l y , the change could r e f l e c t an a l t e r e d p o s t - t r a n s l a t i o n a l event such as an a l t e r e d a d d i t i o n of carbohydrate or c r o s s l i n k i n g of the p r o t e i n s . Although we cannot answer these questions a t present, i t should be p o s s i b l e to o b t a i n answers by biochemical and immunological comparisons of g l y c o p r o t e i n s from s o l i d and a s c i t e s tumors. Surface p r o p e r t i e s of the 13762 a s c i t e s tumor c e l l s . The sublines of the 13762 a s c i t e s carcinoma d i f f e r not only from the s o l i d tumor but a l s o from each other. The d i f f e r e n c e i n membrane s i a l o g l y c o p r o t e i n content between MAT-B1 and MAT-C1 c e l l s i s i l l u s t r a t e d i n F i g . 4. The a c t u a l d i f f e r e n c e between the two i s greater than that shown because of the amounts of membranes loaded onto the g e l s . C e l l surface s i a l i c a c i d r e l e a s e d by t r y p s i n o from the MAT-C1 c e l l s tha t o t a l c e l l s i a l i c a c i d i s only about 40% g r e a t e r . Labeling s t u d i e s w i t h lactoperoxidase and I or with periodate and Hborohydride a l s o show more major s i a l o g l y c o p r o t e i n i n the MAT-C1 cells. The most s t r i k i n g d i f f e r e n c e between these s u b l i n e s i s t h e i r surface morphology, MAT-C1 c e l l s e x h i b i t i n g a very unusual surface s t r u c t u r e . T h e i r surface i s densely covered with m i c r o v i l l i extending from the c e l l body i n t o h i g h l y branched s t r u c t u r e s ( F i g . 5), whereas MAT-B1 c e l l s have a more normal appearance with fewer m i c r o v i l l i , a l l apparently unbranched (Fig. 6). MAT-B1 and CI c e l l s a l s o have d i f f e r e n t c e l l surface p r o p e r t i e s (Table I I I ) . When t r e a t e d with Concanavalin A (Con A), the MAT-B1 c e l l s r e a d i l y a g g l u t i n a t e . Under the appropriate c o n d i t i o n s of temperature and l e c t i n concentration these B l c e l l s a l s o form patches and caps. The MAT-C1 c e l l s do not respond to the l e c t i n treatment under the same c o n d i t i o n s , i n d i c a t i n g a r e s t r i c t i o n of the m o b i l i t y of the c e l l surface l e c t i n receptor s i t e s . I t i s a l s o noteworthy that the MAT-B1 and MAT-C1 c e l l s have d i f f e r e n t 5'-nucleotidase a c t i v i t i e s . This a c t i v i t y i s e s s e n t i a l l y undetectable i n B l c e l l s , even with a h i g h l y s e n s i t i v e radiochemical assay. In view of the l a r g e d i f f e r e n c e s i n c e l l surface p r o p e r t i e s one might expect s i g n i f i c a n t b i o l o g i c a l d i f f e r e n c e s between the B l and CI tumors. However, we have not found t h i s to be t r u e . The growth and k i l l i n g r a t e s of the two tumors are about the same. Tests f o r s t r a i n s p e c i f i c i t y of the tumors have not shown pronounced d i f f e r e n c e s , and p r e l i m i n a r y s t u d i e s suggest that both s u b l i n e s a r e m e t a s t a t i c . Thus i t appears that d i f f e r e n c e s i n c e l l surface p r o p e r t i e s a r e not n e c e s s a r i l y r e f l e c t e d i n the gross p r o p e r t i e s of the tumors. I t i s i n t e r e s t i n g that the p r o p e r t i e s of the MAT-B1 c e l l s f i t the c r i t e r i a o f Kim et aJL. (8) f o r a m e t a s t a t i c tumor under1 2 5


3

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Figure 5. Scanning electron micrograph of MAT-C1 cells. Cells were removed from the animal, fixed in 3% glutaraldehyde in 0.1M sucrose-OJM cacodylate, postfixed with 2% osmium tetr oxide, dehydrated in ethanol, dried in a Polar on critical point dryer, coated with gold/palladium, and examined in a JEOL JSM-35 Scanning Electron Microscope. Note the extensive microvilli, whose branched structures can be readily observed at the periphery of the cell. Magnification, 8160.


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Figure 6.

Membrane

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of Rat Gland

441

Scanning electron micrograph of MAT-B1 cells prepared as noted for Figure 5. Magnification, 8840.


442


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TABLE I I I C e l l Surface P r o p e r t i e s of MAT-B1 and MAT-C1 Mammary A s c i t e s C e l l s

Property Agglutination Patching and Capping 5 -Nucleotidase, umoles/hr*mg p r o t e i n

MAT-B1

MAT-C1

-HHH-H-H-

-

0

2.0

1

going c e l l surface shedding show s t r i k i n g s i m i l a r i t i e two s u b l i n e s , derived from the same s o l i d tumor, may represent two d i f f e r e n t modes of escaping the immune system of the host. Discussion The r e s u l t s presented permit us to supply t e n t a t i v e answers to the questions r a i s e d at the beginning of t h i s a r t i c l e . 1) There does not appear to be a necessary c o r r e l a t i o n i n mammary tumors between metastasis and g l y c o p r o t e i n shedding. However, both g l y c o p r o t e i n expression and metastasis are very complex phenomena, so t h e i r r e l a t i o n s h i p s may a l s o be complex. 2) Tumor environment apparently a f f e c t s g l y c o p r o t e i n expression, as i n d i c a t e d by the d i f f e r e n c e s between the a s c i t e s and s o l i d tumors. These r e s u l t s p o i n t out the problems i n v o l v e d i n e x t r a p o l a t i n g s t u d i e s on a s c i t e s c e l l s to conclusions about s o l i d tumors. However, the s t u d i e s on a s c i t e s c e l l s have t h e i r own inherent v a l u e . In the process of metastasis i n d i v i d u a l tumor c e l l s or small aggregates d i s s o c i a t e from the primary tumor i n t o the body f l u i d s . The s i m i l a r i t y of these d i s s o c i a t e d c e l l s to a s c i t e s c e l l s should be obvious. Since tumors l o s e many c e l l s , the c a p a c i t y of a tumor f o r metastasis must depend on the a b i l i t y of the d i s s o c i a t e d c e l l s to s u r v i v e i n the c i r c u l a t i o n and become e s t a b l i s h e d at a new s i t e . The a b i l i t y of a tumor c e l l to a l t e r i t s surface r e a d i l y i n response to a change i n environment might be a u s e f u l a t t r i b u t e f o r the m e t a s t a t i c tumor. Since the 13762 tumor has t h i s c a p a b i l i t y , f u r t h e r i n v e s t i g a t i o n of the r e l a t i o n s h i p s between the g l y c o p r o t e i n s of the s o l i d and a s c i t e s tumors may provide i n s i g h t i n t o the requirements f o r such changes. 3) The s t u d i e s on the MAT-B1 and MAT-C1 c e l l s c l e a r l y show that c e l l s with very d i f f e r e n t c e l l surface p r o p e r t i e s can be very s i m i l a r b i o l o g i c a l l y . We suspect that these r e s u l t s r e f l e c t the f a c t that d i f f e r e n t tumor c e l l s may use d i f f e r e n t ways of s u r v i v i n g the defense system of the


24.

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ET

AL.

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Glycoproteins

of

Rat

Gland

443

host. However, i t i s a l s o p o s s i b l e that the mechanisms of tumor s u r v i v a l are too s u b t l e to be detected by the methodologies used i n our s t u d i e s . The s t u d i e s presented here have r a i s e d more questions than they have answered. The d i f f e r e n c e s between the MAT-B1 and MAT-C1 c e l l s are p a r t i c u l a r l y i n t r i g u i n g . The abundance and complexity of the m i c r o v i l l i at the surface of the CI c e l l i n d i c a t e the presence of an extensive submembrane c y t o s k e l e t a l network, s i n c e the m i c r o v i l l i c o n t a i n m i c r o f i l a m e n t s . One explanation f o r the extensive, branched m i c r o v i l l i i s that there i s an imbalance i n the turnover of plasma membrane compared to c y t o s k e l e t a l m a t e r i a l . However, these c e l l s a l s o cont a i n an excess of surface s i a l o g l y c o p r o t e i n . Is t h i s a c o i n c i d e n t or a r e s u l t of some type of coupling between the submembrane c y t o s k e l e t o n and extramembrane g l y c o p r o t e i n ? This question assumes greate importanc i vie f th p o s s i b l r o l e s of c e l l surface c e l l u l a r responses. Ou preliminary s i a l o g l y c o p r o t e i n i s not s o l e l y r e s p o n s i b l e f o r i n h i b i t i o n of Con A receptor movements i n the CI c e l l s . There a l s o appears to be a c y t o s k e l e t a l involvement. Regardless of the exact mechanism by which receptor movement i s repressed, the absence of m o b i l i t y may be an important f a c e t i n escape from immune d e s t r u c t i o n . I f m o b i l i t y of the antigens of the target c e l l i s a p r e r e q u i s i t e f o r i t s d e s t r u c t i o n , the immobilization of the e n t i r e surface would be an e f f e c t i v e defense. T h i s might a l s o be considered as an a l t e r n a t i v e mechanism f o r the l a c k of s t r a i n s p e c i f i c i t y of the TA3-HA c e l l s . The decreased nucleotidase and c e l l surface g l y c o p r o t e i n s of the MAT-B1 c e l l s are a l s o i n t r i g u i n g . Can t h i s r e a l l y be explained by a "shedding" mechanism? Or has the c a p a c i t y to synthesize these molecules been l o s t ? I t i s necessary to examine the dynamics of the surfaces of these c e l l s i n order to understand t h e i r behavior. Such s t u d i e s are complex but could provide i n s i g h t i n t o the r e l a t i o n s h i p of c e l l s t r u c t u r e and n e o p l a s t i c behavior. In attempting to understand tumor c e l l behavior i n r e l a t i o n s h i p to i t s c e l l surface p r o p e r t i e s , an important concept i s that tumor c e l l s appear h i g h l y adaptable. Perhaps t h i s a d a p t a b i l i t y i s the key to the success of the tumor i n s u r v i v i n g a h o s t i l e environment and i s the cause of much of the f r u s t r a t i o n i n t r y i n g to e s t a b l i s h d e f i n i t e r e l a t i o n s h i p s between cancer and p a r t i c u l a r c e l l p r o p e r t i e s . This a d a p t a b i l i t y i s not l i m i t e d to c e l l surface p r o p e r t i e s , as evidenced by the a b i l i t y of tumors to develop drug r e s i s t a n c e . However, the c o n t r o l of the c e l l surface would a s s i s t the c e l l i n meeting the r i g o r s of i t s environment. Studies on c e l l surface v a r i a t i o n s should help us understand the r e l a t i o n s h i p of c e l l surface to tumor s u r v i v a l and the more general question of a d a p t a b i l i t y of tumor c e l l s .


444


Acknowledgements This is contribution P-490 of the Oklahoma Agricultural Experiment Station, Oklahoma State University, Stillwater, Oklahoma. This research was conducted in cooperation with the USDA, Agricultural Research Service, Southern Region and supported by the National Cancer Institute (NO-l-CB-33910 and CA 19985), the American Cancer Society (BC-246), a Presidential Challenge Grant from Oklahoma State University and the Oklahoma Agricultural Experiment Station. Literature Cited 1. Hughes, R. C. (1976) in Membrane Glycoproteins, Butterworths, London. 2. Emmelot, P. (1973) Eur J Cancer 9 319-333 3. Nicolson, G. L. 4. Buck, C. A. Glick Biochemistry 9, 4567-4576. 5. Shin, B. C., Ebner, K. E . , Hudson, B. G., and Carraway, K. L. (1975) Cancer Res. 35, 1135-1140. 6. Hynes, R. 0. (1974) Cell 1, 147-156. 7. Hellström, K. E . , and Helström, I. (1974) Adv. Immunol. 18, 209-277. 8. Kim, U., Baumler, A., Carruthers, C., and Bielat, K. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 1012-1016. 9. Friberg, S., Jr. (1972) J. Natl. Cancer Inst. 48, 14631476. 10. Codington, J. F., Sanford, B. H., and Jeanloz, R. W. (1973) J . Natl. Cancer Inst. 51, 585-591. 11. Miller, S. C., Hay, E. D., and Codington, J. F. (1977) J. Cell Biol. 72, 511-529. 12. Hilf, R., Michel, I., and Bell, C. (1967) Recent Prog. Hormone Res. 23, 229-289. 13. Segaloff, A. (1966) Recent Prog. Hormone Res. 22, 351-379. 14. DePierre, J. W., and Karnovsky, M. L. (1973) J. Cell Biol. 56, 275-303. 15. Steck, T. L . , and Wallach, D. F. H. (1970) Methods in Cancer Res. 5, 93-153. 16. Keenan, T. W., Morré, D. J., and Huang, C. M. (1974) in Lactation: A Comprehensive Treatise, (B. L. Larson and U. R. Smith, eds.), Vol. 2, Academic Press, New York, pp. 191-233. 17. Carraway, K. L . , Fogle, D. D., Chesnut, R. W. Huggins, J. W., and Carraway, C. A. C. (1976) J. Biol. Chem. 251, 6173-6178. 18. Beaufay, H., and Amar-Costesec, A. (1976) Methods in Membrane Biol. 6, 1-100. 19. Huggins, J . W., and Carraway, K. L. (1976) J. Supramol. Struct. 5, 59-63.


24. CARRAWAY ET AL. 20. 21.

Membrane Glycoproteins of Rat Gland 445

Warren, L. (1959) J. Biol. Chem. 234, 1971-1975. Warren, L . , Glick, M. C., and Nass, M. K. (1966) J. Cell. Physiol. 68, 269-287.

RECEIVED

March 13, 1978.


AUTHOR INDEX Names and numbers in bold are authors and first pages, respectively, of complete chapters. Numbers in parentheses are reference numbers which indicate that an author's work is referred to, followed by pages of that referral. Numbers in italics show the page on which the complete reference is listed.

Arbogast, B. 148 (44) 144 A r c e , A . 82 (79) 61

A Aashwell,G. 85 (168) 79 Abe, S. 370 (5) 358,359 Abelev, G . I. 339 (1) 326, (4 (5) 326, 327; 340 (40) 330 Abercrombie, M . 376 (7) 376 Ablett, S. 121 (51) 115 Achord, D . T . 149 (64) 146, (65) 146 Adair, W . L . 270 (36) 259, 260, 265 Adam, M . 254 (35) 246, (36) 246 Adelman, L . S. 149 (58) 146 Adinolfi, M . 339 (23) Agostini, R. M . , Jr. 132 (7) 124 Agrawal, B. B. L . 431 (30) 415 Aguilar, H . 225 ( 27 ) 215, 218 Akasaki, M . 40; 134 (16) 131 Akiyama, Y. 270 (32) 259 Alexander, D . 339 (20) 327 Alhadeff, J. A. 8 4 ( 1 4 1 ) 7 3 Allan, C . J . 324(17)312 Allan, D . 270 ( 41) 260, 262, 265 Allen, A. 120 (38) 113 Allen, R. C . 133 (24) 128, (25) 128 Allfrey, V . G . 199 (49) 194 Altenburg, B. A. 403,379 Amar-Costesec, A. 444 (18) 434 Ambrose, E . J . 376 (6) 375, (7) 376 Aminoff, D . 431 (26) 414 Ammon, H . V . 356 (56) 350 Anderson, B. 20 (16) 10; 370 (26) 358 Anderson, J. C . 172 (13) 165; 211, 201 Anderson, S. G . 339 (10) 326 Andersson, P. 199 (39) 188, 190 Ando, S. 80 (26) 52, (27) 52, (28) 52 Andrews, P. 40, 32; 42, 32 A n g l i n J . H . 325 (41) 322 Annese, C . 371 (39) 361 Ansell, N . 324(2)311,312 Anttinen, H . 226 (37) 218, 222, (41) 222,

190, (23) 183, 184, 187, (30) 182, 184, 191; J99 (33) 188 Ariga, K. 341 (68) 332 Arlinghaus, R. B. 180; 198 (20) 182-184, 189, 190, (21) 183, 184, (22) 183, 184, 190, (23) 183, 184, 187, (26) 182-184, 192, (27) 184, 192, (28) 184, 188-190, 192, (30) 182, 184, 191; (33) 188, (34) 188-190, 192, (55) 195, 197; 200 (62) 197 Aro, A. 147 (1) 135, 143, (23) 138 Aronson, N . N . , Jr. 147 (9) 137 Aronson, S. A . 310 (5) 295, 302 Arora,R. C . 85 (160) 77,78 Arstila, A. U . 133 (6) 130, (9) 130 Asofsky, R. 340 (47) 331 Atkinson, P. H . 102 (19) 91, 95 Atsuta, T . 83 (102) 69 Attina, D . 325 (44) 322 A u b J . C . 377 (8) 376 Auger, J. 270 (41) 260, 262, 265 August, J. T . 198 (13) 181, (16) 181 Augustyn, J. 254 (30) 243 Aula, P. 40; 133 (10) 130; 134 (15) 131, (16) 131 A u l l , F . 271 (60) 261,265 Austin, J. 148 (42) 143 Autio, S. 43; 103 (29) 98; 133 (4) 129, 130, (6) 130, (7) 130, (9) 130, (10) 130; 148 (46) 144, (47) 144 Axelrod, D . 271 (79) 268 Ayoub, E . M . 211, 202, 210 B Baeziger, J. 1 0 2 ( 8 ) 8 6 Baghdadi, S. 121 (51) 115 Baig, M . M . 211,202,210

(42) 222, (45) 222 A p f f e l , C . A . 292(16)277

American C h e n ^ I Society Library 1155 16th 8t. N. w . Washington, D. C.

20036


448


Bailleul, V . 220(34) 113 Baker, A . P. 219 (22) 11, (24) 110, 111; 220 (25) 110 Baker, E . A . 84 (138) 73 Baker, J . 227 Balint, J. 158 (10) 153 Baltimore, D . 298 (7) 180, 192, (10) 181, (31) 187; 299 (39) 188, 190, (40) 190, 194 Banatwala, I. 324 (14) 312, 322 Bannikov, G . A. 341 (66) 332, (71) 332 Bargeton,E. 247 (19) 138 Barnett, D . R. 229 (3) 108, (7) 108 Barranger, J . A . 150; 258 (5) 150, 153, 157; 259 (21) 150 Barsness, L . 341 (60) 331 Bartholomew, B. A. 40, 33 Barton, M . 42, 28; 133 ( 23) 128 Baschang, G . 82 ( 46 ) 53 Bass, N . H . 249 (58) 146 Basu, M . 82 (64) 59; 82 (69 61, (87) 61, 64, 65, (88) 61, 66, (89) 61, (90) 61, (92) 64 Basu, S. 82 ( 60 ) 59, (61) 59, (64 ) 59; 82 (66) 59, 61, (67) 61, (68) 61, (69) 61, (71) 61, (72) 61, (73) 61, 66, (74) 61, (80) 61, (82) 61, 66, (84) 61, (86) 61, (87) 61, 64, 65, (88) 61, 66, (89) 61, (90) 61, (92) 64; 83 (105) 71; 84 (124)67 Bauer, C . 403, 378 Baumler, A. 444 (8) 432, 433, 439 Bearpark, T . 148 (28) 140 Beattie, G . 270 (39) 262, 266, 267 Beaufay, H . 444 (18) 434 Becker, F . F . 339 (11) 326, 328, (12) 326, (17) 333; 340 (46) 331, (47) 331; 341 (62) 332, (65) 332-334; 430 (15) 413, 420, 421 Becker, G . W . 341 (64) 332 Behrens. N . H . 40, 22, 23; 43, 23 Beiber, G . F . 84 (131) 69 Beisswenger, P. 226 ( 50 ) 223, 224 Bekesi, J. G . 292 (15) 277 Belchetz, P. 259 (19) 150 Bell, C . 444 (12) 433 Bell, J . E . 40, 32; 83 (103) 69 Bell, P . M . 353 (17) 343 Benassayag, C . 339 (16) 327, 329 Bengali, K. M . 298 (18) 181 Bennett, B. D . 84 (125) 67 Bennett, G . 40, 34, 37; 83 (120) 67 Bentvelzen, R 272 ( 63 ) 261, 265 Beranek, W . E . 43,33 Berenblum, I. 294 (50) 290 Berenson, G . S. 211, 201; 212, 201, 202; 254 (37) 246 Berezesky, I. K. 199 (56) 195

PROCESSES

Berlin, R. D . 430 ( 7 ) 412, 429 B e r m a n , E . R . 432 (28) 415 Bernard, J. 324 (16) 312, 322 Berra, B. 147 (24) 138, 140; 248 (31) 140, (32) 140; 149 (57) 145 Bertrand,I. 247 (19) 138 Bessel, E . M . 354 (37) 348, (40) 348 Beutler,E. 259 (20) 150 Beyer, T . A. 40, 32, 37; 83 (103) 69 Bhadure, S. 299 (42) 190, 194 Bhargava, A . K. 355 (45) 348 Bhargava, P. M . 430 (17) 413 Bhatti, T . 353 ( 20 ) 343 Bhavanandan, V . P. 202 (24) 98; 293 ( 25) 278 Bhoyroo, V . D . 45,22, 76; 269 (20) 258 Bielat,K. 444 ( 8 ) 433,439 Bjorklid, E . 272 (23) 167 Bjorndal H 82 ( 49 ) 56 (50 ) 56 (51) 56 Bladen, H . A. 253 ( 7 ) 227 Blair, H . 258 (18) 150 Blenkinsopp, W . K. 232 (6) 124, (10) 125 B l o b e l , G . 40,26 Bloch, K. J. 370 (16) 358, 359, 361 Bloemendal, H . 199 (41) 190, 194 Bloemers, H . P. J. 299 (41) 190, 194 Blonder, E . 85 (155) 77 Blumenfeld, O. O . 224 ( 5 ) 213 Blumenthal, H . T . 253 (4) 227, 243, (6) 227 Boat, T . F. 108; 229 (1) 108, 112, (21) 110, 111, (23) 110; 220 (26) 110, 112, (33) 113, (40) 113, 114, 116, (42) 113; 222 (49) 115, (53) 118, (55) 116, (56) 111 Butterworth, B. E . 199 (32) 187 Byers, P. H . 226(35)218 Bobbitt, O . B. 370 (14) 358, 359, 369 Bobinski,H. 29(8)6 Boccardi,V. 325 (44) 322 B o c c i , V . 422 (9) 408 Boesman, M . 339 (18) 327, 329 Bogart, B . I . 229 (19) 110 B o g e l , C . L . 339 (28) 328 Bolognesi, D . P. 297 (4) 180, 187; 298 (9) 180, 181, 187, (19) 181; 299 (50) 195 Bonney, R. J. 430 (18) 413 Boorman, K. E . 324 (5) 311 Bornstein, P. 225 (33) 218; 226 (37) 218, 222; 253 (1) 227; 254 (22) 243, 246, (23) 243, (34) 246 Bosmann, H . B. 84 (130) 69, (131) 69, (132) 69; 85 (162) 79; 298 (25) 183 Bouissou, H . 254 (25) 243 Boulding, E . T . 325 (38) 322 Bouma, B. N . 272(6) 164


AUTHOR

INDEX

Bouquelet, S. 148 (28) 140 Bowman, B. H . 119 (3) 108, (7) 108, (18) 110 Boyse, E . 198 (16) 181; 199 (58) 197; 292 (5) 277 Bradfield, J. W . B. 1 3 2 ( 6 ) 1 2 4 Bradley, R. M . 310 (5) 295, 302 Brady, R. O . 83 (96) 66, (109) 71, 76; 150; 158 (1) 150, (5) 150, 153, 157, (15) 152, (16) 150, (17) 154, (18) 150; 159 ( 21) 150, (22) 150, (23) 155; 310 (5) 295, 302 Braidman, I. 159 (19) 150 Branson, H . R. 353 (14) 343, 344 Branton, D . 269 (16) 258 Braun, P. E . 8 1 ( 5 7 ) 5 9 Brennan, P 403, 379 Brennan, S. O . 132 (18) 127 Breslaw, N . 339(10) 326 Breslow, J. L . 119 (8) 108 Bretscher, M . S. 269 (6) 258 Brettschneider, I. 149 (69) 146 Brew, K. 40, 31; 42, 32; 43, 32; 44, 32; 45, 32 Briles, E . B. 411 (19) 410 Brock, D . J . 339 (25) 327 Brockerhoff, H . 211, 210 Brodbeck, U . 41, 32; 83 (123) 67 Brotz, M . 8 0 ( 1 2 ) 4 9 Brown, A. E . 8 4 ( 1 3 1 ) 6 9 Brown, B. D . 147 (1) 135, 143, (22) 138, (23) 138 Brown, J. C . 269 (9) 258; 270 ( 57 ) 261, 265 Brown, M . C . 293 (22) 277, 278, 288, 289, (37) 286, 286, (38) 280, 291, (39) 280, 282, 290; 294 (51) 290, 291, (52) 290, 291 Bruenger,E. 254 (15 ) 231 Brunngraber, E . G . 135; 147 (1) 135, 143, (3) 136, (5) 136, (8) 137, (22) 138, (23) 138, (24) 138, 140; 148 (31) 140, (32) 140, (39) 143; 149 (57) 145, (61) 146, (67) 146, (68) 146, (69) 146 Brims, G . 158(13) 153 Bruns, R. R. 293 (42) 280, 282 Brunyneel, E . 120 (46) 113 Buchbinder, L . 371 (32) 361 Buchhagen, D . L . 198 (17) 181 Buck, C . A. 310 (3) 295; 403, 378; 410 (1) 404; 444 (4) 432 Buck, R. L . 432 Buddecke, E . 211, 201, 202 Bugge, B. 41,23 Bukhari, M . A . 354 (40) 348 Bullis, C . M . 270 (57) 261, 265 Burger, M . M . 270 (56) 261; 377 (9) 376; 430 (3) 412, 425, (4) 412, (10) 412; 431 (38) 425

449

Burke, D . J . 1 0 2 ( 1 1 ) 8 6 Burnet, F . M . 84 (134) 73 Burrowes, C . E . 172 (5) 163 Burtin,P. 355 (48) 350,351 Burton, R. M . 86 (17) 49 Buscher, H . P. 44,37 Butler, W . T . 213; 225 (9) 214, (10) 214, (25) 215, (27) 215, 218, (28) 215, 218, (30) 218, (31) 218, 219; 226 (46) 222, (47) 222, (48) 222 C Calle, R. 325 (46) 319 Candler, E . L . 292 (10) 277 Canellakis, E . S. 270 (58) 261, 265 Canfield, W . M . 173 ( 35) 151 Cantrell, J. L . 325 (35) 320 Cantz M 42; 148 (53) 145; 158 (12) 153 Caputto, R. 82 (79) 61 Carlson, D . M . 20 (15) 6; 40, 36; 119 (21) 110, 111; 120 (40) 113, 114, 116; 177 Carminatti, H . 46, 28 Carraway, C . A . C . 432; 444 (17) 434, 438 Carraway, K . L . 432; 444 (5) 432, 434436, (17) 434, 438, (19) 434, 435 Carrell, R. W . 132 (18) 127, (20) 127 Carruthers, C . 444 (8) 432, 433, 439 Carson, S. D . 119 (7) 108 Carter, H . E . 85 (169) 49 Carter, W . G . 270 ( 38 ) 260, 262, 265, (59) 261, 265 Casals-Stenzel, J. 44, 37 Case, K. R. 84 (131) 69 Casjens, S. 199 (51) 195 Castellani, I. 254 (33) 243 Caterson, B. 227 Cesarini, J. P. 147 (15) 138 Chabaud, O . 84 (127) 67 Chambers, R. E . 80 ( 31) 52; 353 (20) 343 Chan, J. Y . C . 1 7 2 ( 5 ) 1 6 3 Chang, 1 147 (1) 135, 143 Chang, J.P. 430 (13) 412 Chang, Y. H . 353 (22) 343, 348 Chao, H . 83(101)69 Chatterjee, S. 83 (107) Chavanel, G . 355 (48) 350, 351 Cheetham, R. D . 83 (118) 67 Chen, J. H . 199 (45) 192 Cheng, P. 108; 119 (23) 110; 120 (26) 110, 112, (40) 113, 114, 116, (42) 113, (47) 115; 121 (49) 115, (53) 118, (54) 118, (55) 116 Chesnut, R. W . 432; 444 (17) 434, 438 Chien, J. L . 82 (69) 61; (82) 61, 66, (84) 61, (88) 61, 66, (89) 61


450

GLYCOPROTEINS

Childs, R. A . 371 (28) 358 Chipysheva, T . A. 341 (66) 332 Chisaka, N . 341 (67) 332 Chism, S. E . 353 (17) 343 Chou, T . H . 40,31 Chu,G.H. 225(21)215 Chu, T . M . 353 (18) 343; 355 (45) 348, (53) 350 Chung, E . 225 (22) 215 Chung, S . I . 173 ( 30) 170 Ciccarini, C . 102(19)91,95 Claeys,H. 172 (25) 169 Clamp, J. R. 80 (31) 52; 353 (20) 343 Clausen, J . E . 324 (24) 315 Cleland, W . W . 81(62)59 Cline, M . J. 431 (36) 425 Coan, M . H . 172(17)166 Cochrane, C . G . 171 (1) 163; 172 ( 4) 163, (6) 164 Codington, J . F. 277; 292 (1 291, (14) 277, (20) 277, 279 293 (21) 277, 279, (22) 277, 278, 288, 289, (27) 279, (28) 279, 280, 286, 288, (29) 279, 286, 287, (36) 279, (37) 280, 286, (38) 280, 291, (39) 280, 282, 290, (40) 280, 281, 286, 288, 290, (41) 280, 282, 286, (42) 280, 282, (43) 286, 287, (44) 286, 288, (45) 286, 287; 324 (26) 315, (27) 315; 375; 444 (10) 432, (11) 433, 442 Coffee, C . J . 148 (38) 143 Cohen, M . P. 226 (52) 223 C o h n , Z . 158(7)157 Coligan, J. E . 352 (5) 342, (7) 343, (8) 343; 353 (10) 343, (11) 343, 345, (15) 343, (19) 343, 344, (23) 343, 346, 348; 354 (25) 344, (27) 344; 355 (43) 348, (44) 348, (46) 348; 371 (38) 361 Collard, J. G . 430 ( 9 ) 412; 431 (40) 429 Colley, M . 158(14)153 Colman,R.W. 173 (33) 171 Colvin, B. 41, 32 Comb, D . G . 324 ( 21) 314 Conod, E . J. 119 (4) 108, (19) 110 Conover, J. 119 (4) 108, (19) 110 Conrad, H . E . 354 (29) 344 Cook, G . M . W . 83 (100) 69; 376 (5) 375 Cooper, A. G . 293 (22) 277, 278, 288, 289, (37) 280, 286, (38) 280, 291, (39) 280, 282, 290, (40) 280, 281, 286, 288, 290; 294 (51) 290, 291, (52) 290, 291; 370 (22) Corey, R. B. 353 (14) 343, 344 Costello, A. J. R. 149 (68) 146 Cotman, C . W . 271 (67) 266 Cowan, N . J. 40, 26 Cox, B. A . 253 (12) 228, 231, (13) 232, 233; 255 (45) 252

A N D GLYCOLIPIDS I N DISEASE PROCESSES

Cox, D . W . 132 (8) 124,125 Coyle, P. 84 (152) 75, 77; 148 (38) 143 Crawley, J. 159 (19) 150 C r e a n , E . V . 271 (64) 261 Crocker, B. F . 84 (135) 73 Crumpton, M . J. 270 (41) 260, 262, 265, (42) 260, 262, 265; 271 (62) 261, 265 Cudney, T . L . 293 (32) 279, 280 Cullen, S. E . 292 (2) 277; 376 (3) 375 Cunningham, L . W . 225 (9) 214, (10) 214, (13) 214, (27) 215, 218 Cutz, E . 41, 28; 132 (8) 124, 125 Czegledy-Nagy, E . 119 (20) 110 Czichi, U . 40, 28 D D a b a n , A . 325 (46) 319 Dabelsteen, E . 370 (10) 358, 359 Dale, G . 159 (20) 150 Dalferes, E . R. 211 D a l t o n , A . J . 200 (59) 197 Daniels, A. 148 (39) 143 Daniels, J. R. 225 (21) 215 Dankert, M . 43, 23 Danzo, B. J. 271 (61) 261 D a o u d , A . S. 254 (30) 243 Darcy, D . A. 355 (50) 350, 351, (51) 350, 351 Das, I. 120 (43) 113 Das, S. 355 (44) 348 Datta, P. 83 (113) 67 DaussetJ. 324 (16) 312,322 Davidsohn, I. 310 (6) 296; 370 (6) 358, 359, (7) 358, 359 Davidson, E . A. 102 (24) 98; 293 (25) 278 Davie, E . W . 171 (2) 163, (3) 163; 172 (7) 164, (8) 164, (9) 164, (12) 165, (14) 165, (15) 165, 166, 171, (17) 166, (18) 166, (20) 166, 167; 173 (35) 171 D a vies, D . A. L . 324 ( 8 ) 312 Davis, E . M . 276 (6) 276; 412; 431 (22) 414, 418, 419, (31) 415, 429, (32) 426, (35) 423 Davis, J. 121 (49) 115; 197 (5) 186, 195 Davis, L . G . 147 (24) 138, 140; 149 ( 67) 146, (68) 146, (69) 146 Davis, N . L . 198 (8) 180,192 Davis, P. 119 (2) 108, 109, 112 Davis, S. S. 119(11) 109 Dawson, G . 82 (85) 61; 103 (32) 98; 147 (27) 139, 140, 144; 148 (48) 144; 158 (11) 153 Dean, K. J. 84 (145) 75, (147) 75 Dearborn, D . G . 119 (6) 108; 120 (29) 112, 113


AUTHOR

451

INDEX

DeBacker, M . 254 (32) 243 DeDuve, C . 147 (9) 137; 158 (2) 150 DeFries, A. 85 (155) 77 Degand, P. 120 (34) 113, (35) 113, (41) 113, (48) 115 deHaller, R. 119 (14) 109 deHarven,E. 200 (60) 197 Dekaban, A. 159 (23) 155 Del Villano, B. C . 198 (11) 181 Deman, J. 120 (46) 113 Dempo, K. N . 341 (67) 332, (70) 332 de Petris, S. 271 (68) 266, (71) 266, (73) 268 DePierre, J. W . 444 (14) 433, 434 Derbyshire, W . 121 (51) 115 Derouette, J. C . 254 (25) 243 Derouette, S. 254 (31) 243 Desai, P. R. 310 (1) 302; 311; 324 (11) 312, (12) 312, 318, (14) 312 322 (15) 312, 315, 317, 320, (19) 314 314, 320, (25) 315; 325 (28 (29) 320, 322, (35) 320, (42) 322, (43) 322 Desteno, C . V . 226 (47) 222 Deumling, B. 83 (118) 67 D i Benedetta, C . 148 (40) 143 Dicorleto, P. E . 211, 210 Dieleman, B. 46, 39 D i Palma, S. 148 (32) 140 di'Sant Agnese, P. A . 119 (2) 108, 109, 112; 120 (32) 113; 121 (50) 115 Dische, Z. 121 (50) 115; 225 (8) 213 DiScipio, R. G . 172 (8) 164 Distler, J. 149 (63) 146 Dobberstein, B. 40, 26 Dodd, B . E . 324 (5) 311 Doebber, T . 85 (165) 79 Doershuk, C . F . 119(1) 108,112 Domotor, J. J., Jr. 198 (18) 181 Dorfman, A. 148 (44) 144 Dorland, L . 45, 30 Dorval, G . 292 (18) 277 Downey, J. 172 (28) 170 Downs, F . 2 0 ( 1 4 ) 6 Dreslar, S. 198 (29) 184 Drzeniek, Z. 325 (33) 320 Dubois, M . 431 (27) 414 Duesberg, P. H . 199 (37) 188, (44) 192, (48) 194 Dugal, B. 133 (12) 130 Dumsha, B. 224 (4) 213 Durand, G . 120 (35) 113 D u r a n d , P . 45; 149 (55) 145 E Eastoe, J. E . 226 (56) 224 Ebner, K. E . 40, 32; 41, 32; 42, 32; 83 (123) 67; 444 (5) 432, 434-436

Edelman, G . M . 271 (75) 268 Edgar, G . W . F . 147 (18) 138, (19) 138 Edidin, M . 269 ( 2 ) 256 E d s a l l , J . T . 276 (4) 275 Egan, M . L . 352 (4) 342, 346, (5) 342, (7) 343, (8) 343, (9) 343; 353 (19) 343, 344; 354 (25) 344, (32) 344, (39) 348; 355 (46) 348; 356 (57) 350, (58) 350 Egan,W. 172(10)165 E g e l r u d , T . 211,204 Egge,H. 81(44)52 Ehrenreich, B. 1 5 8 ( 7 ) 1 5 7 Eisen, H . N . 354 (38) 348 Eisenberg, S. 211, 201 Elam, D . L . 293 (34) 279 Elgsaeter,A. 269 (16) 258 Emerson, W . A . 133 (21) 127; 270 (40) 260 262 Endo, Y. 340 (38) 330 Enfield, D . L . 172(16) 166 Engelhardt, N . V . 340 (40) 330 Engvall, E . 339 (13) 326; 352 (4) 342, 346; 354 (33) 344, 347, 348; 356 (60) 351, (61) 351 Epstein, J. 119(8)108 Epstein, S. 340 (55) 331 Erickson, J. S. 148 (36) 143 Ericsson, L . H . 172 (16) 166; 173 ( 35) 171 Eriksson, S. 132 (15) 126 Esselman, W . J. 81 (53) 56; 371 (33) 361 Esterly, N . R. 119 (17) 109, 110 Etchinson, J. R. 102 (17) 91 Etchison, J. 41, 24, 31 Eto, Y. 148 (41) 143,(43) 143 Evans, J. E . 80 (20) 51 Evans, L . H . 198 (29) 184 Evans, N . 293 (45) 286,287 Eveleigh, J. W . 353 ( 61) 343 Ewing, C . 132 (17) 127, (19) 127 Eylar, E . H . 41, 36; 83 (100) 69; 198 (25) 183 F Fabre, J. W . 270 (46) 260, 263 Fabre, M . T . 254 (25) 243 Fagerhol, M . 42, 28; 131 (2) 123 Fahey, J. L . 324 (9) 312 Famulare, N . G . 198 (17) 181 Fan, H . 199 (40) 190, 194, (46) 194 Farber, E . 340 (52) 331, (55) 331, (56) 331, (57) 331, (58) 331; 341 (72) 332 Farrell, P. M . 120 (30) 112 Farriaux, J. P. 45; 149 (55) 145 Federico, A. 148 (40) 143


452


Feizi, T . 20 (16) 10; 310 (12 ) 302; 370 (23) 359, (26) 358; 371 (28) 358 Fernlund, P. 172 (10) 165 Ferrand, M . 84 (127) 67 Ferris, B. 270 (55) 261, 265 Fessler, J. H . 225 (34) 218 Fessler, L . I . 225 (34) 218 Fidler, I. J. 147 (15) 138 Fietzek, P. P. 225 (26) 215, 218 Filliat, M 120 (48) 115 Finch, J. E . , Jr. 225 (28) 215, 218, (30) 218, (31) 218, 219; 226 (47) 222 Findlay, J. B. C . 270 (34) 259, 260, 265 Finne, J. 147 (4) 136, (6) 136, (7) 137; 269 ( 26 ) 258 Fischer, A. 411 (8) 408 Fischer, D . 85 (166 ) 79; 149 ( 64) 146 Fischer, K. 325 (36) 322 Fisher, R. L . 132 (11) 125 Fishkin, A . F . 211, 201; 212, 201 Fishman, P. H . 83 ( 96 ) 66, (10 310(5) 295, 302 Fitzgerald, D . K. 41, 32 Fleischer, B. 84 (137) 73 Fliescher, S. 84 (137) 73 Fleisher, T . A . 133 (5) 130 Fleissner, E . 197 (1) 180, 181; 198 (8) 180, 192, (17) 181; 199 (57) 195, (58) 197 Flowers, H . 276 (1) 275 Fluharty, A . 158 (9) 153 Fogle, D . D . 444 (17 ) 434, 438 Folch, K. 403, 382 Foley, E . J . 376 (2) 375 Folk, J . E . 173 ( 30) 170 Fong, J. W . 81 (43) 52 Forbes, M . 325 (45) 322 Ford 225(13 ) 214 Forni, L . 271 (74 ) 268 Forssman, J. 371 (29) 361 Forstner, G . G . 120 (31) 112 Forstner, J . F . 120 (31) 112 Foster, A . B. 355 ( 42 ) 348 Foster, J. A . 254 (16 ) 231 Foster, L . A . 254 (15) 231 Fournet, B. 42; 45, 30; 148 (28) 140; 149 (54) 145, (55) 145 Fournier-Delpech, S. 271 (61) 261 Fowler, M . C . 149 (56) 145 Francis, G . 225 ( 31) 218, 219 Frank, H . 197 (4) 180, 187 Franke, W . W . 83(118) 67 Franzblau, C . 253 (5) 227 Fraser, B. A . 371 (35) 361, (38) 361 Fraser, I. H . 41, 32 Freedman, S. O . 352 (1) 342, (2) 342, (3) 342, (6) 342; 353 (13) 343; 370 ( 27) 358

PROCESSES

Freese, E . 83 (109) 71,76 Fry,M. 199(15)195 Friberg, S., Jr. 293 ( 30 ) 279, 290, (31) 279, (35) 279; 294 (48) 288, (49) 288; 444 ( 9 ) 432 Friedenreich, V . 325 (39) 322 Friedensen, B. 325 (34) 320 Fritz, K. E . 254 (30) 243 Fry, M . 199(15)105 F u h r e r J . P . 310(3)295 Fujikawa, K. 171 (2) 163, (3) 163; 172 (9) 164, (12) 165, (16) 166, (17) 166, (20) 166,167 Fujita,S. 341 (68) 332 Fukuda, M . 102 ( 25 ) 98; 269 ( 21) 258 Fuller, G . M . 119(7)108 Fulling, J. H . 370 (10) 358, 359 Funakoshi, I. 40; 134 (15) 131, (16) 131; 269 (27) 258 (28) 258; 293 (24) 278 75,77; 85 (161) 77,79 Furbish, F. S. 150; 158 (5) 150, 153, 157, (18) 150; 159 (21) 150 F u r i e , B . 172(11) 165 F u r i e , B . C . 172(11)165 Furthmayr, H . 42, 37; 270 (33) 259, (37) 260, 262, 265; 294 (46) 288 Furukawa, K. 370(11) 359, (21) 359 G G a c t o , M . 403,385 Gaerlan,P. 119 (4) 108 Gahmberg, C . G . 269 (10) 258; 310 (4) 295; 371 (37) 361 Gaidulus, L . 132 (17) 127, (19) 127 Gal, A . E . 150; 158 (16) 150, (17) 154; 159 ( 23) 155 Gallop, P . M . 224 (5) 213 Gan, J . C . 132(13) 126 Gardas,A. 82 (95) 64 Gardner, D . A. 82 ( 80 ) 61, (84 ) 61, (89) 61; 83 (105) 71 Garegg,P.J. 403,390 Garguilo, A. W . 370(9) 358, 359 Garner, E . F . 102 ( 22) Garver, F . 44,33 G a s i c , G . 293 (26) 279 G a s i c , T . 293 ( 26 ) 279 Gatt,R. 431 (28 ) 415 Gatt, S. 84 (138 ) 73, (153 ) 77; 85 (154) 77 Gautier, M . 102 (9) 125, (12) 125 Gaver,R.C. 80(8)48 Gazzard, B. 340 (39) 330 G e h l e r J . 148 ( 53) 145 Gelman, R. A. 120(45) 113


AUTHOR

453

INDEX

Gel'Shtein, V . I. 341 (66) 332 Gentry, C . 271 (67) 266 Geren, C . R. 41,32; 42,32 Geren, L . M . 41, 32 Gersten, D . M . 84 (131) 69 Ghosh, H . P. 45, 26 Gielkens, A. L . J. 199 (41) 190,194 Gilbert, G . A. 276 (4) 275 G i l l a r d , G . C . 254 ( 38 ) 246 G i l l e s , K . A . 431 (27 ) 414 Gilliam, E . B. 430 (12) 412, 414, 418, 420, 423; 431 (35) 423 Ginsburg, V . 107 G i r e l l i , G . 325(44) 322 Giro, M . G . 254(33)243 Gitlin, D . 339 (18) 327, 329, (21) 327, 328 Glass, G . B . J . 80 (15) 49,75 G l a z e r , N . 224 (4) 213 Glew, R. H . 84 (152) 75, 77; 132 (7) 124; 148 (38) 143 Glick, M . C . 275; 276 (1) 275 404; 410 (1) 404, (2) 404, (4) 404, (6) 404, 405; 411 (7) 405, 408, (8) 408, (12) 408, (14) 408, 409, (15) 408, (16 ) 408, (21) 406; 444 (4) 432; 445 (21) 437 Glockner, W . M . 269 (25) 258 Go, V . L . W . 356 (56) 350, (58) 350 Gochman, N . 340 (35) 329 G o e b e l , H . H . 148 (33) 140 Gold, P. 339 (8) 326; 352 (1) 342, (2) 342, (3) 342, (6) 342; 353 (13) 343; 370 (27) 358 Goldberg, A. R. 377 (9) 376 Goldman, R. 271 (72) 266 Goldstein, I. J. 102 ( 22); 354 (33) 344, 347,348; 431 (30 ) 415 Goldsworthy, T . 341 (60) 331 Golovtchenko, A. M . 41, 23 Gombos, G . 270 (50) 260, 264 Goodfellow, P. 270 (43) 260, 263 Goodman, M . 225 (18) 215 G o r d , G . 339 (9) 326 Gorin,P.A.J. 81(48)56 Got, R. 83 (98) 69 G o t o h , T . 172 ( 28) 170 Gottschalk, A . 19 (6) 5, 11; 20 (22) 13; 120 (44) 113 Grabar,P. 339 (3) 326 G r a f , L . 371 (49) 363 Granatek, C . H . 292 (10) 277 Grand, R . J . 120(27) 110 Grant, M . E . 226 ( 36 ) 218 Grassman, W . 224 (1) 213, (2) 213, (3) 213 Gray, G . M . 83 (104 ) 71 G r a y , M . E . 84(125) 67 Gray, W . R. 254 (15) 231, (16) 231 Green, M . 199 (42) 190,194

Greenberg, C . S. 411(21)406 Greenspon, S. A . 225 (18 ) 215 Gregoriadis, G . 159 (19) 150 Gregory, W . 270 (31) 259 Griffin, J. H . 171 (1) 163; 172 (4) 163, (6) 164 Grimes, W . J . 83(114)67 Grimley, P. M . 199 (56) 195 Groshman, J. 293 (33) 279 Gross, J. 224 (4) 213; 226 (57) 224 Gruensteun, M . 340 (58) 331; 341 (72) 332 G u a z z i , G . C . 148 (40) 143 Guelstein, V . I. 341 (71) 332 G u g l e r , E . 120 (32) 113 G u i n t o , E . 159 (20) 150 Guminska,M. 147(17) 138 Gurd, J. M . 270 (52) 260, 264 Gurd J W 270 (49) 260 264 266

H Habal,K.M. 172(5)163 Haberland, C . 147 (22) 138; 148 (39) 143 Hadchouel, M . 132(9) 125 H a d d a d , A . 83 (120) 67 Haffenden, G . P. 132 (10) 125 Hafter,R. 224 (2) 213 Hagmar,B. 294 (53) 291 Hagopian, A. 41,36; 198 (25) 183 Haguenau,F. 200 ( 59) 197 Hakkinen, I. 370(17) 358, 359, 361 Hakomori, S. 80 ( 23 ) 51; 81 (38) 52, (47) 53, (54) 56; 82 (93) 64; 270 ( 59 ) 261, 265; 310 (4) 295, (9) 302, (15) 304; 354 (28) 344, (3) 344; 357; 370 (1) 357, (2) 357, (13) 358, (16) 358, 359, 361, (19) 358, 359, 363, 364, 369, (20) 359, (24) 358, 359, 369; 371 (28) 358, (30) 361, (31) 361, (37) 361, (41) 361, (43) 364, (45) 364, (46) 364; 403, 378 391 Halford' M . H . 225(15) 215 H a l l , T . C . 85 (162) 79 Hall, W . J . 132(5) 123 Hallgren, P. 19 (7) 6; 403, 390; 411 (11) 408 Halpern, M . S. 197 (2) 180, 181; 198 (14) 181 Haltia, M . 133 (6) 130, (7) 130 Hamers, M . N . 84 (148) 75 Hamilton, J. K. 431 (27) 414 Hamilton, R. T . 270 (45) 260, 263 Hammarstrom, S. 354 ( 33 ) 344, 347, 348; 356(62) 351 Hanafusa, H . 199 (45) 192, (49) 194 Hanafusa, T . 198 (7) 180, 192


454

GLYCOPROTEINS

Hanaham, D . J. 172 (26) 169 Handa,S. 80(21)51,(22)51 Hanfland,P. 81(44)52 Hanks, J . H . 431 (24 ) 414 Hanna, M . G . , Jr. 198 (19) 181; 292 (10) 277 Hansen, H . J . 355(53) 350 H a r a , A . 371 (34) 361 Hard, W., Jr. 199 (58) 197 Harmon, J. W . 340 (54) 331 Harris, H . 294 (47) 288 Harvey, S. R. 353 (18) 343; 355 (45) 348 H a s c a l l , V . C . 254 (39) 252 H a u p t , H . 173 (32) 170 Hauschka, T.S. 293 (32) 279, 280 Hauser, G . 81 (65) 59, 61, 64, 65; 82 (78) 61 Haverkamp, J. 45, 30 Havez,R. 120 (35) 113 Hawthorne, J. N . 85 (169) 49 Hay, E . D . 293 ( 41) 280, 282 (11) 433,442 Hayashi, H . 85 (164) 79 Hayashi, M . 371 (34) 361 Hay em, A. 120 (34) 113 Hayman, M . J. 370 (42) 260, 262, 265, (43) 260, 263; 271 (62) 261, 265 Haywood, W . S. 199 (47) 194 Heard, D . H . 376 (5) 375 Heath, E . C . 354 (34) 346 Hechtman,P. 85 (158) 77 Hedin, A . 356 (62) 351 Heimburger, N . 173 ( 32) 170 Heldebrant, C . M . 172 (14) 165 Hellerqvist, C . G . 81 (51) 56; 354 (31) 344 Hellstrom, K. E . 444 (7) 432 Helstrom, I. 444 ( 7 ) 432 Henderson, M . 42, 31; 403, 378 Hendricks, J. 270 (44) 260, 263, 266 Hendrix, C . F . 254 (14) 231, 234, (21) 243 Henkart, P. A . 353 (15) 343; 355 (43) 348, (44) 348 Hennigar, G . R. 133 (24) 128 Henson, L . C . 211, 210 Herberman, R. B. 324 (23) 314, 319 Hercz, A . 41, 28; 133 ( 23) 128 Herlant-Peers, M - C . 45 Hermodson, M . A . 172 (8) 164 Hernell, O . 211, 204 Herries, D . G . 42, 32; 46,32 Hers, H . G . 148 (50) 144 Herschkowitz, N . N . 148 (41) 143, (43) 143 Herscovics, A . 41, 23 Hersh, E . M . 292 (10 ) 277 Hershko, A . 199 (52) 195 Hibbert, S. 158 (16) 150; 159 (23) 155


Hickman, S. 85 (167) 79; 149 (59) 146 H i e b e r , V . 149 (63) 146 Hildebrand, J. 81 (65) 59, 61, 64, 65; 82 (78) 61 H i l f , R . 444 (12) 433 H i l l , H . D . 120 (39) 113 H i l l , H . D . J r . 41,36 H i l l , R . C . 173 (29) 170 Hill, R. L . 40, 31, 32, 37; 41, 36; 43, 33, 36, 37; 45, 32, 37; 83 (97) 64-66, (103) 69; 120 (39) 113; 172 (27) 169 Hillegass, L . M . 119 (24) 110, 111; 120 (25) 110 Hirs, C . H . W . 20 (23) 13, 14; 102 (18) 91 Hirsch, J. 120 (30) 112 Hirst, G . K . 376 (4) 375 Hixson, D . C . 276 (6) 276; 412; 431 (31) 415 429 143 H . 147 (1) 135, 143, (3) 136, (8) 137 Hoffman, U . 224 (3) 213 Holdridge, B. A . 293 (32) 279, 280 Holland, J. F . 292 (15) 277 Hollander, W . 253 ( 5 ) 227 Hollinshead, A. C . 324 (23) 314, 319 Holm, M . 8 1 ( 4 2 ) 5 2 Holtermuller, K. H . 356 (56) 350 H o m b u r g , C . 410(5)404 Hondi-Assah, T . 45; 149 (55) 145 H o r m a n n , H . 224 (2) 213 Horowitz, M . I. 19 (2) 5, 13, 14; 81 (37) 52; 84 (142) 73 Horton, R. E . 324 (7) 311, 312, 322; 325 (45 ) 322 H o s k i n s , L . C . 325 (38) 322 Howard, S. P. 432 H s i e h , T . C . Y . 85(171) 49,56 Huang, C . C 102 (6) 86, 87, 95, 98 Huang, C . M . 83 (119) 67; 444 (16) 434 Huang, I.-Y. 325 (30) 320 Hubbard, A . 270 (55) 261, 265 Hubbard, S. C . 102(12) 87 Hubbell, G . J. 212, 201, 202 Hubbell, W . L . 270 (39) 262, 266, 267 H u b e r , C . T . 269 ( 8 ) 258 Hudgin, R. L . 41, 33, 39; 44, 34; 83 (121) 67 Hudson, B. G . 42, 32; 225 (23) 215; 444 (5) 432,434-436 Huggins, J. W . 432; 444 (17) 434, 438, (19 ) 434, 435 Hughes, L . E . 292(17) 277 Hughes, R. C . 19 (5) 5; 276 ( 2 ) 275; 411 (18) 410; 444(1) 432 H u i j i n g , F . 158 (4) 153

Hof,


AUTHOR

INDEX

455

Hunsmann, G . 298 (15) 181, (19) 181 Hunt, L . M . 42,24, 31 Hunt, R. C . 269 (9) 258; 270 (57) 261,' 265 H u p e r , G . 198 (19) 181 Huprikar, S. V . 325 (37) 312, 322 Hurley, N . E . 298 (8) 180,192 H y d e , R . W . 232 (5) 123 Hynes, R. O. 269 (13) 258; 444 (6) 432 I Iammarino, R. M . 341 (73) Ide, H . 270 (48) 260, 263, 264, 266 IhleJ.N. 298 (18) 181,(19) 181 Ikeda, H . 298 (16) 181; 199 (58) 197 Inbar, M . 272 (76) 268, (77) 268 Irimura, T . 293 (29) 279, 286, 287 Iseki, S. 370 (11) 359,(21) 359 Isemura, M . 42, 33 Isenberg, J . N . 122; 133 (3) (8) 130, 131 Ishibashi, T . 82 (70) 61, (83) 61; 83 (102) 69 Ishihara, K. 370 (11) 359, (21) 359 Ishii, M . 222, 201 Ishizuka, H . 342 ( 68 ) 332 Ishizuka,I. 80 (14) 49 Issacs, R. 320 (7) 296, 298, (8) 300, 308 Isselbacher, K. J. 83 (116) 67, 69; 84 (128) 69 I t o , N . 340 (55) 331 Ito, S. 42, 30; 45, 30; 203 (216) 98 Iwamori, M . 80 (19) 51, (24) 51, (25) 51 Iwata, M . 270 (48) 260, 263, 264, 266 Iyer,R. 220 (40) 113,114,116 J Jabbal, I. 42,31; 44,34; 83 (121) 67 Jackson, C . M . 272 (24) 167 Jackson, R. L . 20 (23) 13, 14; 269 (7) 258 Jacob, J. C . 247 (20) 138 Jacob, S . T . 430(17) 413 Jacobs, H . G . 225 (27) 215, 218 Jacobs, M . 255 (45) 252 Jacobsen, A. 272 (28) 170 Jacobson, K. 272 (78) 268 Jacques, P. 258 (13) 153 Jagarlamoody, S. M . 292 (8) 277 Jakstys, M . M . 324 (10) 312 James, A . M . 376 (6) 375 James, R. 355(51) 350,351 Jamieson, J. C . 42,26, 28 Jamjoom, G . A . 298 (20) 182^184, 189, 190, (22) 183, 184, 190, (26) 182-184, 192, (27) 184, 192; 299 (55) 195, 197; 200(62) 197 Jansons, V . K. 270 (56) 261; 430 (10) 412 Jansson,P.E. 82 ( 55 ) 56

Jarasch,E. 83(118)67 Jarlfors,U. 44,32 JarmolychJ. 254 (30) 243 Javaid, J. I. 147 (3) 136, (8) 137, (24) 138, 140; 149 (67) 146, (68) 146 Jayle,M.F. 339 (16) 327,329 Jeanloz, R. W . 3, 42, 23; 220 (41) 113; 225 (15) 215; 292 (14) 277, (20) 277, 279, 280, 288; 293 (21) 277, 279, (22) 277, 278, 288, 289, (27) 279, (28) 279, 280, 286, 288, (29) 279, 286, 287, (36) 279, (38) 280, 291, (40) 280, 281, 286, 288, 290, (43) 286, 287, (45) 286, 287; 294 (51) 290, 291; 324 (26) 315, (27) 315; 370 (16) 358, 359, 361; 403, 378; 444 (10) 432 Jeffery,P.K. 222 (52) 115 Jenner, F . A . 43; 133 (1) 129, 130 Jentoft,N 222 (54) 118 Ji, T . H . 269 (5) 256, 258, 259, 266, 268, (15) 258 Jirgensons, B. 133 (22) 127 Tohansson, B. G . 354 (33) 344, 347, 348 Joklik,W. 199 (43) 190,194 Jolly, R. D . 103 (28) 98; 248 (49) 144 Jones, E . W . 355 (50) 350, 351 Jones, J. 259 (21) 150 Jourdian, G . W . 40, 33, 36; 249 (63) 146 K K a b a t , D . 298 (29) 184 Kabat, E . A. 20 (16) 10, (17) 9; 370 (26) 358 Kadar,A. 254 (27) 243 Kagan, H . M . 253 ( 8 ) 227 Kahan, B. D . 292 (1) 277; 324 (10) 312 Kahane, I. 270 ( 37 ) 260, 262, 265 Kahne,I. 269 (7) 258 Kamimura,N. 342 (68) 332 Kanai,K. 340(38) 330 K a n e d a , H . 342 ( 68 ) 332 Kaneko, A. 342 (67) 332, (70) 332 Kanfer, J. N . 248 (34) 143 Kang, A. H . 225 ( 29 ) 215, 218 Kaplan, A . 85 (166) 79; 249 (64) 146, (65) 146 Kaplan, H . S . 298 (12) 181 Karlsson, K. A . 79 (5) 48, 53; 80 (9) 48; 82 (39) 52, (40) 52, (41) 52, (45) 52; 370 ( 25 ) 359, 361; 371 (36) 361 Karnovsky, M . J. 430 (7) 412, 429 Karnovsky, M . L . 444 (14) 433, 434 Karshin, W . L . 180; 298 (20) 182-184, 189, 190, (22) 183, 184, 190, (23) 183, 184,187,(30) 182,184,191 Kartenbeck, T. 83 (118) 67


456

GLYCOPROTEINS


PROCESSES

Kato,H. 172(12)165 Kiyosawa, I. 42, 32 Katona, E. 42, 28 Klaiber, M. 342 (72) 332 Katz, F. 46, 26 Klebe,R.J. 229 (3) 108 Katzman,R.L. 225 (15) 215 Klein, E. 292 (12) 277, (18) 277; 376 (1) Kaufman, B. 81 (60) 59, (61) 59; 82 (67) 375 61, (68) 61, (71) 61, (72) 61, (73) 61, Klein, G. 292 ( 7 ) 277, (12 ) 277; 293 (22) 66, (74) 61 277, 278, 288, 289; 294 (47) 288, (48) Kaul, K. 85 (170) 49, (171) 49, 56 288, (49) 288; 376 (1) 375 Kawamura, N. 80 (10) 48; 371 (34) 361 Klein, K. M. 340 (46) 331 KawanamiJ. 371 (42) 363 Kleinerman, J. I. 229 (21) 110, 111; 222 Kawasaki, H. 370 ( 5 ) 358, 359 (56) 111 Kawasaki, T. 85 (168) 79 Klemer,A. 222,202 Kay, H. E. H. 370 (8) 358, 359 Klenk,H.D. 298 (17) 181 Kaye,C.I. 324 (10) 312 Klibansky,C. 85 (155) 77 Kean, E. L. 324 (22) 314 Klimmerer, T. W. 84 (131) 69 Keegstra,K. 202 (11) 86 Kline, I. 293 ( 34 ) 279 Keenan, T. W. 83 (111) 67, (119) 67, Klinger, M . 378; 403, 385, 387 Knight, W.S. 352(9) 343 (122) 67; 84 (124) 67; 444 (16 ) 434 Kefalides, N. A. 224 ( 6 ) 213, 215; 225 Knox,W.E. 340 (59) 331 (12) 214, (19) 215, 216, (20 (36) 218, (53) 224 Kellenberg, E. 299 (54) 195 95; 203 (26) 98 Kelly, P. 272 (67) 266 KohnJ. 340 (39) 330 Kempf,S.F. 82(81)61 Kohno,M. 247(14) 137 Kenne, L. 82 (55) 56 Koide,N. 202 (14) 87,91,98 Kennedy, E. P. 82 (62) 59 Konigsberg, W. H. 82 ( 56 ) 59 Kennel, S. H. 298 (11) 181 Koono,M. 85 (164) 79 Kerr, I. M. 299 (35) 188 Koonstra, W. 272 ( 63 ) 261, 265 Kessel, D. 40,31; 42, 31; 403, 378 Kopchick, J. J. 298 (27) 184, 192, (28) Kessler, M. 355 (47) 348, 350; 356 (59) 184,188-190,192 350, 351 Koplitz, M. 342 ( 61) 331 Kessler, S. W. 372 (48) 364 Koppel,D.E. 272 (79) 268 Khalifa, A. 226(52) 223 KoppelJ.L. 84 (135) 73 Khan, M. A. 229 (13) 109; 220 (37) 113 Korant, B. D. 299 (53) 195 Khatra, B. S. 42,32 Kornblatt, M. J. 80(13)49 Khramkova, N. I. 339 (1) 326 Kornfeld, R. 29 (3) 5, 6, 8, 12; 202 (10) Kiely, M. L. 42,26 86; 269 (23) 258, (29) 259, (30) 259 Kihara,H. 258 (9) 153 Kornfeld, S. 29 (3) 5, 6, 8, 12; 45, 24, 26, Kin,N. 203 (30) 98, (31) 98 31; 202 (8) 86, (10) 86, (13) 87; 269 Kindt, T.J. 372 (38) 361 (23) 258, (29) 259, (30) 259; 270 ( 31) King,F. 259 (22) 150 259, (36) 259, 260, 265, (40) 260, 262; King, J. 299 (51) 195 422 (19) 410 King, M. 220 (45) 113 Koscielak, J. 82 (91) 64-66, (95) 64; 370 Kinsky, S.C. 372 (44) 364 (16) 358, 359, 361 Kijimoto, S. 82 (70) 61, (83) 61 Koshland, D. E. 273 (31) 170 Kim,U. 444 (8) 432,433,439 Kosjakov, P. N. 324 (4) 311 Kim, Y. S. 295; 310 (1) 295, 306, (7) 296, Kottgen, E. 403, 378 298, (8) 300, 308, (10) 302, 304, 305, Kovarik, S. 320 (6) 296; 370 (6) 358, 359, (16) 304,305,307, 308 (7) 358, 359 Kishimoto, Y. 80 (19) 51 Kraemer,P. 298 (24) 183 Kisiel, W . 160; 272 (18) 166, (20) 166, Krag,E. 356(56) 350 167, (21) 166,167; 173 (35) 171 Krakower, C. A. 225 (18 ) 215 Kit, S. 403,379 Kramer, R. H. 270 (58) 261, 265 Kitagawa,T. 341 (60) 331 Kramsch, D. M. 253 (5) 227 Kitchen, B. J. 42, 32 Kreese, H. 249 (62) 146; 258 (12) 153 Kivimaki,T. 233 (9) 130 Krupey, J. 352 (3) 342, (6) 342; 353 (13) Kivirikko, K. I. 225 (32) 218, 222; 226 343; 370(27)358 (37) 218, 222, (38) 222, (41) 22, (42) Krusius, T. 247 (2) 136, (4) 136, (6) 136, 222, (43) 222, (44) 222, (45) 222


AUTHOR

INDEX

457

Kuhl,W. 159 ( 20) 150 Lemm, J. 229 (12) 109 Kuhlenschmidt, M. S. 248 (38) 143 Lennarz, W. J. 40, 28; 43, 23, 24; 45, 23; Kuhmichel, A. 370 (14) 358, 359, 369 46, 21-23 Kiihn, K. 224 (3) 213; 225 ( 26 ) 215, 218LeQuire,V. S. 84 (125) 67 Kuhn,R. 84(143 ) 75 Lerch, R. M. 253 ( 8 ) 227 Kundo,S.K. 82 (48) 56 Lerner, M.P. 325 (41) 322 Kundu,S.K. 82 ( 43 ) 52 Lerner,R.A. 298 (11) 181 Kupchik, H. Z. 355 (54) 350 Leseney, A. M. 432 ( 25 ) 414, 420 Kurachi, K. 272 (3) 163, (7) 164 Lessof, M. H. 339 (23) Kurosky, A. 229 (7) 108 Lester, R. L. 85 (170) 49, (171) 49, 56 Letoublon, R. 83 (98) 69 Kusiak, J. 259 (5) 150,153,157 Letts, P. J. 42,34 Kutty,K.N. 247 (20) 138 Lev, R. 276 ( 5 ) 275 Kyriakides, E. 258 (10) 153 Levi, R.I. 222,201 Levin, J. G. 299 (56) 195 Levine, P. 324 (1) 311, 322; 370 (14) 358, LafitteJ.J. 220 (48) 115 359, 369, (15) 358, 369 Lagunoff,D. 258 (3) 153 Levitt, M. H. 342 ( 74 ) 334 Levitzki,A 273 (31) 170 Laine, R. 378 Laine, R. A. 82 (93) 64; 85 372 (45) 364,(46) 364 Lhermitte, M. 220 (48) 115 Laipio, M. L. 233 (10) 130 Lamblin, G. 220 (35) 113, (41) 113, (48) Li, E. 422 (19) 410 Li,S. 85 (159) 77 115 Li,S.C. 80 (34) 52,73 LaMontJ.T. 84 (128) 69 Li, Y. 258 (11) 153 LeMount, J. T. 83 (116) 67, 69 Li, Y. T. 80 (34) 52, 73, (35) 52; 85 (159) Lamport, D. T. A. 29 (10) 6, (11) 6 77 Landsteiner, K. 324 (1) 311, 322 Liang-Tang, L. K. 339 (15) 327 Landy, M. 292 (11) 277 Lieberman, J. 132 (17) 127, (19) 127 Lang,S. 258 (6) 153 Liedgren,H. 82 (55) 56 Langlois, A. J. 298 (9) 180,181,187 Light, D. 85(156) 77 Lankester, A. 377 (8) 376 Lankf ord, B.J. 229 (3) 108, (7) 108 Lillford, P. J. 222 (51) 115 Lantz,R. S. 430 ( 21) 414 Lilliehook, B. 293 ( 35 ) 279 Larriba, G . 3 7 8; 403, 385, 387 Lindberg, B. 82 (49) 56, (50) 56, (51) Larsson, C. 232 (15) 126 56, (52) 56, (55) 56; 354 (31) 344 Laurell, C. B. 42,28; 232 (14) 126 Linsley, K. B. 293 (29) 279, 286, 287 Laurence, D. J. R. 355 (50) 350, 351 Lione,A. 84(131)69 Lautenschleger, J. T. 352 (7) 343, (8) 343 Lippman, M. 292 (19) 277; 293 (34) 279 Lawford, G. R. 42, 26 Lis, H. 269 (17) 258 Lazareva, M. N. 340 (40) 330 Lisowska, E. 325 (33) 320 Leamnson, R. N. 297 (2) 180, 181; 298 Litt, M. 229 (13) 109; 220 (37) 113 (14)181 Liu,T.-Y. 353 ( 22 ) 343,348 Livingston, D. C. 431 (36) 425 Leblond, C. P. 40, 34, 37; 83 (120) 67 Lockhart, L. H. 229 (18) 110 Lee, C. L. 320 (6) 296; 370 (6) 358, 359 Lockney,M. 83 (108) 71 Lee, D. 42, 32 Lodish, H. F. 43, 26; 44, 26, 28; 46, 26; Lee, N. 293 (22) 277, 278, 288, 289 299 (35) 188 Lee, Y. C. 29 (13) 6; 202 (21); 148 (38) Lombardi,B. 342 (73) 143; 431 (42) 424 Long,C. 297 (5) 180,195 Ledeen, R. W. 80 (33) 52; 82 (43) 52 Long, M . M . 227; 253 (10 ) 228, 230; 254 (48) 56 ' (14) 231, 234, (17) 231, 235-237, 240Lees, M. 403,382 242, (18) 237-239, (19) 243, (20) 243, Leffert, H. L. 339 (12) 326; 340 (33), 244, (21) 243; 255 (41) 248, 252, (45) (34), (37) 329 252, (47 ) 247 Legaz, M. E. 272 (9) 164, (12) 165, (14) Lonngren, J. 81 (55) 56 165,(17) 166 Lehman, E. D. 42,32 Loor,F. 272 (74) 268 LeKim, D. 82 (58) 59 Lopez-Vidriero, M. T. 220 (43) 113 Leloir, L. F. 40,22, 23; 43,23 Lorand,L. 172 (28) 170 V

1

V

1


458

GLYCOPROTEINS

Lotan, R. 256; 270 (39) 262, 266, 267; 271 (72) 266 Lote, C . J. 19 (8) 6, (9) 6 Louisot, R. S3 (98) 69 Lowick, J. H . B. 376 (6) 375 Luftig, R. B. 200 (61) 197 Lundblad, A . 29 (7) 6; 42; 43; 103 (28) 98, (29) 98; 148 (46) 144, (47) 144, (49) 144; 403,390; 422 (11) 408 Luner, S.J. 325 (40) 322 M Maccioni, H . J. 82 (79) 61 McClean, R. J. 84 (132) 69 McCluer, R. H . 80 (19) 51, (20) 51 McConnell, H . M . 270 ( 35 ) 259, 260 McCreaJ.F. 84 (134) 73 McCullagh, K. G . 254 (31) 243 McGuire, E . J. 40, 36; 42, 36, 37 37; 83 (112) 67,(121) 67 Mach, J. 355 (49) 350, 351; 356 (55) 350 Macher, B. A . 47; 80 (18) 49; 83 (108) 71 Mclntire, K. R. 339 (27) 328, 332, (28) 328, (29) 328 McKee, P. A. 272 (13) 165, (27) 169; 273 (29) 170 McKhann, C . F . 292 (8) 277 McKibbin, J. M . 82 (41) 52 McKnight, G . S. 42,26 McKusick, V . A. 226 (55) 224 McLellan, W . 298 (13) 181 MacPherson, I. 310 (14) 302 McQuiston, D . T . 324 (17) 312 McSherry, N . R. 229 (5) 108 Magee, S. C . 42, 32; 42, 32 Magnusson, S. 272 (25) 169 Mahler, H . R. 270 ( 49 ) 260, 264, 266; (52) 260, 264 Mahley, R . W . 84(125) 67 Maier, V . 222, 201, 202 Makita, A. 82 (70) 61, (83) 61; 83 (102) 69; 370 (18) 358, 359, 363 Maley, F. 86; 202 (1) 86, 95, (2) 86, 88, 93, 98, (3) 86, (4) 86, (5) 86, (7) 86, 90, 94, (9) 86, 97, (15) 87-89, 98, (16) 87-89, 98, (16) 87, 89, 90, (23) 95, 100; 103 (33) 98, 101, (34) 101; 247 (11) 137,(12) 137,(13) 137 Mallette, M . F . 372 (35) 361 Mallucci, L . 272 (71) 266 Maloney, W . H . 229 (21) 110, 111 Mangos, J. A. 229 (5) 108 Manley, G . 222, 201 Mann, D . L . 324(9) 312 Mann, K. G . 272 (11) 165 Mansson, J. E . 80 (35) 52


Marchesi, V. T . 20 (21) 13, 18, 19; 42, 37; 269 ( 24 ) 258, 259; 270 (33) 259, (37) 260, 262, 265; 294 (46) 288 Marciani, D . J. 199 (38) 188, 194 Marcus, D . M . 371 (50) 363 Mareel, M . 220 (46) 113 MarIer,E. 272 (27) 169 Maroteaux, P. 45; 149 (55) 145 Marsh, W . L . 20 (16) 10; 370 (26) 358 Marshall, R. D . 42, 23, 24, 36 Martens, P. 226 (56) 224 Martensson, E . 80(11) 48 Martin, C . L . 229 (10) 108 Martin, G . R. 226 (35) 218; 253 (7) 227 Martin, G . S. 199 (36) 188 Martin, I. 84 (142) 73 Martin, J. P. 232 (12) 125 Masamune, H . 370 ( 4 ) 358, 359, (5 ) 358, 359 Masseyeff,R. 339 (26) 328 Masson, P. K. 42 Masubuchi, M . 44, 33 Masukawa, A. 370 (4) 358, 359 Matalon, R. 248 (44) 144, (48) 144; 258 (11) 153 Matsubara, T . 403, 378 Matsumura,G. 202 (25) 98 Matthews, L . 229 (12) 109, (21) 110, 111 Mattsson, K. 133 (2) 129 Matuda,Y. 370 (4) 358,359 Matukas,V.J. 225 (16) 215 Mavligit, G . M . 292(10 ) 277 Mawal,R. 42, 32; 42, 32 Mawhinney, T . P. 254 (30) 243 Mayer, H . - E . , Jr. 102(6) 86, 87, 95, 98 Mayo, B . J . 229 (3) 108 Meager, A . 422 (18) 410 Meezan,E. 320 (2) 295 Meier, C . 248 (41) 143 Mellan,P. 299 (44) 192 M e l l a r s , R . C . 298 (16) 181 Mellon, P. 299 (48) 194 Melnick, J. L . 403, 379, 402 Mendicino, J. 44, 33 Merkow, L . 340 (55) 331 Merlin, L . M . 83 (122 ) 67 M e r s k y , H . 233 (1) 129,130 Meyer, F . A. 220 (45) 113 Meyer, R. G . 272 ( 9) 164 Michael, A. F . 226(54) 224 Michaelson, M . 340 (51) 331 Michalski, J. C . 42; 149 (54) 145, (55) 145 Michel, I. 444 (12 ) 433 Migita, S. 293 ( 23 ) 278, 288 Mihalev,A. 339 (30) 328 Mihalik, G . M . 411 (14) 408, 409


AUTHOR

INDEX

459

Miller, D.H. 19 (10) 6 Murakami, W. T. 310 (13) 302; 370 (1) Miller, D. K. 293 (39) 280, 282, 290, (40) 357 280, 281, 286, 288, 290; 294 (51) 290, Muramatsu, T. 42, 30; 43, 30; 45, 33; 202 291,(52 ) 290, 291 (14) 87, 91, 98,(19) 91, 95 Miller, E.C. 340(54) 331 Murgita, R. A. 340 (41) 330, (42) 330 Miller, E. J. 225 (16 ) 215, (17 ) 215, (22)Murphy, C. 40, 31 215, (28) 215, 218, (30) 218 Murphy, E . C , Jr. 180; 198 (28) 184, Miller, J. A. 340 (54) 331 188-190,192; 299 (34) 188-190,192 Miller, M. 19(10)6 Murray, R. K. 80 (13) 49 Miller, S. C, 293 (41) 280, 282, 286; 444 Murray-Lyon, A. M. 340 (39) 330 (11) 433, 442 Murthy, M . S. 320 (11) 302; 311; 324 Minase,T. 341 (70) 332 (15) 312, 315, 317, 320; 325 (29) 320, Miron,T. 371 (47) 364 322, (43) 322 Mitchell, L. W. 255 (41) 248, 252, (45) Myerowitz, R. 249 (63) 146 252,(47) 247 Myllyla, R. 226 (42) 222, (43) 222, (44) Miyazaki, M. 340 ( 36 ) 329 222 Moennig, V. 298 (15) 181 Moertel, C. G. 339 ( 29 ) 328 N Molnar, J. 43, 26; 83 (101) 69 Momoi,T. 80 (27) 52 Montelaro, R. C. 197 (4) 180 (50) 195 Nadler, H. L. 229 (9) 108, (10) 108 Montgomery, P. C. 147 (15) 138 Nagai, Y. 80 (24) 51, (25) 51, (26) 52, Montgomery, R. 20 (20) 11; 202 (6) 86, (27) 52; 324 (13) 312, 314 87, 95, 98, (21), (22); 432 (42) 424 Nager,C. 222,202 Montreuil, J. 29 (4) 5; 43, 21, 24, 26, 33; Nakada, H. 269 (27) 258, (28) 258; 293 45, 30; 248 (28) 140; 249 (55) 145 (24) 278 Mookerjea, S. 42, 32 Nakomori, S. 320 (13) 302 Moreau, R. 324 (16) 312, 322 Nant6,V. 233 (10) 130 Morell,P. 82 (63) 59 Narasimhan, S. 21; 43, 29-31, 34; 45, 29; Morgan, I. G. 270 (50) 260, 264 324 (19) 314, 320; 422 (17) 410 Morgan, W. T. J. 20(18)9 Naso, R. B. 180; 298 (20) 182-184, 189, Morgello, S. 294 (52) 290, 291 190, (21) 183, 184, (22) 183, 184, 190, Moro,S.P. 232 (8) 124,125 (23) 183, 184, 187, (26) 182-184, 192, Morre, D. J. 83 (111) 67, (118) 67, (119) (30) 182, 184, 191; 299 (33) 188, (55) 67, (122) 67; 84 (124) 67, (125) 67; 195 197 444 (16) 434 Nass, M'.K. 445 (21) 437 Morris, H. P. 340 (45) 331 Nathenson, S. G. 202 (19) 91, 95; 292 (2) Morris, J. E. 353 (19) 343, 344; 354 (39) 277; 324 (9) 312; 376 (3) 375 348 Natsu-ume, S. 293(23) 278, 288 Morris, N. P. 225 (34) 218 Nelson, M. M. 339 ( 25 ) 327 Morris, N. R. 229 (3) 108 Nemerson, Y. 172 (19) 166, (22) 166, 167 Morrison, M. 269 (8) 258 Neri, G . 276 (6) 276; 412; 430 (11) 412Morrison, T. G. 43, 26 415, 420, 421, (12) 412, 414, 418, 420, Moscatelli, E. A. 79 (7) 48 423, (14) 412, 414, 415, 424, (19) 413, Moschetto, Y. 254 (32) 243 414; 431 (35) 423 Moscona, A. A. 292 (3) 277, (4) 277 Neufeld, E. F. 85 (167) 79; 249 (59) 146, Moser,H.W. 80(19)51 (60) 146, (66) 146 Moskal, J. R. 47; 82 (80) 61, (84) 61, Neufeld, L. 258 (8) 153 (89) 61; 83 (105) 71, (108) 71 Neurath,H. 272 (16) 166 MoullecJ. 324 (16) 312,322 Neutra, M. 120 (27) 110 Movat,H.Z. 272 (5) 163 Neville, A. M. 355 (50) 350, 351 Mudholkar, G. C. 232 (5) 123 Newman, C. H. H. 147 (21) 138 Mueller, T.J. 269 ( 8 ) 258 Newman, R.A. 269 ( 25 ) 258 Mueller-Lantsch, N. 299 (46) 194 Newton, W. A. 119 (15) 109 Muir, L. 29 (13) 6; 254 (34) 246 Ng, V. L. 200 (62) 197 Muller-Spreer, H. C. 222, 201 Ng Ying Kin, N. M. K. 43; 46; 147 (25) Mullinger, R. N. 222, 201 139, (26) 139; 148 (29) 140, (30) 140 Munro, J. R. 43, 31, 34 Ni,Y. 370(7) 358,359


460


Nichols, M. 339 (11) 326, 328 Nickel, R. 340 (48) 331, (50) 331 Nicol,D. 158(3)153

PROCESSES

Orgebincrist, M. C. 272 (61) 261 0rjasaeter, H. 355 (52) 350, 351 Orr,A.H. 340 (39) 330 Orr,T.W. 340 (53) 331 Nicolson, G . L . 20 (24) 16, (25) 16, 17, (27) 19; 256; 269 (1) 256, 258, (4) Osawa, T. 269 (21) 258; 270 ( 32 ) 259; 256, 258, 268, (5) 256, 258, 259, 266, 293 (29) 279, 286, 287 268, (11) 258, (14) 258, (15) 258, (18) Overkerk, C. H. 248 (45) 144 258, 266, 268; 271 (65) 266, (66) 266, Owens, M. C. 132 (18) 127, (20) 127 (70) 266; 410 (3) 404; 430 ( 2 ) 412, Ownby, C . L . 432 (5) 412, 429, (6) 412, 429; 431 (39) Oyama,K. 370 ( 5 ) 358,359 429; 444 (3) 432 Oyasu,R. 324(10) 312 Ozanne,B. 432 (37) 425 Niemann, H. 372 (28) 358 Nilsen-Hamilton, M. 270 (45) 260, 263 Nilsson, B. 103 (28) 98 P Nilsson, S. F. 270 (47) 260, 263, 265 Pacuszka,T. 82 (91) 64-66 Noonan, K. D. 432 ( 38 ) 425 Pain, P. H. 220 (38) 113 Norberg, T. 403, 390 Norden, N. E. 42; 43; 103 (28) 98, (29) Painter, R.G. 269 (14 ) 258 Pallavicini, C. 222 (50) 115 98; 248 (46) 144, (47) 144 (49) 144 NordinJ.H. 29(12)6 Nordquist, R. E. 325 (41) 32 , Norgaard-Pederson, B. 339 (24) Norman, R. M. 147 (21) 138; 249 (56) 145 Palo, J. 133 (2) 129, (6) 130, (7) 130, (9) 130, (13) 130, 131; 248 (51) 145, (52) Norton, W. T. 80 (12) 49 145 Nosslin, B. 232(14) 126 Panet,A. 198 (7) 180,192 Nowotny, A. 293 (33) 279 Papas, T.S. 199 (38) 188,194 Nunez, E. 339 (16) 327, 329 Parker, S. 340 (58) 331 Nunez, H . A . 47 Parodi, A. J. 40,22, 23; 43,23 Nylen,M. 253 (7) 227 Partridge, S. M. 253 (2) 227 Pascher, I. 81 (40) 52, (41) 52, (42) 52; O 371 (36) 361 Oakes, D. D. 339 (8) 326 Pastan,I. 411(20)410 O'Brien, J. S. 84 (141) 73; 248 ( 35) 143, Patel,V. 148 (33) 140 (36) 143 Patt,L.M. 83(114)67 Ockerman, P. A. 42; 103 (28) 98; 248 (46) Paul,B. 158 (10) 153 Pauling, L. 353 (14) 343, 344 144, (49) 144 Paulson, J. C. 43,33; 44,36, 37 Oda,T. 340(38) 330 Pawson,T. 199 (36) 188 O'Donnell, P. 199-200 (58) 197 Paz, M.A. 224 ( 5 ) 213 Ogata, S. 43, 30 Pearlstein, E. S. 269 (13) 258 Ogawa,K. 342 (70) 332 Peers, M.C. 149(55) 145 Ohashi, M. 80 (29) 52 Ohnishi, T. 253 (11) 228, 237, 240; 254 Pentchev, P. 150; 158 (5) 150, 153, 157, (18) 237-239; 255 (45) 252 (16) 150, (17) 154, (18) 150; 159 (23) Ohno, S. 293 (23) 278,288 155 Oh-uti, K. 370 (3) 357, 359, (4) 358, 359 Perdome, J. M. 310 (7) 296, 298 Oikarinen, A. 226 (41) 222, (45) 222 Perlman, P. 370 (12 ) 359; 403, 378 Okamoto, K. 254 (19) 243, (20) 243, 244, Pernis, B. 271 (74) 268 (21) 243 Perova, S. D. 339 (1) 326 Okita,K. 342 (72) 332 Perret,V. 276 (5) 275 Old, L.J. 292(5) 277 Perrotto, J. L. 84(128 ) 69 Olivecrona, T. 222, 204; 222, 204, 207-210 Peters, J.H. 292(16) 277 Olshevsky, U. 299 (35) 188, (39) 188, 190 Peters, S. P. 84 (152) 75, 77; 148 (38) 143 Onda,H. 342 ( 69 ) 332 Peterson, T. 172 ( 25) 169 O'Neil, P. A. 431 (25) 414, 420 Petterson, B. M. 199 (38) 188, 194 Onoe, T. 341 (67) 332, (70) 332 Philipson, L. 199 (39) 188,190 Opheim, D. J. 84 (139) 73; 247 (10) 137 Phillips, H. J. 430 (20) 413 Oppeheim, J. D. 272 (60) 261, 265 Phillips, S. F. 356 ( 56 ) 350 Oppenheimer, E. H. 229 (17) 109,110 Pieringer, R. A. 84(136) 73


AUTHOR

461

INDEX

Pietropaolo, C. 403, 373 Pietrzykowa, B. 247 (17) 138 Piez,K.A. 226(35)218 Pigman, W. 29 (2) 5, 13, 14; 20 (14) 6; 276 (5) 275 Pilotti, A. 82 (52) 56 Pimlott,W. 372 (36) 361 Pincus, T. 298 (16) 181; 299 (58) 197 Pinter, A. 297 (1) 180,181 Pinteric, L. 42, 34; 44,34; 83 (121) 67 Pinto da Silva, P. 269 (11) 258 Pister,L. 298 (15) 181 Pitlick, F. A. 270 (53) 261, 264 Pitot,H. C. 342 ( 60 ) 331 Pizieux,0. 254 ( 32 ) 243 Pizzo,S. V. 273 (29) 170 Planinsek, J. A. 293 (32) 279,280 Plantner, J. J. 20(15)6 Pless, D. D. 43,23, 24 Plummer, T. H., Jr. 202 (1) 86 (4) 86,(5) 86,(9) 86, 97 Podolsky, D. K. 83 (116) 67, 69; 84 (129) 69 Podrazky, V. 254 (35) 246, (36) 246 Poler, M. 340 (44) 330 Polim,G. 232 (12) 125 Pollitt, R. J. 43; 133 (1) 129, 130, (13) 130,131, (14) 131 Polony, I. 220 (40) 113,114,116 Porter, C.J. 84 (135) 73 Porter, M. 258 (9) 153 Poschmann, A. 325 (36) 322 Poste, G. 269 (5) 256, 258, 259, 266, 268; 272 (78) 268; 420 (3) 404 Potter, J.L. 229 (12) 109 Pouyssegur, J. 422 ( 20 ) 410 Powell, J. T. 43,32; 44,32 Prehn, R. T. 292 (6) 277, (9) 277 Prendergast, R. C. 370 (9) 358, 359 Presper, K. A. 82 (90) 61, (92) 64 Prestegard, J. H. 82 (56) 59 Pretty, K. M. 43; 133 (13) 130, 131, (14) 131 Price, H. C. 82 (43) 52 Price, J. M. 340 (54) 331 Prieels, J. P. 40, 37 Prilzl,P. 258 (3) 153 Princler, G. L. 339 (28) 328 Pritchard, D. G. 353 (12 ) 343; 354 (27) 344, (32) 344; 356 (57) 350, (59) 350, 351 Prockop, D. J. 226 (36) 218, (37) 218, 222, (49) 223 Pusztaszeri, G. 355 ( 49 ) 350, 351; 356 (55) 350 R

Rabb,M.C. 272 (68) 266 Rabinowitz, Z. 410 (4) 404

Radcliffe, R. 272 (19) 166, (22) 166, 167 Radhakrishnamurthy, B. 222; 222, 201, 202; 254 (37) 246 Radin, N. S. 82 (63) 59; 85 (160) 77, 78; 248 (36) 143 Raff, M.C. 272 (71) 266 Raghupathy, E. 83 (99) 69 Ramseur, J. M. 299 (56) 195 Rao, A. K. 44, 33 Rao, G.J. S. 229 (9) 108 Rapaka, R. S. 254 (20) 243, 244 Rapoport, S. 258 (5) 150,153,157 Rapport, M. M. 85 (154) 77; 371 (49) 363 Rauvala, H. 84 (144) 75; 247 (4) 136, (7) 137 Read, R. C. 222, 201-204, 210 Rearick, J. I. 43, 33; 44, 36, 37 Rebers,P.A. 432 (27) 414 Redi K B M 225(24) 215

112, (43) 113; 222 (52) 115 Reinhold, V. N. 225 (15) 215 Reisfeld, R. A. 292 (1) 277 Rekosh,D. 298 (31) 187 Renton,P.H. 324 (18) 312 Reutter, W. 403, 378 Reynolds, J. A. 220 (39) 113 Reynoso,G. 355 (53) 350 Rhodes, R. K. 225 (22) 215 Richard, M. 83 (98) 69 Richards, F. M. 269 (12) 258 Richer, C. 220 (34) 113 Riekkinen, P. 233 (6) 130, (9) 130 Riordan, J. 43, 31, 34 Ripamonte, A. 254 (19) 243 Risteli, L. 225 ( 32 ) 218, 222; 226 (42) 222, (43) 222, (44) 222 Robbins, P. W. 44, 24; 46, 24; 83 (106) 71; 202 (12) 87; 320 (2) 295, (14) 302 Robert, B. 254 (25) 243, (27) 243 Robert, L. 254 (25) 243, (26) 243, (28) 243, (31) 243 Roberts, B. I. 222, 201-204, 207, 210 Roberts, G. P. 220 (36) 113; 292 (17) 277 Robertson, J. 42, 24, 31; 202 (17) 91 Robertson, M. 42, 24, 31 Robinson, D. S. 222, 201 Robinson, G. B. 40, 26 RobozJ.P. 292 (15) 277 Roden, L. 44, 21, 33, 34 Rodman, J. 85 (165) 79; 158(6) 153 Roepstorff, P. 272 (10) 165 Rogentine, G. N., Jr. 324 (9) 312 Rogers, J. 270 ( 31) 259 Rogers, L. A. 272 (27) 169 Roghmann, K. J. 232 (5) 123 Rome, L.H. 249 ( 60) 146 Ronin,C. 84 (127) 67 Ronzio, R. A. 84(126) 67


462

GLYCOPROTEINS A N D GLYCOLIPIDS I N DISEASE

Roosien, J. 431 (40 ) 429 Roseman, S. 40, 33, 36; 44, 34, 36, 37; 81 (60 ) 59, (61) 59, (64 ) 59; 82 (68) 61, (71) 61, (72) 61, (73) 61, 66, (74) 61, (75) 61, 67; 83 (112) 67, (121) 67; 324 (21) 314 Rosenblith, J. 271 (69) 266; 430 (7) 412, 429 Rosenfeld,L. 148 (38) 143 Rosenfelder, G. 370 (13) 358 Rosenthal, A. L. 19 (12) 6 Ross, A. H. 270 (35) 259,260 Ross, R. 253 (1) 227; 254 (22) 243, 246, (23) 243, (24) 243, (26) 243, 246, (29) 243, (34) 246 Rossomando, E. F. 271 (64) 261 Roth, S. 83 (110) 67, (112) 67, (117) 67; 84(133) 69 Rothman, J. E. 44, 26, 28 Roukema, P. A. 148 (45) 14 Roussel, P. 120 (35) 113, (41 115 Roveri,N. 254(19 ) 243 Rubin, D. 226 (57) 224 Rueckert, R. R. 298 (8) 180, 192; 299 (32) 187 Ruizitt,B. 254 (37) 246 Ruoslahti, E. 276 (3) 275; 339 (7) 326, (13) 326, (14) 327; 352 (4) 342, 346; 356 (61) 351; 422 (13) 408 Rutishauser, U. 430 (8) 412 Rutzky, L. P. 324 (10) 312 Ryd,W. 294 (53) 291 Ryd,W. 294 (53) 291 Ryhanen, L. 226 (37) 218, 222, (38) 222, (39), (40) 218 Ryman, B. 258 (14) 153

PROCESSES

Santer, U. V. 420 ( 6 ) 404, 405; 422 (15) 408 Sarvin,A.J. 249 (50) 146 Sass-Kortsak, A. 232 (8) 124,125 Sato T. 44 43 Saunders, T\E. 299 (33) 188 Savolainen, H. 133 (11) 130, (13) 130, 131; 248 (51) 145, (52) 145 Scanlin, T. F. 422 ( 7 ) 405, 408, (12 ) 408 Scanlon, E . F.

311; 324 (12) 312, 318,

(15) 312, 315, 317, 320; 325 (29) 320, 322, (43) 322 Schacter, H .

21; 42, 31, 33, 39; 42, 26, 34;

43, 29-31, 34; 44, 21, 22, 26, 33, 34, 36, 37; 45, 29; 46, 31; 80 (13) 49; 83 (121) 67; 324 (19) 314, 320; 422 (17) 410 Schafer,D.D. 82 (56) 59 Schafer, W. 297 (4) 180, 187; 298 (9) 180,181,187,(15) 181,(19) 181

Scheraga, H. A. 276 (4) 275 Scherrer, M. 297 (5) 180,195 Schiffman,E. 253 (7) 227 Schimke, R. T. 42, 26 Schiphorst, W. C. M. 46, 33, 39 Schlamowitz, M. 431 (29) 415, (32) 426 Schleich, J. 224 (1) 213 Schlesinger, J. 272 (79) 268 Schlesinger, P. 258 (6) 153 Schlesinger, P. H. 85 (165) 79 Schlesinger, S. 45, 24, 26, 31; 202 (13) 87 Schmickel, R. D. 249 (63) 146 Schmid,K. 42,33 Schmidt-Ullrich, R. 270 (44) 260, 263, 266 Schneider, M. 325 (46) 319 Schnute, W. C., Jr. 353 (19) 343, 344; 354 (25) 344, (27) 344; 355 (46) 348 S Schofield, D. J. 226 ( 36 ) 218 Schotz, M. C. 222,210 Saccomani, G. 255 (44) 252 Sachs, L. 272 (76) 268, (77) 268; 420 (4) Schreiber, G. 430 (16 ) 413, 416, 420 Schreiber, M. 430 (16) 413, 416, 420 404; 430 (8) 412 Schultz,A. 82 ( 64 ) 59 Sadler, J.E. 44,36,37 Saito, H. 82 (54) 56; 354 (30) 344; 372 Schut, B. L. 45, 30 Schwarting, G. A. 371 (50) 363 (31) 361 Schwartz, M. L. 173 ( 29) 170 Sajdera,S.W. 254 (39) 252 Schwartz, R. H. 232 (5) 123 Sakamoto, C. K. 430 (10) 412 Schwick,H. 273 (32) 170 Salvaterra, P. M. 270 (52) 260, 264 Schwyzer, M. 42, 36; 44, 37; 45, 37; 83 Sambrook, J. 431 (37 ) 425 Samuelsson, B. E. 82 ( 40 ) 52, (41) 52, (97) 64-66 Scribney, M. 82 (59) 59 (42) 52; 372 ( 36 ) 361 Scrimgeour, J. B. 339 (25) 327 Sandberg, L. B. 254 (15) 231, (16) 231 Seaman, F. 376(5) 375 Sandford, P. A. 354 (29) 344 Segaloff,A. 444(13 ) 433 Sando, G. N. 249 (60) 146, (66) 146 Sanford, B. H. 292 (20) 277, 279, 280, 288; Seglen,P.O. 432 (33) 416 293 (21) 277, 279, (27) 279, (28) 279, Segrest, J. P. 20 (26) 19; 269 (7) 258 280, 286, 288; 324 (26) 315; 444 (10) Seifert,E. 298 (15) 181 Seifter, S. 224(5) 213 432


463

AUTHOR

INDEX

Sell, S.

326; 339 (6) 326, (9) 326, (11)

326, 328, (12) 326, (17) 333, (19) 327329, (20) 327, (31) 328, 330, 331, 334; 340 (32) 328, (34), (35) 329, (43) 330, (44) 330, (45) 331, (48) 331, (49) 331, (50) 331, (51) 331; 341 (61) 331, (62) 332, (65) 332-334, (73), (75) 334 Sen, A. 198 (6) 180,195 Senior, R. G. 46; 103 (30) 98; 148 (29) 140 Seppala, M. 339 (7) 326, (22) 327 Seyer, J. M. 225 ( 29 ) 215, 218 Shander, M. H. M. 197 (2) 180,181 Shanmugam, G. 199 (42) 190,194 Shapira,E. 119 (10) 108 Shapiro, D. 158 (16) 150 Shapiro, L. 85 (167) 79; 158(8) 153 Sharon, N. 269 (17 ) 258; 270 (38) 260 262, 265; 271 (72) 266 Sharp, H. L. 131 (1) 122-125 233 (3) 129,(8) 130, 131 Sheppard, H. W., Jr. 340 (43) 330, (44) 330, (50) 331 Sherblom, A. P.

432

Sherlock, S. 132 (11) 125 Sherr, C. J. 45, 26; 198 (6) 180,195 Shih, C. K. 220 (37) 113 Shiloach, J. 158 (18) 150 Shin, B. C. 444 (5) 432, 434-436 Shincarial, A. 199 (43) 190,194 Shinitzky, M. 272 (76) 268, (77) 268 Shinozuka,H. 342 (73) Shively, J. E .

342; 354 (35) 346; 355 (47)

348, 350; 356 (58) 350, (59) 350, 351, (60) 351 Shotten, D. M. 269 (16) 258 Showe, M.H. 299 (54) 195 Shur, B. 83 (110) 67; 84 (133) 69 Shuster, J. 339 (8) 326

Slomiany, A. 80 (15) 49, 75, (36) 52; 82 (37) 52; 82 (94) 64; 372 ( 39 ) 361, (40) 361 Slomiany, B. L. 80 (15) 49, 75, (36) 52; 82 (37) 52; 82 (94) 64; 371 (39) 361, (40) 361 Sly, W. S. 85 (166) 79; 249 (64) 146, (65) 146 Small, B. 46,26 Smith, A. E. 299 (36) 188 Smith, C. A. 45, 32 Smith, C M . 272 (26) 169 Smith, D. F. 430 (11) 412-415, 420, 421, (12) 412, 414, 418, 420, 423, (13) 412, (14) 412, 414, 415, 424; 431 (35) 423 Smith, E.L. 82(41)52 Smith, F. 202 (22); 354 (36) 348; 431 (27) 414 Smith R.T 292 (11) 277 Snell,E.E. 82 (57) 59 Soloff, M. S. 339 (15) 327 Somers, K. 403, 379 Sonoda,S. 431 (29) 415 Sottrup-Jensen, L. 272 (25) 169 Spector,S. 229 (12) 109 Spielvogel, C. 248 (34) 143 Spik, G. 45, 30; 248 (28) 140; 249 (55) 145 Spiro,M.J. 45,22,76 Spiro, R. G. 29 (1) 5-7, 13, 15; 45, 22, 76; 224 (7) 213; 225 (11) 214, 215, (14) 215, (23) 215; 226 (50) 223, 224, (51) 223; 269 (19) 258, (20) 258; 354 (26) 344 SprangerJ. 42; 248 (53) 145 Springer, G . F. 293 (36) 279; 310 (11) 302; 311; 324 (2) 311, 312, (3) 311,

322, (6) 311, (7) 311, 312, 322, (11) 312, (12) 312, 318, (13) 312, 314, (14) Siddiqui, B. 79 ( 6 ) 48, 49; 295; 320 (10) 312, 322, (15) 312, 315, 317, 320, (19) 302, 304, 305; 371 (30) 361 314, 320, (20) 314, 320, (25) 315, (27) Sillerud, L. O. 82 (56) 59 315; 325 (28) 314, 317, (29) 320, 322, Siminovitch, L. 45, 29; 411(17) 410 (30) 320, (31) 320, (32) 320, (34) Simmons, D. A. R. 370 (12) 359; 403, 378 320, (35) 320, (37) 312, 322, (40) Simmons, J. L. 83 (109) 71, 76 322, (42 ) 322, (43 ) 322, (45 ) 322 Singer, S. J. 20 (24) 16; 269 (1) 256, 258, Squire, R.A. 341 (74) 334 (3) 256 Sramek, S. 378; 403, 385, 387 Sirakov,L. M. 339 (30) 328 Srinivasan, S. R. 211 Six,H.R. 372 (44) 364 Stacey,D. W. 199 (49) 194 Sizaret, P. 339 (10) 326 Stahl, P. 85 (165) 79; 158 (5) 153 Skehel,J.J. 272 ( 62 ) 261,265 Stalder,K. 324 (6) 311 Skelly, H. 339 (19) 327-329; 340 (34) Stallcup, W. B. 173(31)170 Skelly, J. 403, 385 Stanislawski-Birencwajg, M. 339 (3) 326 Slayter, H. S. 293 (40) 280, 281, 286, 288, Stanley, P. 43, 29, 30, 31; 45, 29; 411 (17) 410 290, (44) 286, 288; 353 (10) 343, (11) Starcher, B. C. 253 (10) 228, 230, (12) 343, 345 228, 231; 255 (43) 252 Sloane-Stanley, G. H. 403, 382


464

GLYCOPROTEINS

Starling, J. J.

276 (6) 276; 412; 430 (19)

413, 414; 431 (22) 414, 418, 419, (31) 415, 429 Steck, T. L. 431 (41) 426; 444 (15 ) 433 Steer, C. 159 (21) 150 Stefanko,St. 147 (17) 138 Stegner, H. E. 325 (36) 322 Steigerwald, J. C. 82 (72) 61 Stein, M. 148 (34) 143 Steineman, A. 270 (54) 261, 265 Steiner, D .

378

Steiner, M. 403,379,385 Steiner, S. 403, 379, 385, 387, 402; 411 (10) 408 Steinman, H. M. 41, 36 Stellner, K. 81 (54) 56; 320 (9) 302; 354 (30) 344; 370 (20) 359; 372 (31) 361, (43) 364; 403, 378 Stenflo, J. 272 (10) 165; 173 ( 34) 1718 Stern, R. C. 222 (49) 115 Stetson, B. 46, 24 Stewart, T. H. M. 324 (23) 314, 319 Sticht, G. 81 (58) 59 Stillman, D. 340 (35) 329, (51) 331 Stirling, J. 148 (28) 140 Stockert,E. 199 (58) 197 Stoffel, W. 81 (58) 59 Stoffyn, A. 45, 33; 46, 33 Stoffyn, P. 45,33; 46, 33; 82 (78) 61 Stone, J. D. 84 (134) 73 Stoolmiller, A. C. 82(81) 61 Storm, E. 272 (23) 167 Strand, M. 198 (16) 181 Stratton,F. 324 (18) 312 Strecker, G. 42; 45, 30; 248 (28) 140; 149 (54) 145, (55) 145 Strobach, D. R. 85 (169) 49 Stromberg, K. 298 (8) 180,192 Str0mme, J. 133 (12) 130 Struck, D. K. 45, 23 Stryer,L. 270 (54) 261,265 Sturgess, J. 229 (20) 110; 220 (28) 112 Sturgeon, P. 324 (17) 312 Subba,R.K. 84 (136) 73 Sugahara, K. 40; 134 (15) 131, (16) 131 Sukeno,T. 202 (3) 86 Sullivan, S.J. 299 (50) 195 Summers, D. F. 202 (17) 91 Summers, P. F. 42, 24, 31 Sundblad, G. 354 (33) 344, 347, 348; 356 (62) 351 Sung, S.J. 371 (33) 361 Susz, J. 247 (1) 135,143, (3) 136 SuttieJ. W. 272 (24) 167 Sung, S.-S.J. 80 (32) 52; 82 (53) 56; 84 (140) 73, (146) 75, (147) 75 Suzuki, A. 82 (21) 51,(22) 51 Suzuki, K. 84 (149) 75, (150) 75; 147 (20) 138

A N D GLYCOLIPIDS

I N DISEASE

PROCESSES

Suzuki, M. 80(14)49 Suzuki, Y. 247 (20) 138 Sveger, T. 232 (3) 123,124,128 Svenberg,T. 356(62) 351 Svennerholm, L. 80 (35) 52 Svensson, S. 29 (7) 6; 42; 43; 82 ( 49 ) 56, (50) 56, (51) 56, (52) 56; 203 (28) 98, (29) 98; 248 (46) 144, (47) 144; 354 (31) 344, (33) 344, 347, 348; 403, 390; 422 (11) 408 Sweeley, C . C .

47; 79 (6) 48, 49, (7) 48;

80 (8) 48, (18) 49, (30) 52, (32) 52; 82 (53) 56; 82 (85) 61; 83 (107), (108) 71; 84 (140) 73, (145) 75, (146) 75, 77, (147) 75; 85 (161) 77, 79; 372 (33) 361 Swerdlow, A. 220 (32) 113 Switzer, M.E. 272 (13) 165 Sy,D 43,26 Szigetti, M.

254 ( 25 ) 243 T

Tabas, I. 45, 24, 26, 31; 202 (13) 87 Taber, R. 298 (31) 187 Tager, J. M. 84(148) 75 Tai, T. 45, 33; 202 (20) 95; 103 (26) 98 Takashi,N. 203 (27) 98 Taket,K. 340 ( 36 ) 329 Taketomi, T. 80 (10) 48; 372 (34) 361 Tanaka,H. 84 (150) 75 Tarentino, A. L .

86; 202 (1) 86, 95, (2)

86, 88, 93, 98, (3) 86, (4) 86, (5) 86, 97; (15) 87, 88, 89, 98, (23) 95, 100; 203 (33) 98, 101, (34) 101; 247 (11) 137, (12) 137 Taswell,H.F. 324(17) 312 Tatarinov, Y. 339 (2) 326 Taylor, L. 232 (11) 125 Tchipysheva, T. A. 341 (71) 332 Teasdale, C. 292 (17) 277 Teebor,G.W. 342 (64) 332 Tegtmeyer, H. 324 (13) 312, 314; 325 (37) 312, 322 Temmink, J. H. M. 430 ( 9 ) 412; 431 (40) 429 Terao, T. 270 ( 48 ) 260, 263, 264, 266 Terry, W. D. 339 (14) 327; 353 (15 ) 343; 355 (43) 348,(44) 48 Testas,M. 83 (101) 69 Thatcher, J. 292(17) 277 Thieffry,S. 247 (19) 138 Thistlewaithe, W. 84 (125) 67 Thomas, D. B. 269 (22) 258 Thomas, M. A. W. 20 (22) 13; 220 (44) 113 Thomas, M. W. 432 ( 25 ) 414, 420


AUTHOR

465

INDEX

Thomas, N. R. 226 (56) 224 Thomas, P. 354 (37) 348, (40) 348; 355 (41) 348, (42) 348 Thompson, A. R. 172(9) 164 Thompson, D. M. P. 352 (3) 342 Thompson, W. D. 254 (17) 231, 235-237, 240—242 Tieslau,C. 377 (8) 376 Tingey, A. H. 147 (16) 138, 145, (21) 138; 149 (56) 145 Titani,K. 172 (16) 166 Todaro, G. J. 198 (6) 180, 195; 310 (5) 295 302 Todd, C . W .

342; 352 (4) 342, 346, (5)

342, (7) 343, (8) 343, (9) 343; 353 (12) 343, (15) 343, (19) 343, 344, (23) 343, 346, 348, (24) 344; 354 (25) 344, (27) 344, (32) 344, (35) 346, (39) 348; 355 (43) 348, (44) 48 (46) 348 (47) 348, 350; 356 (57) 350 (59 ) 350,351 Tokura,S. 272 (28) 170 Tom, B. H. 324 (10) 312 Tomasi, T. B., Jr. 340 (41) 330, (42) 330 Tomita, M. 42, 37; 269 (24) 258, 259; 270 (33) 259 Tonegawa,Y. 372 (41) 361 Tonneguzzo, F. 45, 26 Tot, P. D. 370 (9) 358,359 Touster, O. 84 (139) 73; 247 (10) 137 Toyoshima, S. 269 (21) 258 Trayer, I. P. 45, 32 Trefts,P. 340 (43) 330 Trelstad, R. L. 226 (57) 224 Tress, E. 299 (57) 195, (58) 197 Tribulev, G. P. 324 (4) 311 Trier, J. S. 220 (27) 110 Trimble, R. B.

86;

202 (7) 86, 90, 94,

(16) 87, 89, 90, (23) 95, 100; 147 (13) 137 Trowbridge, I. S. 270 ( 45 ) 260, 263 Tsai,W.P. 297 (5) 180,195

Uhlenbruck, G. G. 269 ( 25 ) 258 UhrJ.W. 45,26 UittoJ. 226 (49) 223 Ukena, T. E. 271 (69) 266; 430 ( 7 ) 412, 429 Umenoto, J. 202 (24) 98 Ungkitchanukit, A. 422 (18) 410 Unrau, A. M. 354 (36) 348 Uriel, J. 339 (3) 326 U r r y , D . W . 227; 253 (3) 249-252, (9) 227, (10) 228, 230, (11) 228, 237, 240, (12) 228, 231; 254 (14) 231, 234, (17) 231, 235-237, 240-242, (18 ) 237-239, (19) 243, (20) 243, 244, (21) 243; 255 (40) 252, (41) 248, 252, (42) 249, 252, (44) 252, (45) 252, (46) 229, (47) 247 Ushijima,K. 85 (164) 79 Usui,N 272 (66) 266

Vallette, G. 339 (16 ) 327, 329 Van Beek, W. P. 420 ( 5 ) 404 Van Den Berg, G. 248 (45) 144 van den Bosch, J. F. 431 (40) 429 Van den Eijnden, D. H. 46, 33, 39; 293 (43) 286, 287, (45) 286, 287 VanHoff,F. 248 (50) 144 Van Zaane, D. 299 (41) 190,194 Vanaman, T. C. 40, 31 Vance, D. E. 80 (30) 52 Vance, J. C. 232(5) 123 Vargas, V.I. 46,28 Vehar, G. 272 (15) 165, 166, 171; 173 (36) 171 Velicer, L. F. 83 (107) Venditti, J. M. 293 (34) 279 Via,D.

378; 403, 379

Vicar, G. 370 (26) 358 Vicari,G. 20 (16) 10 Vincendon, G. 270 ( 51) 260, 264 Tsao, D . 295; 320 (1) 295, 306, (16 ) 304, Vitetta,E. 299 (58) 197 Vliegenthart, J. F. G. 45, 30 305, 307, 308 Vogt,P.K. 299 (44) 192 Tsay, D. D. 432 (32) 426 Tsay, G. C. 103 (32) 98; 247 (27) 139, Voight,K.D. 222,201 Volpin, D. 254 (19) 243, (33) 243 140,144; 148 ( 48) 144 Von Den Helm, K. 299 (37) 188 Tsopanakis, A. D. 46, 32 VonFigura,K. 249 (62) 146 Tsukamoto-Adey, A. 297 (3) 180,181 von Kleist, S. 355 (48) 350, 351 Tung, J. S. 299 (58) 197 Turberville, C. 310 (12 ) 302; 355 (50) Voynow,J.A. 422 (12 ) 408 350, 351, (51) 350, 351; 370 ( 23 ) 359 Vuento, M. 276 (3) 275; 356 (61) 351; 422 (13) 408 Turco, S.J. 46,24; 202 (12) 87 Tzingilev, D. 339 (30) 328 W

U

Uemura, K. 371 (44) 364 Ueno,K. 80 (28) 52

Waechter, C. J. 46,21,22, 23 Wagh, P. V . 201; 222, 201-210 Walborg, E. F. 276 (6) 276


466

GLYCOPROTEINS


Walborg, E . F., Jr.

5; 412; 430 (11) 412Wetmore, S. 43, 31, 34 415, 420, 421, (12) 412, 414, 418, 420, Whelan,W. 258 (4) 153 423, (13) 412, (14) 412, 414, 415, 424, White, D. 83(117)67 (19) 413, 414, (21) 414; 431 (22) 414, White, H. J. 222, 201-204, 210 418, 419, (23 ) 414, (25 ) 414, 420, (31)Whitehead, J. 320 (1) 295, 306, (10) 302, 415,429, (32) 426, (35) 423 304, 305 Waldmann, T. A. 339 (27) 328, 332, (29) Whitehead, J. S. 42, 36; 295 328 Whitehead, R. H. 292 (17) 277 Walker, J. M. 354 (40) 348 Wiegandt, H. 82 (46) 53; 84 (143) 75 Walker-Nasir, E. 120 (41) 113 Wiener, F. 294 (48) 288, (49) 288 Wallace, B. H. 370 (8) 358, 359 Wiener, G. 294 (47) 288 Wallace, R. E. 431 (24) 414 Wiesman,U.N. 220 (33) 113 Wallach, D. F. H. 270 (44) 260, 263, 266; Wiesmann,U. 248 (43) 143 431 (41) 426; 444 (15) 433 Wiggins, R.C. 272 (6) 164 Waller, R. K. 370 (14) 358,359, 369 Wigzell, H. 292 (18) 277; 356 (61) 351 Walsh, K. A. 171 (2) 163; 172 (16) 166 Wilchek, M. 372 (47) 364 Waltuck,B. 158 (4) 153 Wile, A. G. 325 (40) 322 Wang,F.F.C. 102(18)91 Williams, A. F. 270 (46) 260, 263 Wang,K. 269 (12) 258 Williams, D. 46, 31 Wang, L. H. 199 (44) 192 Wang, S.-M. 370 (19) 358, 359 369 Wilson, J. R. 21; 42, 28; 46, 31 Wang, S.W. 310(15)304 Wilson, T. 370 (27) 358 Wanner,A. 120 (30) 112 Winzler, R. J. 20 (19) 9; 224 (6) 213, 215; Ward, S.R 247 (21) 138 269 (22) 258; 411 (9)408 Warner, G. 370 ( 20 ) 359; 403, 378 Wirth, D. F. 46, 26; 202 (12) 87 Warner, N. L. 353 (17) 343 Witte, O. N. 297 (3) 180, 181; 298 (12) Warren, C. D. 42, 23 181 Warren, L. 320 (3) 295; 353 (21) 343; Witting, L. A. 248 (39) 143 403, 378; 420 (1) 404; 431 (34) 420; Witz,I.P. 292 (18) 277 444 ( 4 ) 432 : 445 ( 20 ) 434, (21) 437 Wold,F. 259 (25) 157 Wasniowska, K. 325 (33) 320 Wolf, A. 83(106) 71 Watabe,H. 341 (63) 332 Wolf, D. P. 229 (13) 109; 220 (37) 113 Watanabe, A. 340 (36) 329 Wolfe, L. S. 43; 46; 103 (30) 98, (31) 98; Watanabe, I. 148(33) 140; 403,378 247 (25) 139, (26) 139; 248 (29) 140, Watanabe, K. 80 (23) 51; 82 (93) 64; 370 (30) 140 (24) 358, 359, 369; 371 (28) 358, (43) Wolman, S. R. 340 (46) 331, (47) 331 364 Wong,C.G. 80(32) 52 Watkins, W. M. 46,34 Wood, R. E. 229 (1) 108, 112, (23) 110; Watson, K. F. 298 (27) 184, 192, (28) 220 (30) 112 184, 188-190,192 Wood,T.G. 299 (33) 188 Waxdal, M. J. 270 (47) 260, 263, 265 Wrann, M. 276 (3) 275; 353 ( 24 ) 344; Weinberg, R. 299 (39) 188,190 422 (13) 408 Weinstein, I. B. 403, 378 Wrann, W. 356(60) 351 Weinstein, M. J. 272 (14) 165 Wray, V. P. 430 (11) 412-415, 420, 421, Weiser, M. M. 83 (115) 67, (116) 67, 69; (21) 414; 432 (23) 414 84 (128) 69,(129) 69 Wu,E.-S. 272 (78) 268 Weiss, J. B. 29(8)6,(9)6 Wu,H.C. 320 (2) 295 Weiss, L. 293 (32) 279,280 Wu,Y.-C. 202(21) Weissman, I. L. 297 (3) 180, 181; 298 Wusteman, F. S. 254 (38) 246 (12) 181 Wepsic, H. T. 339 (6) 326; 340 (48) 331 Werber, U. 222, 201 Y Wessels, M. 232 (19) 127 Westall,F. C. 42,36 Yahara,I. 272 (75) 268 Westberg, N. A. 226 (54) 224 Yamaguchi, N. 403, 378 Westenbrink, F. 272 ( 63 ) 261, 265 Westwood, J. H. 320 (12) 302; 354 (37) Yamaguchi, Y. 370 (5) 358,359 348, (40) 348; 355 (41) 348, (42) 348; Yamakawa, T. 80 (14) 49, (21) 51, (22) 51, (29) 52 370 (23) 359


AUTHOR

INDEX

Yamashina, I. 40; 134 (15) 131, (16) 131; 147 (14) 137; 269 (27) 258, (28) 258; 293 (24) 278 Yamashita, K. 45, 33; 102 (20) 95; 103 (26) 98 Yamauchi, F. 44,33 Yanagimachi, R. 271 (66) 266 Yanjg, H. J. 310 (11) 302; 325 (30) 320, (31) 320,(32) 320,(34) 320 Yates, S.G. 172 (8) 164 Yeung,K.K. 82(84)61 Yin, H. H. 271 (69) 266; 430 (7) 412, 429 Yip, M.C. M. 82 (76) 61,(77) 61 Yogeeswaren, G. 371 (46) 364 Yoshida, A. J32 (17) 127, (19) 127 Yoshida,Y. 341 (67) 332 Yoshiki,T. 198 (16) 181 Yoshinaka, Y. 200 (61) 197 Yosizawa, Z. 44, 33; 370 ( 4 ) 358 359 YoulosJ. 121(50)115 Young, E. 149 ( 58) 146

467 Young, W . W . , Jr. 310 (15) 304; 357; 370 (13) 358, (19) 358, 359, 363, 364,

369; 371 (44) 364,(45) 364 Yu, R. 80 (28) 52, (33) 52; 81 (56) 59 Yu,S.D. 132(13)126 Yu,S.V. 253 (6) 227 Yu,S.Y. 253 (4) 227,243 Yunis,E.J. 132(7)124 Z

Zambotti, V. 148 (31) 140; 149 ( 57) 145 Zamcheck, N. 355 (54) 350 Zanetta, J. P. 270 ( 50 ) 260, 264, (51) 260, 264 Zeman,W. 148 (33) 140 Zentgraf,H. 83 (118) 67 Zilversmit, D. B. 211, 210 Zinn, A. B. 20(15)6 Ziolkowski C H J 371 (35) 361


SUBJECT INDEX

Abrus precatorius 262 Acceptor specificity of glycosyltransferases 64 Acetates, partially methylated alditol 392 total mass spectra of 393, 395 N-Acetyl derivative 408 N-Acetylgalactosamine 28 4-sulfatase 14 N-Acetyl-D-galactosamine-type oligo saccharides, biosynthesis of serine (threonine)34 a, /8-N-Acetylgalactosaminidase 405 N-Acetylgalactosaminitol 287 N-Acetylglucosamine 287 a-N-Acetylglucosaminidase 144-146 /?-N-Acetylglucosaminidase 137,405 endo137 N-Acetyllactosamine type 26 Acid hydrolase activities of transformed and nontransformed fibroblasts 407 hydrolysis 52,408 phosphatase 405 sialic 177,279 Acidic components, tracheobronchial glycoprotein in sparsely 116 Adenocarcinoma cells, cell surface glycoproteins of human colonic .. 304 Adenocarcinoma, T A 3 , mammary 278 A F P (alphafetaprotein) 335,336 in abnormal pregnancy, increased amniotic fluid 328 and albumin, properties of rat 327 concentrations in rats, serum 333 during fetal development, some proposed functions of 330 as an indicator of early events in chemical carcinogenesis 326 properties of 326 Agaricus bisporus 262 Agglutinability, effect of incubation time on Con A-induced 419 Agglutination, Con A - and W G A induced 413 Agglutination, effect of papain digestion on Con A - or W G A induced 421 Agglutinin titer score, anti-T 318

Agglutinins with human breast gland membrane-cytoplasm preparations, colon carcinomata and controls, anti-blood group M , N , and precursor Albumin, properties of rat A F P and .. Alkaline borohydride treatment of H fucose-labeled gylcoprotein

313 327

3

Alpha-l-antitrypsin 122 Alphafetaprotein (see A F P ) Amides, fatty acid 78 Amidohydrolase, l-aspartamido-/?-Nacetylglucosamine 145 Amidohydrolase, 4-L-aspartylglycosylamine 137 Amino acid(s) analysis of aminoacyl fucosides 388 compositions of epiglycanin, carbohydrate and 281 molar ratios of carbohydrate and . . . 389 sequence(s) 15,217,287 and domanis of glycophorin 18 at N-terminus of C E A 349 Ammonium sulfate fraction, chromatography of 208 Ancillary data 408 Antigen(s) of cancer cells, secreted glycoproteins and cell-surface 275 carcinoembryonic ( C E A ) , a marker of human colonic cancer 342 by ectoglycoproteins, masking of cell-surface 277 glycolipid 367 from human blood group O , M N red cells, preparation of T 3 human carcinoma-associated precursors of the blood group M N 311 major histocompatibility 278 masking 278 tumor specific 277 Antigenic activities 277 Antisera, absorption of 312 ai-Antitrypsin deficiency 28 Aortae, concentration of component sugars of normal and artherosclerotic 203

469


470


Aortic intima, glycoprotein inhibitor of lipoprotein lipase from 201 Ascites cells, cell surface properties of M A T - B 1 and M A T - C 1 mammary 442 Asn-associated glycopeptides 86 Asn-GlcNAc initiation, subcellular site of 26 oligosaccharides, biosynthesis of .... 25 type oligosaccharides, initiation of .. 25 Asparagine -N-acetyl-D-glucosamine linkage 21 glucosaminyl 98 glycopeptides linked to 12 residue 23 structure of heterosaccharides linked JV-glycosically to 11 1-aspartamido-^-IV-acetylglucosamine and amidohydrolase 145 Aspartylglucosaminuria 129,145 4-L-Aspartylglycosylamine amido hydrolase 13 Atherosclerosis 201

Blood group (continued) M N antigens, human carcinomaassociated precursors of the .. M and N glycoproteins, human . . . M , N , and precursor agglutinins with human breast gland membrane-cytoplasm preparations O, M N red cells, preparation of T antigen from human Borohydride treatment of H fucoselabeled proteins, alkaline Bovine milk enzyme Brain Breast gland membrane-cytoplasm preparations, colon carcinomata and controls, anti-blood group M , N , and precursor agglutinins with human

311 316

313 312

3

401 32 135

313

C

B Bacteria and vaccines Bacterium tularensis Battens disease Biosynthesis of acedic glycosphingolipids of blood-group-related glycosphingolipids and catabolism of glycoproteins of neutral glycosphingolipids of sulfo-glycosphingolipids Blood clotting, basic mechanisms involved in coagulation biochemistry of extrinsic pathway of intrinsic pathway of molecular events in proteins of zymogen activation mechanisms in collection and serologic procedures group activities in colonic mucosa, glycolipid carbohydrate chains in human tumors, status of determinants, accumulation of precursor carbohydrates of .. determinants in human cancer, status of -related glycosphingolipids, biosynthesis of

PROCESSES

311 323 146 62 63 21 62 62 160 160 160 166 163 162 161 160 312 305 357 358 359 63

C. parvum C thymidine CEA composition of proposed genetic evolution for protein chains of sequence of amino acids at Nterminus of structural units of polysaccharide portion of C a C l and the pentapeptide, P M R titration data of Calcium ion binding to the elastin matrix, neutral-site Cancer carcinoembryonic antigen ( C E A ) , a marker of human colonic cells, secreted glycoproteins and cell-surface antigens of glycoprotein-associated A B O ( H ) glycoproteins in colonic of masking by endogenous glycoproteins, relevance to human .. status of blood group determinants in human Carbohydrate(s) and amino acid compositions of epiglycanin and amino acids, molar ratios of .... of blood group determinants, accumulation of precursor chains, O-glycosyl-linked chains in human tumors, status of blood group 1 4

323 72 345 349 349 347

2


241 229 342 275 303 288 359 281 389 358 279 357

SUBJECT

Carbohydrate (s) (continued) composition of the membrane fraction of normal and cancerous colonic mucosa 298 content of galactoprotein I 307 content of invertase preparations... 90 diseases affecting the levels of collagen-associated 223 material, N-glycosyl-linked 287 metabolism, genetic disorders of complex 107 moieties of the collagens in health and disease 213 moiety, structure of the 214 relative to mannose, molar ratio of .. 299 residues, terminal 76 Carcinogenesis, alphafetaprotein as an indicator of early events in chemical 326 Carcinogens, hypothesized target chemical toxins and chemica Catabolism, glycoprotein 39,135 Catabolism, glycosphingolipid 71 Cell(s) agglutinability of normal and malignant rat liver 418 aminoacyl fucosides of N R K and MSV-NRK 380 cell-surface glycoproteins of normal and malignant rat liver 412 cell surface properties of M A T - B 1 and M A T - C 1 mammary ascites 442 components, lectin affinity chromatography of nerve 264 disease, I136 electron micrograph of T A 3 - H a ascites 283,284 -free product, gel electrophoresis of the 191 -free synthesis of an unglycosylated precursor 188 hepatoma 413 with H fucose, pulse-labeling of NRK 400 -mediated immune response, measurement of 314 membrane components, lectin affinity purification of lymphoid 262 membranes, sialic acid contents of tumor 436 papain digestion of 414 preparation of T antigen from human blood group O, M N red 312 role for fucosylation in membrane functions of mammalian 407 surface (s) antigens of cancer cells, secreted glycoproteins and 275 3

471

INDEX

Cell (s) (continued) surface(s) (continued) antigens by ectoglycoproteins, masking of electron micrograph of T A 3 - H a ascites epiglycanin on glycoconjugates, synthesis of glycoproteins of human colonic adenocarcinoma cells lectin receptors, dynamics of properties of the 13762 ascites tumor properties of M A T - B 1 and M A T C l mammary ascites cells .... effect of sodium butyrate on the aminoacyl fucosides of M S V N R K cells effect f sodiu butyrat

277 285 289 68 304 266 439 442 381

uniqueness of fucose in mammalian 408 viability 413 Ceramide, synthesis of 59 Ceroid lipofuscinoses, neuronal146 Chitobiosyl, di-N-acetyl 86 Cholesterol 145 Chromatographic comparison of "disaccharide" and "glucosyl fucitol" 397 Chromatographic retention times of permethylated compounds, gasliquid 392 Chromatography affinity 343 of ammonium sulfate fraction 208 gas-liquid 387,404 gel filtration 286 ion exchange 343 of tryptic digests 186,189,193 lectin affinity 258 of components from other cell types 264 of nerve cell components 264 Chromobacterium violaceum 322 Cirrhosis 122 Clostridium perfringens 75 Clotting, basic mechanisms involved in blood 160 Coacervate, electron micrographs of tropoelastin 249 Coacervation blocked a-elastin temperature profiles of 250 polypentapeptide temperature profiles of 251 process of 247


472

GLYCOPROTEINS

Coagulation blood biochemistry of extrinsic pathway of intrinsic pathway of molecular events in proteins of zymogen activation mechanis ns in disorders mechanism involving proteolysis in the termination of Collagen(s) -associated carbohydrate, diseases affecting the levels of enzymatic steps in the biosynthetic attachment of hexose to in health and disease, carbohydrate moieties of structures and characteristics interstitial types, occurrence in different Colon carcinomata and controls, antiblood group M , N , and precursor agglutinins with human breast gland membrane-cytoplasm preparations Colon, glycoconjugates alterations in malignant and inflammatory disease of Colonic cancer, carcinoembryonic antigen ( C E A ) marker of human Concanavalin A Core, complex oligosaccharide Core sequence Corynebacterium parvum Covalent linkage of saccharides to peptide chain Cystic fibrosis, mucous glycoproteins in Cytoagglutination lectin-induced assay of Cytoplasm preparations, colon carcinomata and controls, anti-blood groups M , N , and precursor agglutinins with human breast gland membraneCytostructural integrity

160 160 166 163 162 161

PROCESSES

Di-N-acetyl chitobiosyl 86 Difucohexaose, mass spectrum of permethylated 55 Digestion, lysosomal 151 Digests, ion-exchange chromatography of tryptic 186,189,193 Disaccharide component, acid hydrolysis of H-labeled 399 "Disaccharide" and "glucosyl fucitol," chromatographic comparison of .. 397 Disease(s) affecting the levels of collagenassociated carbohydrate 223 Battens 146 carbohydrate moieties of the collagens in health and 213 of the colon, glycoconjugates alterations in malignant and inflammatory 295 3

160 160 170 223 221 213 21 215

313 295 342 136 87 86 323 6 108 412 412 414

313 415

D Dansylated substrates Deficiency, multiple sulfatase 6-Deoxyhexose, internal Detergents on the glycoprotein binding efficiencies, effect of increasing concentrations of


98 143 394 286 267

glycoprotein storage Gaucher's I-cell infection and degenerative liver lung lysosomal enzyme deficiency Maroteaux-Lamy Niemann-Pick's Sandhoff's Tay-Sachs Disorders, coagulation Dixon plot Dolichol phosphate sugars Dolichol pyrophosphate oligosaccharide Dolichos biflorus

122 143,152 136 177 123 123 135 143 145 138-140 139,140 160 206 22

E Eccrine glands, electrolyte composition of Ectoglycoproteins, endogenous Ectoglycoproteins, masking of cellsurface antigens by 4-Eicosasphingenine Elastin and elastin repeat peptides, proteoglycan interaction with ion titration of the repeat hexapeptide of pentapeptide of a-Elastin calcium elemental maps C D spectra of secondary ion image temperature profiles of coacervation, blocked X-ray spectrum


22 262

109 278 277 48 253 238 235 232 248 232 250 232

SUBJECT

473

INDEX

Electrolyte composition of eccrine sweat glands 109 Electron micrograph of polypentapeptide 249 Electron micrographs of tropoelastin coacervate 249 Electrophoresis, gel of the cell-free product 191 of mature viral proteins 182 of native invertase 92 polyacrylamide 405 of surface membranes 406 of R-MuLV specific glycoproteins .. 185 Elongation with the Golgi apparatus .. 29 Emphysema 122 Endo-D 87 Endo-H, composition of compounds tested as substrates for 97 Endo-H, substrate speficity of 99 Endo-L 8 substrate speficity of 9 Endo-y8-N-acetylglucosaminidase-H (endo-H) 86,93,137 Endo-y8-glucosaminidase 136 Endocytosis of lysosomal glycosidases 78 Endoglycosidase, hen oviduct 100 Endoglycosidase treatment 90 Enterobacteriaceae

323

Enzyme(s) bovine milk 32 deficiency diseases, lysosomal 135 degradation 404 into lysosomes, incorporation of exogenous 150 replacement 150 trials in Gaucher's disease 154 Enzymatic steps in the biosynthetic attachment of hexose to collagen 221 Epiglycanin 278, 282,287 carbohydrate and amino acid compositions of 281 on cell surfaces 289 isolation of 286 physicochemical characterization of 287 Escherichia coli

73, 322

Ester groups, sulfate143 Eukaryotic organisms, glycolipids of .. 47 Exocytosis 146 of lysosomal glycosidases 78 Exoglycosidases 71, 74, 404 F

Fatty acid amides Ferritin, polycationic Fetal development, some proposed functions of AFP during Fiber, molecular pathology of vascular elastic

78 282 330 227

Fibrin formation of cross-linked Fibroblasts acid hydrolase activities of transformed and nontransformed ... human Fibronectin Filtration analysis of "disaccharide" material from glycoprotein and from FL4a, gel FL4a gel filtration analysis of "disaccharide" material from Fluid mosaic model of membrane structure Focusing, isoelectric Forssman glycolipid, induction of "incompatible" determinants and

160 169 146 407 408 408

398 391 398 17 343

361

-polyacrylic hydrazide, synthesis of "poly-glycolipid" 365 reactivity of purified ceramide pentasaccharide fractions 368 status of Taiwan tumor cases 365 Fucopeptide profile, Sephadex G-50 383,384 Fucose -containing components, low molecular weight 409 -labeled glycoprotein fraction, alkaline borohydride treatment of H 396 -labeled proteins, alkaline borohydride treatment of H 401 in mammalian cells, uniqueness of .. 408 pulse-labeling of NRK cells with H 400 L-Fucose 404 a-Fucosidase 137,144 a-L-Fucosidase, rat testis 405 Fucosides, aminoacyl amino acid analysis of 388 of MSV-NRK cells, effect of sodium butyrate on 381 of NRK and MSV-NRK cells 380 from rat tissue, purification of 386 Fucosidosis 136,137,144 a-Fucosidosis 98 Fucosylation 404 in membrane functions of mammalian cells, role for 407 role of 410 3

3

3

G

Galactoprotein I carbohydrate content of Galactose -containing unit, structure of


307 287 217

474


Galactosidase 405 £-Galactosidase 137,140,146 Galactosyltransferase 31 Ganglioside(s) 138-140,145, 304 composition of murine sarcoma virus-transformed N R K cells, effect of sodium butyrate on .... 382 Gangliosidosis, G M 1 98,136-140,146 Gangliosidosis, G M 2 98,136-138 Gaucher patients, glycolipid accumulation in livers of 156 Gaucher's disease 143,152 enzyme replacement trials in 154 Gene order of the proteins 187 Gene products by tumors, oncodevelopmental 329 Genetic disorders of complex carbohydrate metabolism 107 Genetic evolution for the protein chains of C E A , proposed 34 G l c N A c residue 2 Globopentaglycosylceramide 61 Globotetraglycosylceramide 61 C N M R spectrum of 60 Globotriglycosylceramide 61 Glucocerebrosidase 143 Glucocerebroside 155 0-/?-D-Glucopyranosyl (1 -> 3 ) - 0 - « - L fucopyranosyl-L-threonine, structure of 388 D-Glucosamine 405 Glucosaminyl asparagine 98 Glucose containing unit, structure of .. 217 D-Glucose 405 ^-Glucuronidase 146 Glycoasparagine sequences 131 Glycoconjugates alterations in malignant and inflammatory disease of the colon 295 in expression of transformed phenotype, role of 378 synthesis of cell surface 68 Glycoglycerolipids 47-49 Glycolipid(s) accumulation in livers of Gaucher patients 156 antigens 367 blood group activities in colonic mucosa 305 of eukaryotic organisms 47 extracted from normal and colon mucosa, complement fixing reactivities of 362 induction of "incompatible" determinants and Forssman 361 macro64 membrane-associated 375 methods of analysis of 49 1 3

PROCESSES

Glycolipid (s) (continued) radioimmunoassay of Forssman 366 structure of H 360 structure and metabolism of 47 Glycopeptide(s) 86,135,139,412 accumulation of 38 Asn-associated 86 bonds 7 cell-surface 413 gel filtration of 422 lectin receptor activities of 423,424 gel filtration of 421 linked to asparagine 12 linked to serine or threonine, structures of 8 of the normal and cancerous mucosa, hemagglutination inhibition by the membrane 298 papain-labile 413 3

and domains of 18 Glycoprotein^) 138 -associated A B O ( H ) glycoproteins in colonic cancer 303 associated with the celle matrix, high molecular weight 276 binding efficiencies, effect of increasing concentrations of detergents on 267 biosynthesis 21 of a membrane-bound 27 catabolism 21,39,135 and cell-surface antigens of cancer cells, secreted 275 into cellular membranes, integration of 16 comparison of membrane 433 discharge of H-labeled Ill discharge of S0 -labeled Ill fraction, alkaline borohydride treatment of H fucose-labeled 396 gel electrophoresis of R - M u L V specific 185 gel filtration analysis of "disaccharide" material from 398 human blood group M and N 316 of human colonic adenocarcinoma cells, cell surface 304 using immobilized lectins, isolation and characterization of membrane 260 inhibitor of lipoprotein lipace from aortic intima 201 intracellular viral specific 183 lectin affinity purification of R B C .. 259 during malignant transformations .. 375 membrane 256, 404, 405, 435 3

35

4

3


SUBJECT

475

INDEX

Glycoprotein (s) (continued) molecules, endogenous 277 mucous 14 abnormal milieu for 112 altered physiochemical properties of 113 in cystic fibrosis 108 of normal and malignant rat liver cells, cell-surface 412 porcine intimal 203 (lipolipin) sutdies on 202 of rat mammary gland, membrane .. 432 of rat mammary tumors, membrane 438 relevance to human cancer of masking by endogenous 288 secretory 26 in sparsely acidic components, tracheobronchial 116 storage disease 122 structure, current concepts o studies on human intimal 20 Glyccsaminoglycans 138 Glycosidase(s) 297 activities in normal and cancerous colonic mucosa 303 endocytosis of lysosomal 78 exocytosis of lysosomal 78 physiological significance of 79 purification of several 73 substrate specificities of 73 Glycosphingolipid(s) 47-49,74 biosynthesis 59 of acidic 62 of blood group-related 63 of neutral 62 of sulfo62 catabolism 71 Glycosyl asparagine derivatives 96 O-Glycosyl-linked carbohydrate chains 279 N-Glycosyl-linked carbohydrate material 287 Glycosylation, factors controlling hydroxylysine formation and subsequent 222 Glycosylation, intercellular 67 Glycosylhydrolases 405 Glycosyltransferase (s) 297 acceptor specificity of 64 activities in human colonic mucosa 299 activities in normal and cancerous colonic mucosa 301 localization 67 regulation of 69 Goblet cells 34 Golgi apparatus 146 elongation within the 29

H H-labeled glycoproteins, discharge of Health and disease, carbohydrate moieties of the collagens in Hemagglutination inhibition by the membrane glycopeptides of the normal and cancerous mucosa Hen oviduct endoglycosidase Heparan-N-S-sulfatase Hepatocytes lectin-induced agglutination of adult mechanically and enzymatically dispersed morphological comparison of rat binding of Con A to cell-surfac morpholog f

3

111 213 286 298 100 144 415 413 417 413 426 428

Hepatoma cells, papain digestion of .. 414 Heterosaccharide (s) information content of 9 linked N-gylcosically to asparagine, structure of 11 linked glycosically to serine or threonine, structure of 6 moieties on peptide, distribution of 13 Hexapeptide 239 of elastin, ion titration of the repeat 238 Hexosaminidase 140 /^-Hexosaminidase 138,146 Hexose to collagen, enzymatic steps in the biosynthetic attachment of 221 to procollagen a chains, biosynthetic attachment of 218 terminal 394 Human carcinoma-associated precursors of the blood group M N antigens 311 colonic adenocarcinoma cells, cell surface glycoproteins of 304 colonic adenocarcinomatous tisues, studies in 296 fibroblasts 408 Hunter 144 cells 143 Hurler 144 Hydrazide, structure of Forssmanpolyacrylic 366 Hydrazide, synthesis of "poly-glycolipid," Forssman polyacrylic 365 Hydrolysis, acid 52 of H-labeled disaccharide component 399 3


476


Hydrolysis of oligosaccharides, rates of Hydroxylsine formation and subsequent glycosylation, factors controlling Hydroxylysines and lysines, sequences around 4-D-Hydroxysphinganine Hyperplasia atypical of liver Hypersecretion, mucous Hypersensitivity, measurement of delayed-type "recair Hypertrophy

88 222 219 48 109 334 336 109 314 109

I a-Iduronidase a-L-Iduronidase Immune response, measuremen cell-mediated Immunogenic Immunoresistant Incubation time on Con A-induced agglutinability, effect of Infected cells, pulse-chase experiment in R - M u L V Infection and degenerative diseases .. Intracellular steps in the biosynthesis of procollagen Intracellular viral specific glycoproteins Invertase electrophoresis of native preparations, carbohydrate content of stability of native and carbohydrate-depleted Ion complexes, water titration of strontium, calcium, and magnesium L-Isomer

144 14 31 279 279 419 186 177 220 183 91 92 90 92 239 408

L Lacto-N-tetraose, mass spectrum of permethylated Lamy disease, MaroteauxLectin(s) affinity chromatography of components from other cell types of nerve cell components purification of lymphoid cell membrane components purification of R B C glycoproteins -binding sites, cryptic cell-surface ..

54 143 412 258 264 264 262 259 425

PROCESSES

Lectin (s) (continued) concanavalin A (Con A ) and wheat germ agglutination ( W G A ) .... 412 -induced agglutination of adult hepatocytes 415 -induced cytoagglutination, assay of 414 isolation and characterization of membrane glycoproteins using immobilized, 260 of oligosaccharide side chains, possible binding region for 267 receptor activity 413 of cell-surface glycopeptides .423,424 receptors, dynamics of cell surface .. 266 Vicia graminea 280 Leukemia 404 virus membrane proteins, biosynthesis of murine 177 Leukodystrophy, Metachromatic 143 Lipofuscinoses, neuronal-ceroid 146 Lipopolysaccharides 321 Lipoprotein lipase from aortic intima, glycoprotein inhibitor of 201 Liver(s) 335 atypical hyperplasia of 336 cells agglutinability of normal and malignant rat 418 cell surface glycoproteins and normal and malignant 412 normal 413 papain digestion of 414 disease 123 of Gaucher patients, glycolipid accumulation 156 injury, hepatotoxic 329 storage material 125 L u n g disease 123 Lysines, sequences around hydroxylysines and 219 Lysosomal accumulation 153 blockage 150 digestion 151 enzyme deficiency diseases 135 uptake 153 Lysosome (s) 137,146 incorporation of exogenous enzymes into 150 M Macrosialoglycopeptides Malignant transformation glycoproteins during Mammalian cells Mannoglycopeptides


412 412 375 277 138

SUBJECT

477

INDEX

Mannose 287 molar ratio of carbohydrate relative to 299 /3-Mannose core 404 a-Mannosidase 98,144, 405 Mannosidosis 144 Mannosyl oligosaccharides 98 Mannosyl residues 98 Maroteaux-Lamy disease 143 Masking 364 antigen 278 of cell-surface antigens by ectoglycoproteins 277 by endogenous glycoproteins, relevance to human cancer of .. 288 in the T A 3 tumor, further evidence for 288 Matrix, high molecular weight glycoproteins associated with the cell 276 Matrix, neutral-site calcium io ing to the elastin 22 Meconium ileus 108 Megalosaccharide 10 Membrane alterations 412 -bound glycoprotein, biosynthesis of 27 components, lectin affinity purification of lymphoid cell 262 -cytoplasm preparations, colon carcinomata and controls, antiblood group M , N , and precursor agglutinins with human breast gland 313 flow, hypothesis 68 functions of mammalian cells, role for fucosylation in 407 glycoproteins 256, 404,405 using immobilized lectins, isolation and characterization of 260 hypothetical structure of plasma 257 polyacrylamide gel electrophoresis of surface 406 structure, fluid mosaic model of 17 Metabolism, genetic disorders of complex carbohydrate 107 Metachromatic Leukodystrophy 143 Metastasis 432 Methanolysis 52 iV-Methyl-N-acetyl-3,6-di-0-methyl1,4,5-tri-O-acetygalactosaminitol, mass spectrum of 57 N-Methyl-N-acetyl-4,6-di-0-methyl1,3,5-tri-O-acetylgalactosaminitol, mass spectrum of 57 N-Methyl-N-acetyl-3,4,6-tri-0-methyl1,5-di-O-acetylgalactosaminitol, spectrum of 57 Methylation analysis 349 Mice, pneumonia virus of 282

Mice, strain A 288 Microbes, T-specific substances in .... 321 Micrograph, electron 440,441 of rat hepatocytes 427 of T A 3 - H a ascites cell 283 surface 285 of TA3-St ascites cell 284 Microheterogeneity 128 Microscopy, dark field light 282 Microscopy, electron 282 Morphology, surface 439 Mouse, A . C A 288 Mucins, ovine submaxillary 35 Mucins, porcine submaxillary 35 Mucolipidoses 145 Mucopolysaccharidoses 144 Mucosa, colonic carbohydrate composition of the membrane fraction of 298 glycolipid blood group activities in glycosidase activities in glycosyltransferase activities in human hemagglutination inhibition by the membrane glycopeptides of .... sialytransferases of Mucosa and tumor tissue, perchloricacid extractable fraction of normal Mucous glands glycoproteins abnormal milieu for altered physiochemical properties of hypersecretion Murine sarcoma virus-transformed rat ( M S V - N R K ) cells Multiple sulfatase deficiency

305 303 301 299 298 301 360 34 14 112 113 109 378 143

N Nascent peptide 26 Neuraminidase 73,286 Neuronal-ceroid lipofuscinoses 146 Neutralization theory, neutral site/ charge 228 Niemann-Pick's Disease 145 Nodule, neoplastic 335 5'-Nucleotidase 434 Nucleotides, sugar 177 O Oligomannoside type 26-29 Oligosaccharides 135 accumulation of 38 biosynthesis of serine (threonine )N-acetyl-D-galactosamine type 34



478

Oligosaccharides (continued) core, complex 87 dolichol pyrophosphate 22 initiation of 21 Asn-GlcNAc type 25 mannosyl 98 processing 24-28 rates of hydrolysis 88 side chains, possible binding region for lectins of 267 Olive oil 209 Oncogenic transformation 357 Osmium tetroxide 53 Ovine submaxillary mucins 35 Ozonolysis 53

P Papain digestion on con A - or WGA-induced agglutination, effect of of hepatoma cells of liver cells Papain-labile glycopeptides Pathology of vascular elastic fiber, molecular Pentaglycosylceramide Pentapeptide of elastin P M R titration data of C a C l and .... Pentasaccharide fractions, Forssman reactivity of purified ceramide .... Peptide chain, covalent linkage of saccharides to distribution of heterosaccharide moieties on maps of the envelope-related protein nascent proteoglycan interaction with elastin and elastin repeat Perchloric acid extractable fraction of normal mucosa and tumor tissue Perfusion, collagenase Permethylated compounds, gas-liquid chromatographic retention times of Permethylation Phenotype, role of glycoconjugates in expression of transformed Phosphoglycoprotein Phosphohexosyl Phospholipid Physiocochemical properties of mucous glycoproteins, altered .... 2

421 414 414 413 227 58 236 235 241 368 6 13 190 26 253 286 360 43 392 52 378 146 146 145

PROCESSES

Pick's Disease, Niemann145 Pioncytotic 146 Plasma membrane 146, 434 hypothetical structure of a 257 Pneumonia 280 "Poly-glycolipid," Forssman-polyacrylic hydrazide, synthesis of .... 365 Polyhexapeptide, cross-linked 245 Polypentapeptide cross-linked 244 electron micrograph of 249 temperature profiles of coacervation 251 Polysaccharide portion of C E A , structural units of 347 Porcine intimal glycoprotein 203 (lipolipin), studies on 202 pancreatic ribonuclease 14 A F P in abnormal 328 Procollagen a chains, biosynthetic attachment of hexose to 218 Procollagen, intracellular steps in the biosynthesis of 220 Protein(s) alkaline borohydride treatment of H fucose-labeled 401 biosynthesis of murine leukemia virus membrane 180 of blood coagulation 161 -bound oligosaccharides, processing of 24 chains of C E A , proposed genetic evolution for 348 gel electrophoresis of mature viral 182 gene order of 187 interactions, l i p i d 77 model for synthesis and processing of R - M u L V 190,193 model for synthesis and processing of viral envelope 194 peptide maps of envelope-related .. 190 of Rauscher murine leukemia virus, virion 181 viral 187 Proteoglycan interaction with elastin and elastin repeat peptides 253 Proteolysis 160,286 in the termination of coagulation, mechanism involving 170 Proteolytic activity, residual 94 Prothrombin activation 167 Pseudomonas vaccine 323 Pulse-chase experiment in R - M u L V infected cells 186 Pulse-labeling of N R K cells with H fucose 400 3

3

113


SUBJECT

479

INDEX

Purification of R B C glycoproteins, lectin affinity Purification of glycosidases

259 73

Rabbit tracheal explants 117 Radioimmunoassay of Forssman glycolipid 366 Rat(s) A F P and albumin, properties of 327 hepatocytes 413 binding of Con A to 426 cell-surface morphology of 428 electron micrograph of 427 liver cells, agglutinability of normal and malignant 418 liver cells, cell-surface glycoproteins of normal and malignan mammary gland, membrane proteins of 432 mammary tumor, tissue 435 membrane glycoproteins 438 ( M S V - N R K ) cells, murine sarcoma virus-transformed 378 serum alphafetaprotein concentrations in 333 testis a-L-fucosidase 405 tissue, purification of aminoacyl fucosides from 386 Rauscher murine leukemia viral proteins, synthesis and process of 193 virus 282 envelope proteins, synthesis of .... 196 virion proteins of 181 "Recall" hypersensitivity, measurement of delayed-type 314 Recall skin reactivity, delayed-type .... 319 Receptor activity 412 assay of Con A and W G A 415 Ribonuclease, porcine pancreatic 14 Ricinus communis 296, 306 R N A , translation of fractionated R - M u L V genomic 190

S. abortus equi 322 S. typhi 320 Saccharides to peptide chain, covalent linkage of 6 Sachs disease, T a y 138-140 Salmonella milwaukee 322 Sandhoff's disease 138-140 Sanfilippo A and B 143-156 Secretory glycoproteins 26

Sephadex G-50 column effluents, analysis of 414 elution profile 413 fucopeptide profile 383, 384 Serine structures of glycopeptides linked to 8 structure of heterosaccharides linked glycosidically to 6 (threonine) -N-acetyl-D-galactosamine-type oligosaccharides, biosynthesis of 34 Sialic acid 177,279,287 contents of tumor cell membranes .. 436 residue 33 Sialoglycopeptide (s) 135,140,146 cell-surface 413 papain-labile 413 Sialtransferases 33 Skin reactivity, delayed-type recall .... 319 S0 -labeled glycoproteins, discharge of HI Sodium butyrate on the aminoacyl fucosides of M S V - N R K cells, effect of 381 Sodium butyrate on ganglioside composition of murine sarcoma virustransformed N R K cells, effect of 382 Spectrum (a) of a-elastin, C D 248 of globotetraglycosylceramide, C NMR 60 IR 230 of N-methyl-N-acetyl-3,6-di-0methyl-l,4,5-tri-0-acetylgalactosaminitol, mass 57 of N-methyl-N-acetyl-4,6-di-0methyl-l,3,5-tri-0-acetylgalactosaminitol, mass 57 of N-methyl-IV-acetyl-3,4,6-tri-0methyl-l,5-di-0-acetylgalactosaminitol 57 of partially methylated alditol acetate, total mass 393, 395 of permethylated difucohexaose, mass 55 of permethylated lacto-N-tetraose, mass 54 Sphinganine 48 4-Sphingenine 48 Sphingomyelin 145 Sphingomyelinase 145 Staphylococcus aureus 364 Storage disorders 154 Strain specificity 290 Streptococcus 361 Streptomyces plicatus 86-88, 93

35

4


1 3

480


Substrate specificities of glycosidases 73 Sugar(s) deoxy 408 dolichol phosphate 22 of normal and atherosclerotic aortae, concentration of component 203 nucleotides 177 Sulfatase deficiency, multiple 143 heparan-N-S144 sulfoiduronate 144 Sulfatase A 143 Sulfatase B 143,144 4-Sulfatase, ^-acetylgalactosamine .... 144 Sulfate-ester groups 143 Sulfatides 143 Sulfoiduronate sulfatase 144 Surface morphology 429 T Tay-Sachs disease 138-140 Temperature profiles of coacervation, blocked-elastin 250 Temperature profiles of coacervation, polypentapeptide 251 Tetrahymena pyriformis 101 Therapy 128 Threonine permethylated synthetic glucosyl fucosyl 391 structures of glycopeptides linked to 8 structure of heterosaccharides linked glycosidically to 6 Thrombin 160 Tissue carcinomatous and control 311 normal and lactating mammary 435 rat mammary tumor 435 studies in human colonic adenocarcinomatous 296 Titer score, anti-T agglutinin 318 Titration data of C a C l and the pentapeptide, PMR 241 data, C M R 242 of the repeat hexapeptide of elastin, ion 238 of strontium, calcium, and magnesium ion complexes, water 239 Torpedo californica 264 Toxins and chemical carcinogens, hypothesized targets for chemical 337 Tracheal explants, rabbit 117 Tracheobronchial secretions 113 Transformation, oncogenic 357 Triantennary 404 2

PROCESSES

Tropoelastin coacervate, electron micrographs of tropoelastin Tumor cases, Forssman status of Taiwan .. cell(s) membranes, sialic acid contents of metastatic surface properties of the 13762 ascites development fractions, nucleotidase activities of mammary gastric growth membrane glycoproteins of rat mammary specific antigens status of blood group carbohydrate further evidence for masking in .. tissue, perchloric-acid extractable fraction of normal mucosa and

249 365 404 436 277 439 331 436 358 331 438 277

288 360

U Unglycosylated precursor, cell-free synthesis of an

188

V Vaccines, bacteria and 311 Vaccines, microbial 321 Vibrio cholerae 75, 312, 320 Vicia graminea 279 lectin 280 Virion proteins of Rauscher murine leukemia virus 181 Virus assembly 195 mammary tumor 282 membrane proteins, biosynthesis of murine leukemia 180 particles 180 Rauscher leukemia 282 transformation 404 -transformed N R K cells, effect of sodium butyrate on ganglioside composition of murine sarcoma 382 -transformed rat ( M S V - N R K ) cells, murine sarcoma 378 virion proteins of Rauscher murine leukemia virus 181 Z

Zymogen activation mechanisms i n blood coagulation


160

Glycoproteins and glycolipids in disease processes

Glycolipids

Glycopeptides and glycoproteins: synthesis, structure, and application

Functional and Structural Proteomics of Glycoproteins

Functional and Structural Proteomics of Glycoproteins

Proteoglycans in Lung Disease (Lung Biology in Health and Disease)

Markov Processes, Ray Processes and Right Processes

Resveratrol in Health and Disease (Oxidative Stress and Disease)

Environmental Stressors in Health and Disease (Oxidative Stress and Disease)

Nutraceuticals in Health and Disease Prevention (Oxidative Stress and Disease)

Soy in Health and Disease Prevention (Nutrition and Disease Prevention)

Resveratrol in Health and Disease

Micronutrients in health and disease

Micronutrients in Health and Disease

Huntington's Disease (Genes and Disease)

Carotenoids in Health and Disease (Oxidative Stress and Disease)

Phytochemicals in Health And Disease (Oxidative Stress and Disease)

Vitamin K in Health and Disease (Oxidative Stress and Disease)

Nanomedicine in Health and Disease

Epigenetics in Health and Disease

Centrosomes in Development and Disease

Carotenoids in Health and Disease

Androgens in Health and Disease

Stress in Health and Disease

Flavonoids in Health and Disease

Alcohol in Health and Disease

Epigenetics in Health and Disease

Immunoendocrinology in Health and Disease

Glycoproteins II, Volume 29 (New Comprehensive Biochemistry)

Glycoproteins I, Volume 29 (New Comprehensive Biochemistry)

Molecular Biology in Plant Pathogenesis and Disease Management Disease Management

Glycoproteins and glycolipids in disease processes

Glycolipids

Glycopeptides and glycoproteins: synthesis, structure, and application

Functional and Structural Proteomics of Glycoproteins

Functional and Structural Proteomics of Glycoproteins

Proteoglycans in Lung Disease (Lung Biology in Health and Disease)

Markov Processes, Ray Processes and Right Processes

Resveratrol in Health and Disease (Oxidative Stress and Disease)

Environmental Stressors in Health and Disease (Oxidative Stress and Disease)

Nutraceuticals in Health and Disease Prevention (Oxidative Stress and Disease)

Soy in Health and Disease Prevention (Nutrition and Disease Prevention)

Resveratrol in Health and Disease

Micronutrients in health and disease

Micronutrients in Health and Disease

Huntington's Disease (Genes and Disease)

Carotenoids in Health and Disease (Oxidative Stress and Disease)

Phytochemicals in Health And Disease (Oxidative Stress and Disease)

Vitamin K in Health and Disease (Oxidative Stress and Disease)

Nanomedicine in Health and Disease

Epigenetics in Health and Disease

Centrosomes in Development and Disease

Carotenoids in Health and Disease

Androgens in Health and Disease

Stress in Health and Disease

Flavonoids in Health and Disease

Alcohol in Health and Disease

Epigenetics in Health and Disease

Immunoendocrinology in Health and Disease

Glycoproteins II, Volume 29 (New Comprehensive Biochemistry)

Glycoproteins I, Volume 29 (New Comprehensive Biochemistry)

Molecular Biology in Plant Pathogenesis and Disease Management Disease Management

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