ADVANCES I N
Immunology VOLUME 7
CONTRIBUTORS TO THIS VOLUME R. E. BILLINGHAM SYDNEYCOHEN ROSELIEBERMAN JOHN
P. MER...
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ADVANCES I N
Immunology VOLUME 7
CONTRIBUTORS TO THIS VOLUME R. E. BILLINGHAM SYDNEYCOHEN ROSELIEBERMAN JOHN
P. MERRILL
CESARMILSTEIN
MICHAELPOTTE~ DARCY B. WILSON JOHN
B. ZABRISKIE
ADVANCES I N
Immunology EDITED B Y
F. J. DIXON, JR.
HENRY G. KUNKEL
Division of Experimental Pathology
l h e Rockefeller University N e w York, New York
Scripps Clinic and Rerearch Foundotion
La 10110, Californio
VOLUME 7 1967
ACADEMIC PRESS A Subsidiary of Harcourt Brace Jovanovich, Publishers
New York
London Toronto Sydney San Francisco
COPYRIGHT 1967, HY ACADl;nfI Types of light chain 'I
Fd Ip(ab')? Fab' Heavy chain Light chain y 01
p
I. 11, .I,c, s 111, €3, F
.Ipiece 5 S divaleiit fragment Uiiiv:dent frngnieiit H, A L, B
Chain Chain Chain
tl Challl I< or A cliaiiis I, or A chaiiih
1, 11, A
From B d l . World Health Otgan. 30, 447 ( 1964).
Sedimentation and viscosity measurements of rabbit immunoglobulin ( I g ) ( Noelken et al., 1965) have shown that the enzymatically separable fragments are more compact than the whole molecule and are presumably linked by a relatively extended area of the heavy chain (Fig. 1). This general molecular configuration is supported by the results of electron microscopy studies. Preparations of rabbit and human IgG examined by negative contrast appear as essentially symmetrical particles 80-120A. wide and about 34A. thick (Feinstein and Rowe, 1965). Pictures of ferritin and antiferritin systems taken at antigen excess show antibody molecules of similar appearance attached to antigen. At antigen antibody ratios nearer to equivalence, a number of Y-shaped strands are observed cross-linking antigen molecules and having a maximum length of 200A. which is twice the length of the intact antibody molecule ( Feinstein and Rowe, 1965); but see Fig. 3 (Valentine and Green, 1967). Examination of pepsin-treated antibody has shown that the base of the Y-shaped strand comprises the Fc fragment (Feinstein and Rowe, 1965; Rowe, 1966). These observations suggest that when antibody is crosslinked to antigen the Fab portion of the molecule can open to varying
4
SYDNEY COHEN AND CESAR MILSTEIN
FIG. 2. Comparison of reduced nlkylated humnn IgG ( l ) ,human IgM ( 2 ) , dogfish 17 S Ig ( S ) , and dogfish 7 S Ig ( 4 ) . Electrophoresis was performed on a starch gel in 8 M urea-formate buffer. (From Marchalonis and Edelman, 1966b.)
degrees and form a link twice as long as the original compact molecule (Fig. 3).Normal IgM examined by similar techniques shows a complex stellate structure made up of rods with a diameter of about 25A. and a maximum overall dimension of 230 to 250 A. (Feinstein and Munn, 1966, 1967). Anti-Salmonella IgM antibodies which visibly agglutinate were observed to crosslink flagella. At higher antibody concentrations, noncross-linked IgM antibodies appeared increasingly along the length of flagella and were seen in profile as staplelike structures with a maximum length of only 140 A. (Fig. 4).
A. ANTIGENICCLASSIFICATION The concept of immunoglobulins as a family of related but nevertheless heterogeneous molecules has arisen to a large extent from the study of their antigenic properties. Several degrees of antigenic difference between the immunoglobulins of a single species have been described (Oudin, 1960; Dray et al., 1962). These can be classified as ( 1 ) isotypic
STFtUcIzTRE AND A(;TIVITY OF IMMUNOGLOBULINS
5
speciiicities, common to all individuals of the same species, which differentiate classes and types of immunoglobulins; ( 2 ) allotypic specificities which distinguish polymorphic forms of immunoglobulin not present in all members of a given species; ( 3 ) idiotypic specificities which characterize individual antibodies (and myeloma proteins ) .
B. ISOTYPIC VARIANTS Different classes of immunoglobulins have been identified on the basis of physicochemical properties and the distinct antigenic specificities of their heavy chains; types within a single class of heavy chain show varying degrees of cross-reaction. This distinction is confused by the fact that heavy chains of different classes may, in fact, have common antigenic determinants. Thus rabbit antisera against human F( ab’) absorbed with light chains fail to react with Fc fragments, but show strong reactions with isolated y chains; such antisera, which are apparently specific for the Fd portion of the y chain, react with some IgA myeloma proteins (Kunkel et al., 1965) and also with some IgM macroglobulins (Kunkel et al., 1965; Harboe and Deverill, 1966). Similarly, antisera absorbed to show individual specificity for a particular IgG myeloma protein fail to react with many other proteins of the same class (Grey et al., 1965), but do react with certain IgM macroglobulins and also with a proportion of normal IgM molecules ( Seligman et al., 1965). The difficulty of antigenic classification is probably associated with the fact, discussed in detail below, that all classes of heavy chain seem to be derived from a common ancestor and have certain structural similarities. In addition, each has a portion which is relatively constant in structure and a part which is highly variable ( Fig. 1) . The variable portions considered collectively must contain a very large number of antigenic sites; their occurrence in different combinations probably accounts for some cross-reactions between heavy chains of different classes. The constant portions show a relatively restricted number of variants, and antisera which distinguish these will delineate clear-cut classes and types of immunoglobulins. The distinction between classes and types, as originally applied to whole immunoglobulin molecules, was based upon somewhat arbitrary differences. However, the terms will be retained here to indicate degrees of divergence in structural and biological properties, Classes of immunoglobulin are those having distinct antigenic properties which reflect major structural differences in C-terminal halves of heavy chains and are associated with discrete biological properties. Each class may contain several related types (or subclasses) which are not allelic products
STRUCTURE AND ACTIVITY OF IhihiUNOGLOBULINS
7
(C) FIG. 3. A. Electron microscopy of rabbit anti-DNP IgG saturated with a divalent DNP hapten (bisdinitrophenyl octamethylene diamine). Magnification: X500,OOO. Much of the antibody is linked to form closed rings with regular shapes (triangles, squares, pentagons). The hapten is at the mid-point of each side and the projections at each corner are at the middle of antibody molecules. Individual antibody molecules are therefore Y-shaped;- the angle between the- two arms may vary from 0" to 180". Each arm is about 60 A. long and about 35 A. wide. B. The antibody-hapten complex shown in (A) after peptic digestion. F c fragments a t each corner of the closed rings have been removed. C. The complex shown in (B) after reduction with dithiothreitol to convert F(ab')' to univalent Fab fragments. (From Valentine and Green, 1967.)
since they occur in all individuals. A given type of chain is defined by the amino acid sequence of its C-terminal half which is invariant except for polymorphic forms determined by ailelic genes.
1. Classes of Immunoglobulins The physical, chemical, and biological properties of the four main classes of human immunoglobulin have been established to a large extent by studying monoclonal proteins which characteristically belong to a
8
SYDNEY COHEN AND CESAR MILSTEIN
FIG. 4. Electron microscopy of flagellae of Salmonella paratyphi agglutinated in an IgM antibody at concentration 8 times the agglutination point. Negatively stained with sodium phosphotungstate p H 7. Magnification: X286,OOO. Bar = 0.1 p. (From Feinstein and Munn, 1966.)
STRUCTURE A N D ACTIVITY OF IMMUNOGLOBULINS
9
single antigenic class. The well-characterized human proteins are consequently regarded as prototypes for those in other species (Table 11). Differences between classes of heavy chains apart from those indicated in Table I1 include (1) molecular weights which appear to be characteristic for homologous chains in mammals as well as lower species (Table 111); ( 2 ) amino acid composition-comparative analyses have been recorded for human (Chaplin et al., 1965; Bernier et al., 1965), rabbit ( Lamm and Small, 1966), and bullfrog ( Marchalonis and Edelman, 1966a) heavy chains; ( 3 ) peptide maps which in human (Frangione and Franklin, 1965; Bernier et al., 1965) and rabbit (Lamm and Small, 1966; Cebra and Small, 1967) suggest dissimilarity in amino acid sequences of different heavy chain classes. The interspecies homology between chains of the same class is shown by their molecular weights (Table 111) and striking similarity of C-terminal sequences (see Table XX). The molecnlar weight of IgM is about 900,000 (Table 111). The molecule is split by reducing agents and reducing enzyme systems (Micheli and Isliker, 1966) into 4-chain nnits having molecular weights of 180,000 (Table I11 ) . IgM is, therefore, probably made up of five 7 S units linked by inter-p-chain disulfide bonds (Chaplin et al., 1965; Miller and Metzger, 1965) which may be formed between C-terminal cysteine residues (Doolittle et at., 1966). In some lower vertebrates, a protein which appears analogous to serum IgM occurs in the form of 7s monomers as well as larger polymeric forms (Marchalonis and Edelman, 1965; Clem and Small, 1966, 1967); a similar situation has been observed with certain horse (Sandor, 1962) and human antibodies (Rothfield el al., 1965; Stobo and Tomasi, 1966). The purification of IgA from normal human (Vaerman et al., 1965) and rabbit (Onoue et al., 1966) sera presents considerable difficulties. IgA can be more readily isolated from various seromucous secretions which contain a relatively high concentration of this immunoglobulin (see Schultze and Heremans, 1966). Exocrine and senim IgA show certain characteristic differences: 1. The sedimentation coefficient of IgA from saliva and colostrum (Tomasi et al., 1965) and bronchial secretions ( Masson and Heremans, 1966) is predominantly 11 S; over 90%of normal serum IgA, on the other hand, is 6.9 S (Tomasi et al., 1965). 2. The polymeric form of exocrine IgA is associated with an antigenically distinct fragment (transport or T piece) which is not present in 7s colostral IgA or in the polymeric IgA's of myeloma and normal sera (Tomasi et al., 1965). A similar fragment is excreted in the saliva
TABLE I1
PROPERTIES OF HUMAN IMMUKOGLOBULINS Properties
Biological Serum conc. (mg. %) (1)" 800-1680 Synthesis rate (mg./kg./d.) 20-40 (3) Catabolic rate (% I.V. pool/d.) 4-7 (3) Distrihution (% in I.V. pool) 48-62 (3) Ailtibody activity Complement fixation Placental passage (7) (14) Presence iri cerehrospinal fluid (8) Selective seromucnus secretionb 0 Skin sensitization heterologous species (9) homologous species 0 Immunological Light-chain types Kl Heavy-chain classes Y typesb 4 Allotypes, Gm IllV Ph ysicocheniical S?O.W 6 5-7.0 Ammonium sulfate precipitation (molar conc.) 1.49-1 64 (19) Total carbohydrate (%) (12) 2.9 Hexose (%) 1 10 Acetylhexosamine (%) 1 30 Sialic acid (%\ 0 30 Fucose (%j 0.20
+ + + + +
+ +
NOTE:
See footiiotes on faring page.
Igh
IgG
IgM
IgD
50-190 3.2-16.9 (5, 6 ) 14-25 (5, 6 ) 6 5 1 0 0 (5, 6 )
0.3-40 ( 2 ) 0.03-1.49 (6) 18-60 (6) 63-86 (6)
+
0 0
0
(b
t-
0
0 (10) ?
0 (8) 0 K,
x
140-420 2.7-55 (4) 14-34 (4) 40 (4)
+
0 0
+ +
Kt
K,
a
M
x
(2)"
d
2
2
0
+
0
+
0 (11)
7, 10, 13, 15, 17
18-20, >30 1.64-2.05 ( I S ) 11.8 5.4 4.4 1.3 0.7
6.2-6.8 (11)
7.5 3.2 2.3 1.8 0.2'2
11
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
of agammaglobulinemic patients lacking detectable IgA ( South et al., 1966b). The T piece dissociates from IgA of rabbit (Cebra and Small, 1967) and human colostrum (South et al., 1966a) in the presence of 5 M guanidine (Fig. 5B). The dissociated material has a molecular weight of about 50,000 and contains light-chain determinants which are presumably present either on the T piece (Hong et al., 1966) or on dissociated light chains (Cebra and Small, 1967). It has been postulated TABLE I11 MOLECULARWEIGHTSof IMMUNOGLOBULINS AND THEIRCHAINS
I3 at hit
Bullfrog (.5)
Dogfish (6)
y
Chain IgA (milk)
140,000 ( I , 2)" 53,000 (I, 2) 370,000 (4)
Chain IgM 7 S units of IgM p Chain Light chain
64,000 (4) 85&900,000 (3) 180,000 (3) 70,000 (3) 22-23,000 ( 1 , 2)
54,000 72,000 20-22,000
980,000 72,000 20,000
Immunoglot~ulin
IeG
01
-
' Key to references: ( 1 ) Pain (1963). ( 2 ) Small and Lamm (1966). ( 3 ) Lamm and Small (1966).
( 4 ) Cebra and Small (1967). ( 5 ) Marchalonis and Edelman ( 1966a ) ( 6 ) Marchalonis and Edelman ( 1966b).
.
that human colostral IgA (molecular weight 510,000) consists of three 4-chain IgA units noncovalently linked to the transport piece (Hong et al., 1966). The molecular weight of rabbit colostral IgA, on the other hand, is about 370,000 and the molecule is apparently made up of two 4-chain units and the transport piece (Cebra and Small, 1967). 3. After mild reduction, the IgA's from normal and myeloma sera are converted into 3.5 S units (Franklin, 1962; Fahey, 1963a; Deutsch, Key to references in Table 11: ( 1 ) Fahey and McKelvey ( 1965). ( 2 ) Rowe and Fahey ( 1965). ( 3) Cohen and Freeman ( 1960). ( 4 ) Solomon and Tomasi ( 1964). ( 5 ) Wochner et al. ( 1963) . ( 6 ) Rogentine et al. ( 1966). ( 7 ) Citlin et ol. (1963).
( 8 ) Schultze and Heremans ( 1966). ( 9 ) Ovary and Karush ( 1961). ( 10) Franklin and Ovary ( 1963). ( 11 ) Hansson et al. ( 1966). ( 12) Heimburger et al. ( 1964). ( 1 3 ) Olesen et al. ( 1905). ( 1 4 ) Kohler and Farr (1966).
See text. Myeloma IgDs are predominantly of Type L (Hobbs et al., 1966; Hansson el a/., 1966).
12
SYDNEY COHEN AND C E S A R MILSTEIN
1964), whereas exocrine IgA remains undissociated (Tomasi et al., 1965; Onoue et al., 1966). An additional class of immunoglobulin ( IgE ) has been postulated to account for the properties of human reaginic antibodies. The association of such antibodies with IgA was previously suggested by immune precipitation and by the observation that purified IgA (Ishizaka et al., 1963) or its isolated CY chain (Ishizaka et at., 1964) blocked the skin-sensitizing activity of reaginic sera. More recently, Ishizaka and colleagues have shown that serum reaginic activity may be retained after precipitation with antisera specific for IgG, IgA, IgM, or IgD, but was removed by coprecipitation with IgGanti-light-chain complexes ( Ishizaka and . activity was also Ishizaka, 1966; Ishizaka et al., 1 9 6 6 ~ )Skin-sensitizing removed with a rabbit antiglobulin which does not react with IgG, IgA, IgM, IgD, or with human light chains (Ishizaka et al., 1966a,b), but which precipitates with a protein having ?,-globulin mobility. This protein was designated IgE and was thought to contain light chains on the basis of radioimmunodiffusion tests using an anti-IgG antiserum absorbed with Fc. In several sera from patients sensitive to ragweed pollen extract, skin-sensitizing antibody was almost quantitatively removed by precipitation with anti-IgE. In addition, sensitizing activity and IgE were similarly distributed on diethylaminoethyl cellulose ( DEAE ) chromatography, gel filtration, and density gradient centrifugation of serum; the sedimentation coefficient of both was estimated to be 7.7 S. In some human subjects, therefore, reaginic activity is apparently associated with a fraction having the features of a distinct immunoglobulin class. It remains possible, however, that reaginic antibodies are heterogeneous and associated with different immunoglobulins in different patients Reid et al., 1966). Anaphylactic antibodies closely analogous to human reagins have been described in the rat (Mota, 1964; Binaghi and Benacerraf, 1964; Binaghi et al., 1964, 1966; Oettgen et al., 1966), the rabbit (Zvaifler and Becker, 1966), and the dog (Patterson et al., 1963, 1964; Rockey and Schwartzman, 1966). The sensitizing antibodies in these three species resemble human reagins in many respects; they are present in extremely low concentrations, have p mobility on electrophoresis, are destroyed by heating at %"C, have high affinities restricted to homologous tissues, their sedimentation coefficients are greater than 7 S and their skinsensitizing activity i destroyed by reduction and alkylation. 2. Types of Immunoglobulins a. Light-Chain Types. The occurrence of distinctive isotypic specificities within a single class of immunoglobulin led to the recognition of
STRU-
AND ACTJYITY OF IMMUNOGLOBULINS
13
two types of light chains in human (Mannik and Kunkel, 1963a; Fahey, 1963b) and mouse immunoglobulins (Potter et al., 1964; McIntire et al., 1965). Both human light chains occur on all classes of immunoglobulin and the ratio of Type K : L is about 2:l. Mouse light chains, on the other hand, occur with different frequencies on distinct immunoglobulins (see chapter by Potter and Lieberman); those designated K (previously A ) are found predominantly on yG-Bel, yG-Be2, y-A, and y-F myeloma proteins and a distinct type, h (previously K ) has been described in association with a yM-macroglobulin ( McIntire et al., 1965). Antigenic analysis of human light chains indicate that normal K and h chains each contain at least two and probably more subtypes, but it has not proved possible to separate these into clear-cut groups or to correlate observed antigenic differences with the nature of the N-terminal amino acid (see Table XV) or with InV specificity of K chains (Stein et al., 1963; Williams, 1964; Epstein and Gross, 1964; Solomon et a,?., 1965; Kunkel et al., 1965; Nachman et al., 1965; Putnam et al., 1966). Human monoclonal immunoglobulins that fail to react with antisera to K and h chains, may, nevertheless, contain one or other of the light chains in normal yield after reduction and alkylation (Rowe and Fahey, 1965; Osterland and Chaplin, 1966). These proteins apparently have light-chain antigenic determinants which are inaccessible in the intact molecule; partial screening of Iight-chain determinants has previously been described (Nachman and Engle, 1964; Epstein and Gross, 1964). When tested with certain antisera, light chains may behave as univalent antigens (Franklin, 1963) and for this reason Fab fragments of myeloma proteins may fail to precipitate with anti-light-chain antisera (Franklin et al., 1966). This may be of practical importance in typing samples which have undergone spontaneous fragmentation during storage. Two types of light chain have been demonstrated in the guinea pig ( Nussenzweig et al., 1966). Certain antibodies, notably, those against dinitrophenyl-bovine y-globulin ( DNP-BGG ) have light chains of a single antigenic type (designated Type K ) . After absorption with light chain from purified DNP-BGG, a rabbit antiserum against guinea pig light chains was specific for Type L immunoglobulins. Specific anti-K antisera were obtained directly by immunization with light chains from anti-DNP-BGG antibodies. About two-thirds of normal guinea pig y2globulin molecules have K chains and one-third have h chains; both have similar molecular weights as judged by gel filtration but differ somewhat in electrophoretic mobility so that the h-chain content of slow y,-globulin is only 10%and that of fast 7,-globulin, about 40%. b. 7-Chain Types. Types of the humar, y chain have been identified using rabbit antisera to purified human myeloma proteins (Grey and
TABLE IV TYPESOF HUMANIgG Nomenclature"
% Total (6) y2 yl
73
r4
"
(1)
Ne We Vi Ge
(2)
(5)
Y Z ~ 72%
yzC
C Z
T?d
Key to references: (1 ) Grey and Kunkel ( 1964 ). (2)Terry and Fahey (1964). , 3 ) Ballieux et al. ( 1964).
(4)
% Type K (4)
% Type L
(4)
11 77 9
5.8 54.5 4.7
4.2
3
2.6
0.5
100%
67.6%
-
Skin sensitization (5)
Location of type specificity
5.2 22.5
___ 32.470
( 4 ) Terry et al. (1965)-analysis of 191 IgC myeloma proteins. ( 5 ) Teny (1965).
( 6 ) Unified notation proposed by Kunkel et al. ( 1967).
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
15
Kunkel, 1964) or fragments of heavy-chain ( BalIieux et al., 1964; Takatsuki and Osserman, 1964) or monkey antisera to pooled human IgG (Terry and Fahey, 1964). These antisera made specific by absorption with selected myeloma proteins identified four types of IgG which have been variously designated (Table IV). IgG types are present in all normal human sera but techniques for their isolation have not been devised. There is evidence that the normal serum concentration of IgG types is influenced by the genotype of the individual (Yount, Kunkel, and Litwin, 1967). IgG myeloma proteins belong to one or other type, the commonest being yJl, ( W e ) (Table I V ) ; each may be associated with either K or chains, although available data suggest that Y chains may be commoner in y.,, than in yJ,, or -prproteins (Table IV). The typespecific determinants of y.,, and y?,, are located on the Fc part of the y chain (Terry and Fahey, 1964). Those of yZc appear on either Fc or F(ab')' depending on the antiserum used for testing; type specificity may, therefore, be distinguished in an analogous fashion by sera that react with determinants on different parts of tha chain. The Fc fragments from various types of human y chains have different peptide fingerprints ( Frangione and Franklin, 1965; Frangione et al., 1966b). The fragments of y..,, yl13, and y 2 . appear to be closely related, whereas y2,, has a greater degree of strnctural individuality. Differences observed in C-terminal amino acid sequences are only partially related to type specificity. Thus, y.n and y2,, have identical C-terminal peptides, and amino acid substitutions ( possibly related to allotypic specificity ) occur among individual yLLproteins (see Table XX). Guinea pig serum contains two immunoglobulins, referred to as yland yl-globulin (Yagi et al., 1962; Benacerraf et al., 1963; White et al., 1963). Whether these should be regarded as distinct Ig classes or types of IgG is uncertain. Both have sedimentation coefficients of 7 S and the same hexose content (0.74%)and both are transmitted from maternal to fetal circulations (Bloch et al., 1963b) so that y,-globulin is not regarded analagous to human IgA (Oettgen et al., 1965). The two guinea pig 7-globulins differ in their biological properties, only y l being capable of sensitizing guinea pigs to local and systemic anaphylaxis whereas y2 is involved in cytophilic activity for macrophages, complement fixation, and in complement-dependent phenomena such as cell lysis and the Arthus reaction (Ovary et al., 1963, Bloch et al., 1963a; Berken and Benacerraf, 1966). The specific determinants that distinguish y,- and yJglobulins are localized on the Fc portions of their heavy chains (Nussenzweig and Benacerraf, 1964; Thorbecke et al., 1963); the light chains and Fd fragments of both types are antigenically indistingurshable ( Nussenzweig and Benacerraf, 1966a ) .
16
SYDNEY COHEN AND CESAR MILSTEIN
Mouse serum also contains a y,-globulin which is capable of transferring passive cutaneous anaphylaxis in the mouse and does not fix complement (R. S. Nussenzweig et al., 1964; Ovary et al., 1965). The similarity of yl-globulins of mouse and guinea pig is also shown by the fact that their sensitizing activity is not inactivated by heating at 56°C. or by reduction and alkylation (R. S. Nussenzweig et al., 1964). It is of interest that reaginic antibody has not been detected in either species and that, conversely, distinct y ,-antibodies are not found in those species ( man, rabbit, rat) that have reaginic antibodies. In the bovine there appear to be two types of IgG with electrophoretic mobilities of 7,- and 7,-globulin. On serological testing, the light chains and Fab fragments of IgG, and IgG, are identical, but the Fc portions of their respective heavy chains give reactions of partial identity. IgG, sensitizes skin in the homologous species but in contrast to the y I of mouse and guinea pig, the bovine fraction fixes complement; IgG, is associated with precipitating antibody but fixes complement very poorly (Pierce and Feinstein, 1965, 1967). Horse serum contains what appear to be three variants of IgG having antigenically distinguishable heavy chains and common light chains [IgG(a), I g G ( b ) , and I g G ( c ) ] (Rockey et al., 1964; Klinman et al., 1965, 1966). An additional antigenically distinct immunoglobulin has a relatively high carbohydrate content, faster electrophoretic mobility, fails to fix complement and was designated IgA (Klinman et al., 1966). This may correspond to horse immunoglobulin which was recognized by van der Scheer et al. (1940) as being distinct from IgG and named T-globulin. This protein, on the basis of its yl electrophoretic mobility (Smith and Gerlough, 1947), carbohydrate content (Schultze, 1959), and distinct antigenic determinants (Jager et al., 1950) has been regarded as analogous to human IgA. More detailed comparisons of horse IgG and T-globulin have shown that the light chains and Fab fragments are antigenically indistinguishable and that the Fc fragments, although antigenically different, show some cross-reaction ( Weir and Porter, 1966) and have identical C-terminal sequences (Table XX). These chemical and antigenic similarities suggest that horse T-globulin is, in fact, an IgG type rather than a protein analogous to human IgA. The interspecies variation of immunoglobnlins and the difficulty of defining analogous classes or types on the basis of overall chemical and antigenic properties are emphasized by this work. c. chain Types. The occurrence of two antigenically distinguishable types of human IgA has been demonstrated using goat antisera to chains of a myeloma protein (Vaerman and Heremans, the isolated (Y
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
17
1966) and monkey or rabbit antisera to myeloma IgA’s (Kunkel and Prendergast, 1966; Terry and Roberts, 1966; Feinstein and Franklin, 1966). One type includes about 10 to 15%of myeloma proteins examined and is antigenically deficient when compared by gel diffusion with the major type. Absorption of antisera with antigenically deficient proteins leaves an antiserum which reacts only with the major group; attempts to produce a specific antiserum for the minor subgroup by direct immunization have been unsuccessful, nor has it been possible to demonstrate this subgroup in normal IgA, presumably because it constitutes a minor component. Subgroup determinants appear to be located on chains and are unrelated to the polymeric forms of IgA since both subgroups include these and since antigenic specificity is unaltered after conversion to 7 S units by reduction and alkylation. d. p-Chain Types. Antigenic heterogeneity has been observed with human IgM antibodies (Deutsch and MacKenzie, 1964; MacKenzie and Deutsch, 1965) and with monoclonal IgM’s. However, the first possible differentiation of two antigenic classes of IgM in patients with macroglobulinemia was made by Harboe et al. (1965a). A rabbit antiserum to a macroglobulin absorbed with normal serum gave a precipitin reaction with 8 out of 21 macroglobulinemic sera and failed to react with the remaining 13 as judged by direct precipitation or inhibition tests. The antiserum did not precipitate with either heavy or light chains but absorption tests indicated that its specificity was directed against determinants on the p chains. Since normal sera varied in their ability to inhibit the typing serum, it is possible that the observed specificity represents a genetic factor rather than a subtype of IgM present in all normal individuals. (Y
C. ALLOTYPIC VARIANTS Those antigenic specificities present on immunoglobulin molecules which differ between individuals of the same species, are referred to as allotypes. This phenomenon was first observed in rabbits (Oudin, 1956) and man (Grubb, 1956) and has since been described in the guinea pig (Oudin, 1958; Benacerraf and Cell, 1961) mouse (see chapter by Potter and Lieberman), baboon (Kelus and Moor-Jankowski, 1962), pig (Rasmusen, 1965), chicken (Skalba, 1964), and rat (Rarabas and Kelus, 1967) . 1 . Human Allotypes
The genetic polymorphism of human immunoglobulins detected on the basis of serological differences has been the subject of several recent
18
SYDNEY COHEN AND CESAR MILSTEIN
reviews (Steinberg and Polmar, 1965; Mhtensson, 1966; Steinberg, 1966; Oudin, 1966a,b). A t present over twenty allotypes have been identified on human immunoglobulins. A new terminology for these Gm and InV factors has been proposed (Table V ) but its general adoption is delayed TABLE V NOTATIONOF HUMAN ALLOTYPES' Specificities
Gm
Original a, X
[f"" and b2 b and b' c or "Gm-like" r e P
New 1 2 3 4 5 6
Original
New
tP
10 11 12 13 14 15 16 17 21 22 24
I? b? I-, 3
b4
7 8
si
9
g
t
Y n
InV
I a
b
1' 2 3
b
From Bull. World Health Organ. 33, 721 (1965). Does not produce a detectable antigen. InV( 1) is found whenever InV( a ) is present, but may also be found in the absence of InV ( a ).
by the expectation that recent work may lead to a more rational basis for a revised terminology. The distinct identity of certain Gm specificities is a matter of controversy and it has been claimed, for example, that G m ( b w ) and G m ( f ) are the same (Steinberg, 1965; Steinberg and Polmar, 1965) . Studies on the distribution of allotypic factors on immunoglobulins have shown that Gm groups occur only on IgG molecules; InV groups, on the other hand, are found on IgG, IgA, IgM, and Bence-Jones proteins (tests for InV activity of IgD do not appear to have been recorded). As would be expected from this distribution, Gm activity is associated with the y chain of IgG and InV with light chains (Polmar and Steinberg, 1964; Lawler and Cohen, 1965). The InV activity is, therefore, confined to the Fab fragment of the molecule which contains the light chain. The
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
19
distribution of Gm activity on enzymatic fragments of IgG varies with different specificities. Gm( a ) , Gm(x), Gm(b), Gm(b3), Gm(y), and Gm( n ) are located on the Fc portion of the y chain, whereas Gm(f) and Gm( z ) are associated with the Fd fragment and are, therefore, present on Fab (Steinberg and Polmar, 1965; Litwin and Kunkel, 1966). Gm(f) specificity is dependent on the quaternary structure of the molecule (Polmar and Steinberg, 1964). Thus, specificity cannot be detected on separated heavy or light chains, but is restored when chains are recombined provided that the y chain comes from a Gm(f+) IgG. On the other hand, light chains of either Type K or L and derived from IgGs of various Gm specificities are equally effective in restoring Gm(f) activity in the recombined molecule (Steinberg and Polmar, 1965). In a preliminary report, Litwin and Kunkel (1966) state that Gm( z ) specificity is partially dependent on quaternary structure. This may be true for many allotypic factors since the serological activity of isolated chains and fragments is frequently less than that of the parent molecule; this is especially the case for InV determinants on isolated light chains. Analyses of myeloma proteins have shown that each genetic factor is associated with a particular type of peptide chain. InV activity is present only on K chains and is not detectable on X chains (Terry et al., 1965); the reported association of InV(b) activity with a X chain was attributable to the use of an unreliable typing serum (Lawler and Cohen, 1965). The available data indicate that only those K chains associated with yZbor yzcheavy chains carry recognizable InV specificity; twelve yZRor yZa proteins of Type K were all negative for InV factors (Terry et al., 1965). The significance of this observation remains to be determined but it may be that InV determinants are masked by the quaternary structure in these types of molecules. The G m ( a ) , Gm(x), G m ( f ) , Gm(z), and Gm(y) occur only on yZh proteins, whereas Gm(b'), Gm(b3), and Gm(b4), Gm(c), Gm(s), and Gm(t) are found only on y2? (Kunkel et al., 1964b; Mirtensson and Kunkel, 1965; Terry et al., 1965; MArtensson, 1966) (Table VI). The recently described allotypic specificity, Gm ( n ) which is detectable in a precipitating system using primate antisera, is associated only with y?., proteins (Kunkel d al., 1966). Another allotypic specificity, Gm( g ) is probably the true allele of Gm( b ) (Natvig, 1966). Specificities of the Gm system are inherited in certain fixed combinations which differ from race to race (Table VII), and each set behaves as a unit of inheritance in family studies, i.e., no recombination of specificities is observed. It appears, therefore, that each set of specificities is determined by a cluster of sites in 1 chromosome. These sites
TABLE VI DISTRIBUTION OF ANTIGENIC AND ALLOTYPIC SPECIFIC~IES IN IgG MYELOMAPROTEINS
Gm Type
Caucasians
wa y2b
-
+ +
Y2c
Negroes 72s
Y2b
Y2C
YZd
Chinese Y2b
7%
+-
-
+-
-
-
-
+
-
a Gm( a ) and ( x ) are closely associated and occur together in about 50%of Caucasian (1965), Litwin and Kunkel (1966), and Kunkel et al. (1966).
Y2b
-
+
myelomas. Data from Terry et d.
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
21
have been regarded as constituting a single gene (defined as a unit of inheritance) with sets of specificities comprising a series of alleles (Steinberg, 1965). However, the fact that every Gm factor is confined to one type of heavy chain has suggested to several investigators that there are four closely linked genetic loci each directing the synthesis of one type of chain (Kunkel et al., 1964a,b; MHrtensson, 1964; Fudenberg et al., 1966). According to this theory, the y2b locus controls the synthesis of Gm(a), ( f ) , ( y ) , and ( z ) , the y Z c locus that of Gm(b) and Gm(g) (Natvig, 1966), and the palocus controls synthesis of Gm(n). TABLE VII SOMEGm SPECIFICITIES WHICHBEHAVEAs UNITSOF INHERITANCE’
‘I
Froiii Steinberg ( 1966 ).
It seems likely that the varying relationships of these genetic antigens in different races together with their molecular locations will provide a means of mapping the genes that control the synthesis of human heavy chains (see Mgrtensson, 1966). For example, the invariable association of Gm( n-) with Gm( a+z+) in Negroes and Caucasians suggests that genes controlling yZa and yZb are adjacent (Table VIII). The YZb immunoglobulins carry Gm specificities on both the Fd [Gm(z) and Gm(f)] and the Fc [Gm(a) and Gm(y)] portions of the heavy chain (see Table VIII). In Y2b myeloma proteins from Caucasians or Negroes these are invariably paired and such proteins are either Gma+, zf or Gmf+, y+ (Litwin and Kunkel, 1966). This pairing of specificities on the same myeloma protein implies that both are present on the same y chain. The two halves of the Y2b chain, therefore, appear to be inherited as a single genetic unit and this does not support the view
22
SYDNEY COHEN AND CESAR MILSTEIN
TABLE VIII TENTATIVE ARRANGEMENT FOR CHROMOSOMES CONTROLLING THE SYNTHESIS OF
COMMONLY OCCURRING y CHAINS“ Chromosomes
yZ b
(We)
Caucasians
Negroes
‘See data of Kunkel et al. ( 1966). The linear order of the genes is arbitrary.
frequently put forward (see Cohen and Porter, 1964) that Fc and Fd are, in fact, separate peptide chains. The Fd fragment probably contains a portion of the constant region and this is likely to include Gm(f) and Gm( z ) specificities. However, if these are localized in the “variable” N-terminal part of the y chain, then this must be a product of the same gene that codes for the relatively constant C-terminal part, and any mechanism involving gene fusion is excluded (see Section VI1,B).
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
23
In the Chinese a different relationship between the genetic factors of y r b myeloma proteins has been observed. Such proteins were shown by
Mkrtensson and Kunkel (1965) to be Gm( a+f+)-a combination never observed in Caucasians or Negroes-and more recently two such myeloma proteins were found to be Gm(a+f+y+) (Litwin and Kunkel, 1966). This indicates that Gm(a) and Gm(z) are carried on different molecules in Mongoloids, and this is in accordance with the observation that many Chinese sera are Gm(a+), Gm(z-). The fact that Gm(a) can remain associated with Gm(y) suggests that although both are on Fc, they do not occupy exactly homologous positions in the y chain (Table VIII). Considerable progress has been made in establishing the structural basis of InV specificity; InV(b+) and InV(a+) light chains have been shown to differ by the substitution of valine for leucine at residue 191 (see Table XII). Other substitutions which would account for InV( 1) specificity or for K chains without detectable allotypy, have not been reported. Peptide differences observed on Fc fragments of y chains were originally thought to correlate with the presence of Gm(a) and Gm(b) specificities (Meltzer et al., 1964; Fudenberg et al., 1964; Frangione and Franklin, 1965). The peptide regarded as characteristic of Gm(b) was later found in all Gm(a-) myeloma proteins of the pa,yZb,and y p c subtypes, but not in those of Xzd (Fudenberg et al., 1964; Thorpe and Deutsch, 196%; Frangione et al., 196%). It appears that these distinctive peptides characterize proteins which are Gm( a+ ) and those which are Gm( a - ) in three of the subclasses. Thorpe and Deutsch (1966b) obtained similar results and in addition found the following sequences for the peptides isolated from two Y.'b proteins: Gm(a +): Thr-Leu-Pro-Pro-Ser-Arg-Asp-Glu-Leu-Thr-Lys Gm(a - ) : Thr-Leu-Pro-Pro-Ser-Arg-Met-Glu-Glu-Thr-Lys
Multiple amino acid differences between what appear to be allelic products have previously been observed in sheep hemoglobins A and B (Huisman et al., 1965; Boyer et al., 1966). Although the Fc fragment produced by trypsin from y chains retains Gm(a) and Gm(b) activity (Lawrence and WiIliams, 1966), the tryptic peptide isolated from completely reduced Gm( a + ) proteins did not show allotypic activity (Thorpe and Deutsch, 1966b) so that the specific configuration may be dependent upon a longer sequence. A single amino acid substitution has been observed in the C-terminal peptides of ysr chains and may correlate with the allelic Gm( b ) and Gm(g) specificities (see Table XX).
24
SYDNEY COHEN AND CESAR MILSTEXN
2. Rabbit Allotypes Several rabbit allotypes have been described (Table IX); the molecular location and apparent genetic relationships of six of these have been studied in detail (reviewed by Oudin, 1966a,b; Kelus and Gell, 1967). Those designated A l , A2, and A3 appear to be controlled by alleles at one locus "a" and are located on the Fd portion of the heavy chain (Feinstein et al., 1963; Stemke, 1964). The specificities A4, AS, and A6 are controlled by alleles at a second locus "b" and are located only on TABLE IX ALLWIYPESOF RABBIT IMMUNOGLOBULIN
Ig Clitss
Specificity
IgG, IgM, IgA
Light, chain
IgG, ?IgM ?IgA
Light, chaiii Fc
IgG
"
Molecular locat ion
Key to references: ( 1 ) Hamers et al. ( 1964). ( 2 ) Oudin (1960). ( 3 ) Dray et al. (1963a).
Specificities occurring together
4: A;' AS A:' AS" A: A:
( 4 ) Kelus and Gell (1965). ( 5 ) Sell (1966). ( 6') Dubiski and Muller ( 1967).
light chains ( Wilheim and Lamm, 1966). An additional specificity (A9) appears to be related to the "b" locus, since in heterozygous rabbits A9 is associated with only one other specificity of the "b" group, whereas rabbits homozygous for A9 have no other allotypes of this group (Dubiski and Muller, 1967). Some antisera used for the detection of a specific allotype reveal several specificities systematically found together; these are designated with a prime or double prime (Table IX). The allotypic specificity designated A8 has been identified on rabbit IgG molecules some of which do not appear to carry A l , 2, or 3 (Hamers
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
25
et al., 1964). This specificity is present on the Fc portion of the heavy chain, has not been detected on IgM and is thought to identify a distinct subclass of rabbit IgG controlled at a locus closely linked to “a” (Hamers and Hamers-Casterman, 1965; Hamers et al., 1966). The specificity “e,” study of which was discontinued through lack of immune serum, is probably controlled by an allele closely linked to the “a” locus (Oudin, 1966a). The allotype P, on the other hand, appears to be determined at a locus distinct from “a” and “b,” whereas the genetic control of specificity T has not been established (Dray et al., 1963a). Individual molecules may carry specificities determined by different loci, but allelic specificities are found on separate molecules (Oudin, 1962; Dray and Nisonoff, 1963; Dray et al., 196313; Stemke, 1965). Up to 20% of molecules may have no allotypic specificity determined by the “a” locus (Dray and Nisonoff, 1963; Stemke, 1965) and a similar proportion have no “b” locus allotypes (Dray and Nisonoff, 1963; Oudin and Bornstein, 1964; Bornstein and Oudin, 1964; Stemke, 1964). Specificities controlled by both “a” and “ b loci have been detected on serum IgG and IgM (Todd, 1963; Stemke and Fischer, 1965) and have also been identified on both IgG and IgA in rabbit colostrum (Feinstein, 1963; Sell, 1967). However, Cebra and Robbins ( 1966) were unable to identify A1 or A2 factors on the IgA isolated from colostrum of five individual rabbits, although these specificities were readily identifiable on IgG from the same animals. Similarly, A 1 specificity present on serum IgG was not detectable on IgM (Lamm and Small, 1966). These discrepancies have not been resolved but may be due to differences in typing sera and reflect a complexity of rabbit allotypes which has not been adequately defined. An allotypic specificity confined to IgM (MS-1) was detected by Kelus and Gel1 (1965) who used a typing serum raised by immunization of an A3, A4, A5 recipient with anti-Proteus antibody from an A3, A4 donor. This antiserum reacted on gel diffusion with serum of the donor taken before immunization, with serum from 15 out of 40 of the donor’s offspring and 8% of unrelated rabbits. The specificity appeared to be localized on IgM as judged by immunodiffusion, ultracentrifugation, and gel filtration analyses. A second specificity present on rabbit IgM, but not on IgG and absent from a serum containing MS-1 has since been identified and designated MS-2 (Sell, 1966). The genetic relationship between MS-1 and MS-2 and between these and the specificities controlled by the “a” and “ b loci have not been determined. Several studies have been concerned with the structural basis of allotypic specificity in rabbit immunoglobulins. Data on the molar-
26
SYDNEY COHEN AND CESAR MILSTEIN
combining ratios of anti-A4 Fab fragments and A4 IgG from homozygous rabbits, suggest that light chains have three or four A4 determinants (Mage et al., 1966). Light chains isolated from the IgG of A4 and A5 homozygous donors have different amino acid compositions (Reisfeld et al., 1965) and show several distinct peptides on fingerprinting (Small et al., 1965). The heterogeneity of these preparations makes it difficult to assess the relationship of chemical differences to A4 and A5 specificities. Heterogeneity of the Fd fragment prevents its effective examination by the fingerprint method but, nevertheless, heavy chains with A2 specificity were distinguished from A 1 or A 3 chains by the presence of a yellow spot and absence of a brown spot (Small et al., 1966). Whether these are attributable to differences in amino acid composition or to distinct carbohydrate moieties in the heavy chains has not been determined.
D. IDIOTYPICVARIANTS Myeloma proteins and macroglobulins have been known for some time to possess individual antigenic specificity ( Kunkel, 1965). Antisera detecting such specificity do not react with normal immunoglobulin. Nevertheless, in the majority of instances, continued absorption with pooled immunoglobulin causes a progressive removal of the specific antibody; this suggests that normal immunoglobulin contains molecules analogous to most of those present in the spectrum of myeloma proteins. Determinants responsible for individual specificity are always associated with the Fab fragment and may be localized either on Fd or on light chain or be manifest only when these are recombined (Grey et al., 1965; Seligman et al., 1966). More recently, an apparently similar form of antigenic individuality, referred to by Oudin (1966a) as idiotypic specificity, has been demonstrated on a number of isolated antibodies (reviewed by Gel1 and Kelus, 1967). Antisera detecting a given idiotypic specificity react only with the individual antibody used for immunization and appear to have identical specificity when raised in different animals. Such antisera do not react with normal immunoglobulin, with preimmunization serum, with antibodies of other specificities from the donor, nor with antibodies having the same spectrum of specificities but raised in other animals. Idiotypic specificity, therefore, appears to be located in a variable region of the Fab fragment associated in some way with combining specificity. This variable stretch could be responsible for modulation of the combining site or for specific association of heavy and light chains (see Section V ) .
STRUCTURE AND ACTMTY OF IMMUNOGLOBULINS
Ill.
27
Enzymatic a n d Chemical Fragments
The IgG molecules can be split by a variety of enzymes which act at different sites, hut all within a limited and as yet incompletely defined area of the y chain (reviewed by Fleischman, 1966; Cohen, 1966). From molecular weights (Table X ) , the ability of univalent fragments to reform dimers, and sequence data (see Table XXI) it seems that papain splits the IgG molecule at the N-terminal side of the inter-heavychain disulfide bond (Fig. 1); pepsin, on the other hand, splits the y chain at a point nearer to the C-terminus. In accordance with this view, papain digestion of the peptic fragment F(ab’) 2, together with mild reduction and alkylation releases peptides which contain approximately TABLE X
MOLECULARWEIGHTSOF Enzlme ~
~~~~~
Papain Insoliible papaiii and S.D.S. Pepsin
THE
ENZYMATIC FRAGMENTS OF RABBIT IgG
Fragments
Molecular weight
Ref.
~
Fr Fab F(ab)? F(ab’)’
48,000 42,000 84,000 91,000
Soelken et al. (1965) Soelken et al. (1965) Jaquet and Cehra (1965) Jaquet and Cebra (1965)
one blocked sulfhydryl group per Fab fragment (Mage and Harrison, 1966). Analyses of such peptides have been carried out in rabbit IgG to define the structure of the 7-chain area which contains an inter-heavychain disulfide bond and is sensitive to proteolytic hydrolysis (see Section IV,C,3). Analysis of N-terminal peptides isolated from rabbit Fc has shown that the susceptible section of the chain has a high proline content ( 8 out of 18 residues) and that papain may act at several different peptide bonds in this region (Hill et al., 1966b). At acid pH, papain is able to hydrolyze the isolated rabbit Fc fragment releasing a C-terminal peptide of 113 residues; this is apparently devoid of certain biological activities associated with whole Fc (Prahl, 1967) and is similar to a fragment isolated after peptic digestion ( Utsumi and Karush, 1965). Another fragment of papain digestion (Fc’) has been isolated and may be part of Fc ( Poulik, 1966). The heavy chain is also susceptible to cleavage by cyanogen bromide. In the presence of 0.3 M HCl this reagent splits about half the methionyl residues of rabbit IgG and liberates a bivalent antibody fragment together with several smaller peptides ( Cahnmann et aE., 1965). Compari-
28
SYDNEY COHEN AND CESAR MILSTEIN
son of molecular weights and amino acid compositions indicates that the cyanogen bromide 5 S fragment is somewhat smaller than F(ab’)’. Reduction of the cyanogen bromide fragment releases two pieces resembling Fab and on reoxidation about 50% of the original bivalent fragment is reformed (Cahnmann et al., 1966). It seems likely that, in rabbit IgG, cyanogen bromide splits the y chain between the sites of cleavage by papain and pepsin perhaps at residue 221 from the Cterminus (see Table XXI) . Particular interest attaches to the isolation of the Fd fragment of the heavy chain in view of its likely association with the antibody-combining site. This fragment has been isolated from rabbit IgG, horse T-globulin, and a human myeloma IgG by gel filtration of reduced Fab under conditions that favor dimerization of Fd (Fleischman et al., 1963; Press et al., 196613; Weir and Porter, 1966). Isolation of normal human Fd’ from a fraction of F( ab’)2 soluble in 18%Na,SO, has been reported (Heimer, 1966). Preparations of IgG contain molecules which vary in their susceptibility to papain digestion; brief digestion of rabbit IgG releases Fab fragments (3.5 S ) and a 5 S fraction which appears to be an intermediate product containing an intact y chain linking one Fab and one Fc fragment (Nelson, 1964; Goodman, 1965). In addition, there remains a 6.9 S fraction with IgG specificity which is relatively resistant to redigestion with papain; its hexose content is almost twice that of the total IgG and this difference is confined to Fab (Goodman, 1965). Types of human IgG react differently to papain digestion. Human y Z Dappears to be unusually susceptible and its Fc fragment is hydrolyzed more rapidly than that of Y?b (Takatsuki and Osserman, 1964; Poulik, 1964; Frangione and Franklin, 1965; Thorpe and Deutsch, 1966a). Horse IgG and T-globulin (which appears to be a type of IgG-see Section II,B,2,b) differ markedly in their products of papain digestion in the presence of 0.01 M cysteine (Weir and Porter, 1966). IgG gives the expected 3.5 S fragment, whereas the T-globulin gives a divalent 5.6 S fragment (molecular weight 97,000) together with smaller peptides. This difference is associated with the presence of an additional disulfide bond linking the Fd fragments of T-globulin; the divalent fragment is converted to the univalent piece by reduction with 0.1 M mercaptoethanol. Guinea pig yl- and y2-globulins behave similarly on papain digestion; however, if both are dialyzed against phosphate buffer pH 7.6 in the cold, a crystalline fraction of the y,-Fc fragment is obtained while y2-Fc remains in solution (Nussenzweig and Benacerraf, 1964). Only limited information is available concerning the enzymatic
STRUCTURE AND ACTIVITY O F IMMUNOGLOBULINS
29
cleavage of IgA molecules (Deutsch, 1963, 1964; Bernier et al., 1965). As far as IgM is concerned, reports by Petermaim and Pappenheimer (1941) and by Deutsch et al. (1961) have suggested that fragments analogous to F(ab’)’ could be obtained by enzymatic digestion. In a more recent study Miller and Metzger (1966) observed that human macroglobulin or its 7 S units underwent a progressive cleavage when reacted with trypsin; after 18 hours incubation, about 52% of the IgM was recovered in the form of two fragments. One had a molecular weight of about 47,000, contained a single interchain disulfide bond, and both p - and light-chain ( K ) determinants which were separable by reduction and gel filtration in N-propionic acid. A second fragment with a molecular weight of 114,000 appeared to be a dimer of the first, linked by an inter-p-chain disuKde bond. These fragments apparently correspond to Fab and F(ab‘)?, respectively, and were designated F ( a b ) p and F(ab’)?p. Unlike the fragments of IgG, the divalent piece of IgM is converted to the univalent form by further tryptic digestion and without cleavage of a disulfide bond. URINARY FRAGMENTS OF IMMUNOGLOBULINS Human urine is known to contain low molecular weight proteins antigenically related to immunoglobulin and apparently identical to free light chains (reviewed by Cohen and Porter, 1964). More recently, fragments of light chain have been identified in the urine of several myeloma patients with Bence-Jones proteinuria ( Cioli and Baglioni, 1966; Williams et d., 1966; Baglioni and Cioli, 1966; Solomon et d., 1966). In some cases peptide fingerprints showed that the fragment consisted of the variable N-terminal portion of the corresponding Bence-Jones protein. Since the invariant C-terminal half was not detected in the same urine samples, this finding raised the possibility that the light chain is made up of two separately synthesized units (Cioli and Baglioni, 1966; Baglioni and Cioli, 1966). In other patients, however, immunological tests showed that low molecular weight, urinary fragments corresponded to either the variable or the constant portion of the light chain (Solomon et al., 1966). In one case the C-terminal peptide from such a urinary fragment was missing, but the common iiitrachain disulfide bridge peptides were detected in fingerprints (Milstein, 1966b). It now appears likely that these fragments arise through enzymatic splitting which occurs when light chain is incubated with serum (Baglioni, personal communication), but not readily on incubation with urine (Fagelman et al., 1966) . A similar explanation probably accounts for the presence in urine of
30
SYDNEY COHEN AND CESAR MILSTEIN
heavy-chain fragments antigenically related to Fc (Turner and Rowe, 1966). In the rare syndrome originally described by Franklin, on the other hand, fragments of heavy chain, having structural features of Fc and present in serum and urine, seem to be synthesized de nouo (Franklin, 1964). This has been thought to signify that the heavy chain consists of two separately synthesized units, but in fact there is no independent evidence for this ( see Section VI1,B). Antibody activity which has frequently been reported in urine is confined mainly to IgG (Turner and Rowe, 1964; Hanson and Tan, 1965). However, activity has also been found in fractions thought to have molecular weights of about 10 to 15,000 (Merler et al., 1963; Hanson and Tan, 1965). Such fragments have not been fully characterized; they appear to contain determinants of Fab, and their precipitating activity is lost on reduction (Merler, 1966). IV.
Structure of Immunoglobulin Chains
A. SEPARATION OF PEPTIDE CHAINS The separation of partially reduced immunoglobulin chains by the method of Fleischman et al. (1962) gives a yield of about 25% light chain and preparations of heavy chain partially contaminated by light chain (Porter, 1962; V. Nussenzweig et al., 1964; Nelson et al., 1965; Haber and Richards, 1966). The degree of dissociation of reduced chains in a given solvent may, therefore, vary among different immunoglobulin molecules. Human IgG and IgA molecules with h chains dissociate at higher p H than those with K chains; this difference, which was not observed with IgM, provides a means of partially separating normal human K and h chains (Cohen and Gordon, 1965). Heavy chains uncontaminated by immunological or chemical criteria have been isolated by repeated gel filtration of reduced Ig (Haber and Richards, 1966) or by reduction and fractionation in the presence of denaturing agents or detergents. Such complete separation of extensively reduced rabbit chains can be achieved by gel filtration in the presence of 0.03 to 0.05 M sodium decylsulfate ( Utsumi and Karush, 1964), 6 M urea (Frangk and Zikhn, 1964), or 5 M guanidine-HC1 (Small and Lamm, 1966) ; with these methods the yield of light chain is about 33%of the total IgG and 22%of IgM (Lamm and Small, 1966). Separation of mildly reduced guinea pig y,-globulin by gel fiItration in 4 M guanidine-HC1 gives biologically active chains uncontaminated by immunologically criteria and a yield of about 30%light chain (assuming equal extinction coefficients for both chains) (Lamm et al., 1966). Heavy chains which are soluble in neutral
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
31
Volume of e f f l u e n t h l )
(A)
I
40 I
Volume of effluent (rnl)
(B)
FIG.5. A. Gel filtration of rednced rabbit colostral IgA in the presence of 5.0 M guanidine HCl. The arrows show the elution volumes from the Sephadex G-200 column of the following reduced and alkylated materials: p = heavy chain of rabbit IgM; BSA = bovine s m u n albumin; y = heavy chain of rabbit IgC; L = light chain of rabbit IgM or IgG. B. Gel filtration of rabbit colostrnl IgA dialyzed against 5 M guanidine-HC1-0.01 M iodoacetamide, and passed through a column of Sephadex G-200 equilil~rateclwith 5 M guanidine-HC1. (From Cebra and Small, 1967.)
32
SYDNEY COHEN AND CESAR MILSTEIN
aqueous media, have been isolated by reduction of previously succinylated ( Lenard and Singer, 1966) or polyalanylated immunoglobulin (Fuchs and Sela, 1965). Isolation of the peptide chains of exocrine IgA is complicated by the association of these molecules with a fragment referred to as “transport piece” or T chain (Cebra and Small, 1967; Hong et al., 1966). After extensive reduction and gel filtration on Sephadex G-200 in the presence of 5 M guanidine-HC1, 29%of rabbit colostral IgA is eluted in the position of light chain (Fig. 5 A ) . This fraction contains a mixture of light and T chains, and these have not, as yet, been quantitatively separated. Unreduced IgA dissociates in the presence of 5 M guanidine and separates into three components on gel filtration (Fig. 5B). The first has a sedimentation coefficient of 7.2 S and after reduction can be separated into LY chains and light chains, the latter comprising 20.5%of the total fraction. The second component (molecular weight about 50,000) contains a mixture of T and light chains partly separable on DEAE chromatography; the third peak contains monomeric light chains. It appears, therefore, that the T chain is noncovalently bound to and light chains and, perhaps, stabilizes the IgA 4-chain dimer. The partial dissociation of light chains from the unreduced molecule in the presence of guanidine is difficult to explain in terms of the conventional, covalently linked, 4-chain structure unless disulfide interchange occurs under the experimental conditions used. (Y
B. HETEROGENEITY OF PEPTIDECHAINS Heterogeneity of peptide chains is the most characteristic feature of immunoglobulin molecules. The variability associated with differences in isotypic and allotypic specificities has been discussed above. Differences in peptide fingerprints of Fc fragments especially of y Z r molecules may be associated with unrecognized allotypes or technical artifacts (Frangione et al., 1966b). Definite heterogeneity within a single type of chain is shown by carbohydrate analyses (Thorpe and Deutsch, 1966a; Clamp et al., 1966) and more especially by distinct peptide patterns of Fd portions of heavy chains. Tryptic digests of rabbit (Small et al., 1965) and human (Frangione and Franklin, 1965; Frangione, Prelli and Franklin, 1966a, 1967) heavy chains characteristically show fewer peptides than the number expected from lysine and arginine contents of these chains. Although digests contain an unknown number of core peptides this discrepancy was attributed to variability of the Fd fragment as judged by comparing the peptide maps of iritact heavy chains and their corresponding Fc fragments. In the case of heavy chains
STRUCTURE AND ACT M T Y OF IMMUNOGLOBULINS
33
from whole rabbit IgG, only about 5 spots instead of the expected 13 could be associated with Fd, indicating that many peptides are either in a core or present in concentrations too low to be detected (Nelson et al., 1965; Small et al., 1965). Human myeloma proteins of a single type show 6-15 spots, apparently originating from Fd; about one-third of these peptides were common to several proteins, but the remainder had different distributions indicating a unique primary structure for the Nterminal part of each heavy chain (Frangione and Franklin, 1965; Fragione et al., 1967). Variations in the primary sequence of ,U and CY chains have also been suggested by differences of fingerprint patterns of monoclonal chains (Frangione and Franklin, 1965a). In accordance with these findings, the soluble tryptic peptides obtained in high yield from rabbit 7 chains are derived almost completely from the Fc fragment (Nelson et al., 1965; Hill et al., 1966a,b). Heterogeneity within a single heavy chain type has also been demonstrated by gel electrophoresis of y chains from horse IgG and T-globulin (Weir and Porter, 1966). Similarly human 7 chains isolated from monoclonal ySarp,,,y.,., or y>d proteins show multiple components on gel electrophoresis (Terry et al., 1966) but fewer than are present in pooled human y chain (Rejnek et al., 1966; Sjoquist, 1966; Sjoquist and Vaughan, 1966). Electrophoretic heterogeneity of the Fc fragment of the 7 chain arises, at least in part, from progressive degradation by papain; fractions of greater mobility are almost entirely absent from a 5-minute digest of rabbit IgG and increase progressively as hydrolysis proceeds ( Paraskevas and Goodman, 1865). The chemical heterogeneity of light chains is discussed in the following section. Such heterogeneity presumably accounts for the fact that on chains are reelectrophoresis in urea-glycine starch gels, both K and ,i solved into about ten bands (Cohen and Gordon, 1965), each differing by a unit net charge (Feinstein, 1966). On the basis of type specificity and allotypic and electrophoretic variation, there must be at least fifty different chemical forms of the human light chain. That the actual number is far greater (see Section VI1,B) is shown by the fact that distinct tryptic peptide fingerprints were obtained from human K chains of identical electrophoretic mobility and allotypic specificity ( Gordon and Cohen, 1966). Peptide chains isolated from antibodies of restricted specificity frequently show a degree of heterogeneity almost indistinguishable from that of the total immunoglobulin (Cohen and Dresser, 1965; Choules arid Singer, 1966; Reisfeld and Small, 1966; Lanckman, 1966). However, some antibodies from human, rabbit, and other species have relatively restricted heterogeneity (see Fleischman, 1966) especially when judged
34
SYDNEY COHEN AND CESAR MILSTEIN
by the distribution of antigenic and allotypic determinants and by the electrophoretic properties of heavy (Roholt and Pressman, 1966) and light chains (see Cohen and Dresser, 1965). A few antibodies have been reported which show an unusual degree of homogeneity at least in regard to certain properties of their constituent chains. The antibodies associated with chronic cases of cold agglutinin disease are always IgM molecules, and, in over ninety recorded cases, these have had Type K light chains (Mannik and Kunkel, 1963b; Franklin and Fudenberg, 1964; Harboe et al., 196%; Costea et al., 1966; Harboe and Lind, 1966). Some IgG erythrocyte autoantibodies are also associated with monospecific light chains, but these may be either Type K or L (Leddy and Bakemeier, 1965). Anti-DNP-BGG antibodies in guinea pigs may be either y,- or p-globulins, but in both types over 99%of molecules have Type-K light chains ( Nussenzweig and Benacerraf, 1966b). The proportion of h chains in anti-DNP antibodies is appreciably higher when the hapten is coupled to a different protein antigen; other evidence shows that properties of the carrier antigen used for immunization may influence the heterogeneity of antihapten antibodies (Sela and Mozes, 1966). The striking homogeneity of peptide chains from monoclonal immunoglobulins is widely recognized (see Fleischman, 1966) . Available data suggest, in fact, that the chains of such proteins have unique amino acid sequences (see below). It is possible, therefore, that the electrophoretic heterogeneity observed for both heavy and light chains of myeloma proteins (Terry et al., 1966; Melchers et al., 1966; Sjoquist and Vaughan, 1966) is not attributable to differences in amino acid structure. Heterogeneity of carbohydrate content has been demonstrated in a human myeloma IgA (Clamp et al., 1966) and in K chains isolated from a mouse myeloma protein. Three fractions of the latter were separable on starch-gel electrophoresis; all had the same amino acid composition, and charge differences resulted from variations in sialic acid content (Melchers et al., 1966). In the case of mouse myeloma proteins, electrophoretic heterogeneity has been shown to occiir after protein synthesis, either during the process of secretion or after exposure to serum (see Section VI).
C. SEQUENCESTUDIESON IMMUNOGLOBULIN CHAINS 1 . Light Chains
Heterogeneity has been a major difficulty in sequence studies of normal and antibody light chains. The use of homogeneous myeloma proteins Ied to a breakthrough in understanding the primary structure of
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
35
light chains and provided valuable information concerning the heterogeneity of normal immunoglobulins and their relationship to monoclonal proteins. Bence-Jones proteins are accepted as being free light chains (Edelman and Gally, 1962; Putnam, 1962; Schwartz and Edelman, 1963) which are of one or other antigenic type. Fingerprints of human K and h light chains show very few common peptides (Putnam et nl., 1963a,b; Schwartz and Edelman, 1963) and each type has a characteristic Cterminal sequence which seems to be common to all individual proteins of the same type ( Milstein, 1965). In fact the two types of chain have completely different sequences although an evolutionary relationship can be recognized (Milstein, 1966d; Hood et al., 1966; Milstein et al., 1967; Wikler et al., 1967). The most surprising finding emerging from sequence studies of Bence-Jones proteins has been that in both K and h chains the N-terminal halves are highly variable in structure whereas the C-terminal sequences are almost invariant for each type. Hilschmann and Craig (1965) first compared the partial sequences of two K chains and showed that, with the exception of a single residue, the C-terminal halves were identical. More detailed analysis of a third protein of the same type confirmed the invariant structure of the C-terminal half of the chain with the exception of the same single residue (Titani et al., 1965). These results were confirmed on large stretches at both extremes of the C-terminal half of several other proteins ( Milstein, 1966a,c). Results obtained with mouse myelomas on the corresponding chain type (Bennet et nl., 1965; Gray et al., 1967) and on human X chains (Milstein, 1966d) also indicated that the C-terminal half of the molecule remains essentially invariant. Differences in sequences of individual proteins were confined to N-terminal halves. a. The C-Terminal IZalf. Table XI shows the sequences of the C-terminal halves of human and mouse K chains and of human X chain. So far, the only well-established variation observed in this region among individual proteins of the same type is at residue 191 of human K chains. Valine is always present in that position in I n V ( b + ) , and leucine in InV(a+) proteins (Table XII). Eleven proteins have been analyzed so that the correlation is highly significant. Simple chemical techniques based on peptide patterns have been developed to distinguish the two allotypic forms (Baglioni et al., 1966; Milstein, 1966b,d). However, the chemical difference between the closely related InV(1) and InV( a ) antigens remains obscure. The important question of whether there are other differences in the
TABLE XI C-TERMINAL HALF OF LIGHTCHAINS“. *
I30
145
140
w
m
150
155
’4 Vol-L$-Asn-Alo‘///, , I l e -$$3y
////
‘/
A I a -
I95
/
210
I
//I///// '///:
////////
' ~ [ ~ L - A /I O
- ~ ~ ~ ~ -/L y s - - T h r - S e r - T h r - ~ / o l - I l-Val e
/////,
'/////
7//z
/C y ! i / d k - " ~ ~ - ~ h/ ~ /~ ~ ~ ~ - t l u - /, /~/ ~G Iu-dl-Thr~ S ~/ ~ T Valh r Alo-Pro-T - ~ ~ l - hr$u-&iSer
//y
L
////////
///
//A
75%
'//
/
/(//h
" Composite picture from several studies in man and mouse. Human K chains: Hilschmann and Craig ( 1965); Hilschmann ( 1966); Titani et al. ( 1965); Milstein ( 1966a,c). Mouse K chains: Gray et al. (1967). Human A chains: Milstein ( 1966d; Milstein et d.,1967; Wikler et al., 1967). Residues 140, 141 of human K chains are as indicated by more recent results (Putnam, personal communication). Residues 184, 185 of k chains are as determined by Milstein et al. (1967) but differ from those reported by Wikler et al. ( 1967 ). Common residues have been shaded.
38
SYDNEY W H E N AND CESAR MILSTEIN
C-terminal half of the molecule remains unanswered. An apparent difference in position 122 (an Asp for Asn) ( Milstein, 1966c) could result from technical reasons. However, the number of proteins fully investigated is not large. Studies based on amino acid composition and sequence of peptides are available for three human K chains and for large stretches in several others, and for two mouse K chains (see references, Table XI). Soluble tryptic peptides of the aminoethylated C-terminal half of K chains have been identified by their position and staining properties on peptide TABLE XI1 InV CHARACTERS AND RESIDUE 191 Protein
InV
Residue 191
Roy
-k
Leu VSl Leu Leu Val Val Val Leu Val
B-J4 B-J26
Val Leu
CU B-J
Ker Itad Day Man Fr 4
t
OF
HUMANti CHAINS Ref.
Hilschmanii arid Craig (1965)
Milstein (1966a,c)
t
Titaiii et al. (1966) Baglioni ct nl. (1966)
maps. By this method no further difference was detected in twenty-four Bence-Jones proteins, one macroglobulin light chain ( Baglioni and Cioli, 1966), and five other Bence-Jones proteins (Easley and Putnam, 1966), all of type K. The constancy of the C-terminal half of mouse K chains is indicated by almost complete sequence data of two proteins (Gray et al., 1967) supported by earlier fingerprint studies. The evidence on the human A chain is largely based on fingerprints and recent sequence studies on three human chains (Milstein et al., 1967; Wikler et al., 1967). Another important point concerns the position at which the invariant region starts. Variations have not been observed beyond residue 107, but residues 108 to 115 have been investigated in only three human and two mouse K chains and in three human X chains. However, it seems unlikely that variations will occur beyond residue 110 in view of the identity of residues 111-113 in the human K and A chains and in the mouse K chain. The C-terminal half of the molecule contains three half-cystine resi-
M o NOMER
"
'I
DlMER
IMMUNOGLOBULIN
----t--
-(NH~)
I
4
---7
H Chains
(-0Oc)
'----( N H ~ ) c ~ s ( c w - )
H Chains
4 (-0Oc) e--
f
t-i7
-4- - - -
FIG.6. The disulfide bridges of light chains in Bence-Jones proteins (monomer and dimers) and in immunoglobulins. The broken lines indicate the N-terminal half where multiple variations in amino acid sequences have been observed. (From Milstein, 196613.)
TABLE XI11
N-TERMINAL SEQUENCEAND SEQUENCE AROUND CYS 23 TYPE-K BENCE-JONESPROTEINS
'%
cu Rad Roy
Ag Ker
B-J Rad Day Mall Fr 4
Ale
SEVERAL
Sequence
Protein
Ker B-J 3 12
IN
Reference"
Asx-Ile-Gln-Met-Thr (Glx,Pro,Ser,Ser)Ser-Leu-Ser-lla-Ser-Val-Gly-hsp-Arg 10 Asp-Ile-G!n-Met-Thr-(:ll1-Pr~~-~~-Ser-Ser-~~i-Ser-.lla-Ser-~'a~-~!~-~~~p-Ar~ Asx(Ile,Glx)(Pulet,Thr,C;!ii,Pro,Ser,Ser,~r,LeujSer-.~la-Ser-T.'al-Gly-Asp-Arg Asx (Val,G lx)Met -Thr-G 111(Pro,Ser,Sex ,Ser,Leu) (Ser,Ala,Ser,Val,G 1y ,Asp)Arg ,lsp-Ile-Val-Leu-Thr-Gln Glu-Ile-Val-Val-Thr-Gln G Ix Glx Val(Thr,Ile,Thr,C?s,C;ls,~lla,Ser,(;lx,~l~~,Ile,Ser) Ile-Phe-Leu 20 30 Val-Thr-Ile- Th~-Cyi-C;ln-~I~tSer-C~l~i(.\.~x,Ile, Ser, dsu,Phe)Lea lsp-Ile- Lys(hw,Phe) Ile - Thr-Ile - Thr-Cys-Glii-.lIa-Ser-(:iliiVal-Thr-I!e - Thr-C) s-Glii-.~l~~-Ser-~lii-.l~p-Ilehsn-Lys (Tyrj Alla-Thr-Leu-Ser-Cys-Alrg-.lla-Ser-C;:li-Vd- Ser-Ser- .\sii-Ser-Tyr Val(Thr,Ile, Thr.Cys,Glx ,-lla,Ser,Glx. Aiu, Ile. Ser, -liu,Phe Leu Ile-Ser-Cys--Arg (Ala,Thr,Leu,Ser,Cys,Arg) Val(Thr,Leu,Thr,Cys,hrg)
Key to references: ( 1 ) Hilschmann and Craig (1965); Hilschmann ( 1966 ). ( 2 ) Putnain et al. (1966). ( 3 ) Milstein ( 1 9 6 6 ~ ) .
(4)Milstein (1966a). ( 5 ) H o o d e t d . (1966). ( 6 ) Milstein (unpublished). ( 7 ) Pink and Milstein (unpublished).
STRUCTURE A N D ACTIVITY OF II\.II\IUNOGLOBULINS
41
dues giving a pattern of disulfide bridges similar in both K and chains (Milstein, 1966b,d). The half cystines in positions 134 and 194 form an intrachain disulfide bridge which has been identified in several individual proteins. The C-terminal or next to C-terminal disulfide bridge is an interchain bridge, linked to a homologous sequence in the BenceJones dimer and to a lone hali-cystine in the monomer (Fig. 6 ) . The blocking of the free SH of the monomer could result from disulfide interchange during catabolism or represent a specific reaction occurring during assembly of the chains; in vitro labeling experiments should provide an indication of the mechanism involved. Direct evidence that light and heavy chains of IgG and IgM are joined through the same half-cystine has been obtained by isolating a cystine peptide made up of a half-cystine peptide from the C-terminus of either K or h chains and a half-cystine peptide from either or p chains (Pink and Milstein, 1967). The sequences shown in Table XI are very similar in some stretches, notably those between rcsidiies 110-140 and 160-180. In spite of this, only 60%of all the residues of the two K chains (mouse and human) are identical, as compared, for instance, with the 84% of identical residues in chains of human and rabbit hemoglobins. The similarity between the human h and K chains approximates to that observed in human and mouse K chains. This may indicate that the requirements for tertiary structure and association with heavy chains (as well as other biological functions) are localized in the conservative stretches, whereas the residues of other stretches alter in the course of evolution at a faster rate than occurs in the LY chain of hemoglobin (Zuckerkandl and Pauling, 1962), in cytochrome c (hfargoliash, 1963), or in pancreatic ribonuclease ( Epstein and Motulsky, 1966). I?. The N-Terminal Half. In contrast to the constant sequences of C-terminal halves, Rence-Jones proteins of the same type contain multiple variants in the N-terminal halves. This variation has been studied extensively only in the N-terminal stretch and around the cysteine residues of human K chains (Tables XI11 and XIV) and for this reason the overall degree of variation is still speculative. The results obtained (see also Table XV) suggest the presence of conservative residues either isolated (e.g., residues 2, 20, 23, 95) or occurring in stretches (e.g., residues 5-6, 25-27, 86-90, 97-98). Variation also occurs either in isolated positions (e.g., residue 96) or in stretches (e.g., residues 92-94). However, the distinction between conservative and variable residues is nncertain because of the restricted number of available sequences. Residues may be very conservative and yet variants appear when a suificient number of proteins are analyzed; for instance, rccidue I, was found to differ (Y
SEQUENCEAROUND CYS 88
Protein Roy
Ag Ker
B-J Cu Itad
TABLE XIV SEVERAL TYPE-KBENCE-JONESPROTEINS
IN
Sequence
Ref:
Leu-Gln-Pro-Glu-Asp-Ile-Ala- Thr-Tyr-Tyr-Cys-Gln-Gln- P he-Asp-Asn-Leu-Pro-Leu-Thr-Phe-Gly-Gly-Gly-Thr-Lys ( 1 ) 80 90 100 Leu-Gln-Pro-Glu-Asp-Ile-Aln-Thr-Tyr-Tyr-Cys-Gln-Gln- Tyr-Asp-Thr-Leu-Pro-Arg-Thr-Phe-Gly-Gln-Gly-Thr-Lys (8) Tyr-Tyr-Cys-Gln-Gln- Tyr-Asp-Asp-Leu-Pro-Pro-Thr-Phe-Gly-ProGly-Thr-Lys (3) Tyr-Tyr-CysGln-Gln- Ty r-Glu-Asn-Leu-Pro-Tyr (3) Val- Glx-Ala- Glx-AspVal- Gly-Val- Tyr-Tyr-CysGln-Met-Arg-Leu-Glu-IlePro-Tyr-Thr-Phe-Gly-Gln-Gly-Thr-Lys ( 1 ) Leu-Glu-Pro-Glu-Asp-Phe-Ala- Val- Tyr-Tyr-C ys-Gln-Gln- Tyr-Glu-Thr-Ser- Pro-Thr-Thr-P he (3)
* Key to references:
( 1 ) Hilschmann and Craig (1965); Hilschmann (1966). ( 2 ) Titani et al. (1966). ( 3 ) Milstein ( 1 9 6 6 ~ ) .
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
43
in only one out of thirteen proteins investigated. The large stretches without recorded variants may, therefore, reflect the insufficiency of data rather than real invariability. Some sequences may depart considerably from those included in Table XIII; for example, the N-terminal tryptic peptide present in a considerable number of proteins, as judged by fingerprints, is absent from others (Easley and Putnam, 1966; Raglioni and Cioli, 1966). Similarly, some cysteine peptides are so different from those more commonly found that they cannot a t present be related to any of the others studied (Milstein, 1966a), although observation of a larger number of sequences may disclose similarities. Some positions, on the other hand, seem to contain either one of two residues. This is well illustrated by the N-terminal residue (either Asp or Glu) and by the third residue (Gln or Val). Position 4 is either Met or Leu in ten out of eleven proteins investigated. The importance of all these observations lies in the possibility of using sequence data as a means of understanding genetic mechanisms (see Section VI1,B). Studies on the N-terminal stretch of mouse K chains shows a similar situation to that observed in the human (Hood et al., 1966; Perham et al., 1966). A blocked Glu has been reported in the N-terminal position of one protein (Perham et al., 1966). Almost complete amino acid sequences of two mouse K chains have been reported by Gray et al. (1967). Multiple differences were found localized in the N-terminal half of the chain (more precisely in the first 94 residues out of the 214 of the molecule, see Table X V ) . Differences between the two mouse N-terminal halves were larger than between a selected mouse and human pair. The two mouse proteins studied had a different total length, resulting from an insertion of four residues which could be confidently placed between residues 27 and 28 (see Table XV). In human K chains, residues 25/27 seem to be very conservative whereas a variation in residue 28 has been observed (see Table X V ) . Knowledge of the variable portion of h chains is more restricted. The N-terminal sequences of several h chains are presented in Table XVI. Sequences around the cysteine residue of human Bence-Jones proteins which appear to be related to the cysteine in position 22 are also shown. Again a difference in length seems to emerge from this comparison, probably related to a deletion at the N-terminal position. Table XV gives a summary of the variability observed in the N-terminal halves of human K and h chains and in mouse K chains. The comparison emphasizes the occurrence of conservative stretches within light chains. Homologous regions such as those occurring in the C-terminal halves of human K and h chains (Milstein, 1966d) are also present in the N-
VARIABILITY OF
THE
TABLE XV N-TERAIISALHALF OF HUMAN AND MOUSE LIGHTCHAISS"-'
Glu Val Val Leu 6 0-1 6 3-4 I(
Human
A la 1-2 Pro Ser Ser Ser Leu Ser Alo Ser Val Gly Asp Arg Val 1-4 1-4 1-4 2-4 2-4 3-4 3-4 3-4 3-4 3-4 3-4 4 5
-
_ 1
Val Leu
5 K
Mouse
Alo Val
Gin
A l a Thr
3
Asp I l e Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly Glu Arg V a l Ser 5 6 3 3 2 2 2 2 2 2 2
GlP
A Human
Alo
Val
K
Humon
25
Ile K
Mouse
Ser
Glu Ser Gly
Leu Thr
Cys Arg A l o Ser Gln Asx Ile
2
A Humon
30
2
2
2
Ile
35
Phe Met Asn
Phe?
Lys
Gly
Gly Ser Leu Ser Asx Trp Leu Glx Glx Gly Pro Asx Glx 1-2 1-2 2 ? 1-2 7 1-2 2
2m
46
F]e
C
5
k
I
n Pro
1
-
K
TY r
Gln
2
2
Leu Glu
2
Gly Gly Thr Lys Vol
Human
4
95
90
4
4
2
Ile Arg 2
Asp Phe Lys
4
2
100
I05
__ Glx K Mouse
Phe Cys Leu Glx
2
1-2
Ser Lys Glu
Val
Tyr Ala
Ser Pro Trp Thr Phe Gly
Ser
2
2
2
2
2
F l y Gly Thr L y s Leu Glu Ile
2
2
2
2
2
2
2
Lys 2
" Numbers outside the boxes are the numbering of residues from the N-terminus. Numbers within boxes show the number of ptoteins which have been described containing that particular residue; two numbers indicate that some results are inferred from composition of peptides: no number in the box means that it was found in only one protein. Glp = pyrrolidone-carboxylic acid. ' Shaded residues are those found in at least 5 out of 6 of the proteins investigated. ' In mouse the top row shows variants of protein 70 as compared with protein 41. Data from Perhani et al. ( 1966) are also included. In human K chain the bottom row i\ protein Roy. ' References-see Tables XI, XIII, XIV, and XVI. ' Many of the substitutions listed in the table are based on sequences of isolated peptides and the residue position is placed on comparative grounds. Aclditions or deletions may occur in the middle of the chain and this consideration should be borne in mind when making use of these results.
N-TERMINAL SEQUENCE AND SEQUENCE
TABLE XVI Fmsr CYSTEINE
AROUND THE
OF
SEVERAL HUMANk CHAINS ~~
Chain
Seqwnce
5
Sh
s
H BJ2 HBJ7 HBJR HBJ11 BJSS Sh
X IIil 3Ie BJ98
Reference.
10
15
Ser-C:lu-Leu-Thr-~~lii-.-Zsp-Pro-.-Zla-Val-Ser-Val-Ala-Leu-Gly-Gl~i-Thr-Val-ArgT y r-.-Zsp-Leu-Thr-Gln-Pro-Pro-Ser-Val-Ser-Val-SerPro-Gly-Gln-Thr-Ala-SerGlp-Ser-Ala-Leri-Thr-Gln-Pro-Pro-Ser-Al~-Ser-Gly-Ser- Pro-Gly-Cln-Ser- Val-Thr Glp-~er-V~~-Leri-Thr-Gln-Pro-Pro-Ser-.\ln-~r-Gly-~hr-Pro-Gly-Gln-Gl~-~a~-~hr Glp-,%r-a41a-Leii-.~a-Gln-Pro-Aln-Ser-Val-~r-Gly-Ser- Pro-Gly-Gln-Ser- Ile- ’I’hr Glp-Ser-Val-Leu (;lp(Ser,Val, Leu)Thr(Glx.Pro, Pro, Ser,\’al)Ser-Ala-~~la(,~s~,Gly) (C;lx,.ila)Val-Thr20 25 -Ile-Thr-Cys-C:Ii1-C;ly-Asp-Ser- Leu-;\rg-Cily-1le-Thr-Cys-Ser- Gly-.\sp-Lys-Leii-Gly-Asp -1le-Thr-Cy s-Gly-Gly- Asp-Glx -1le-Ser- Cys-Ser ( ~ l ySer) , Ser- Ser-.\sn-lIet Ser-Ile- Cys
J!
~
‘’ Key to references: ( 1 ) Milstein et al. (unpublished). ( 2 ) Hood et al. ( 1966). ( 3 ) Milstein (unpublished), ( 4 ) Wikler et al. (1967). ( 5 ) Baglioni ( 1967).
~~
STRUCTURE AND ACTIVITY O F IMMUNOGLOBULINS
49
terminal halves (Hood et al., 1966). This is indicated by residues at some positions (e.g., 5, 6, 7, 9, 12, 16) and by the position of Cys 23 when a deletion in the h chains is assumed (numbering is made with reference to K chains to give maximum homology). In spite of such striking similarities and of the variations observed in each chain, the available results strongly suggest that the variable stretch of h and K chains are recognizable as belonging to each type (Hood et al., 1966; Milstein, 1966d; Cioli and Baglioni, 1966). In other words, sequences shown in Table XI11 seem to be definable as belonging to K chains and the sequences of Table XVI as belonging to h chains. This distinction can be made, for example, on the basis of the N-terminal residue (Asp or Glu in K chains, Tyr, Ser, or a blocked Glu in h chains), residue 2, and the sequences around Cys residues. Thus, in K chains a recurrent sequence Cys-Gln ( o r Arg)-Ala-Ser-Glu has been found, while in h chains the equivalent position is the less conservative, Cys-Ser( or Gly ) -Gly-Asp (o r Ser)-Ser(or Lys or Glx). Similar observations apply to other residues in the N-terminal position (Table XV), to the sequences around other Cys residues (Milstein, unpublished), and to certain features of the fingerprint patterns ( Baglioni and Cioli, 1966). In all the proteins investigated so far, two cysteine sequences have been found in the N-terminal half of human K chains. This seems to be the case also in the mouse. Amino acid analyses indicating a lower number (Putnam et al., 1963b) ha\ e not been substantiated by consecutive work on the same protein. This is not surprising because of the known difficulties in accurate estimation of cysteine content. Evidence that the two cysteines present in the variable stretch are linked by an S-S bond has been presented in three proteins (Milstein, 1966b). Disulfide bridge peptide maps of several others suggest a similar arrangement. As in K chains, the N-terminal half of several h chains contains one disulfide bridge ( Milstein, unpublished). However, there are some proteins that contain three Cys residues in this stretch and one of them has been shown to occur as a free SH (Feinstein, 1966). 2. Bence-Jones Proteins and Normal Light Chains
If Bence-Jones proteins represent single species of the normal lightchain population, then peptides common to a large number of these myeloma light chains should be present in significant quantities in digests of normal pooled light chains. The peptides of K chains should be present in larger amounts than those of h chains. A quantitative estimate of the recovery of the C-terminal peptide of K and h chains (Table XVII)
50
SYDNEY COHEN AND CESAR MILSTEIN
showed that, within experimental error, normal pooled light chains contain one or the other in expected proportions (Milstein, 1965). Other soluble peptides from K chains, located by fingerprinting techniques (Putnam and Easley, 1965), may account for a large proportion of the C-terminal half of normal K chains starting at residue 146. Furthermore, the common intrachain disulfide bridge of Type-K Bence-Jones proteins has been found in good yields in normal light chains by the diagonal electrophoretic technique and by amino acid analysis of the peptides; these include the cysteines at positions 134 and 194 (Milstein, 1966b). YIELD OF
TABLE XVII C-TERMINAL TRYPTIC PEPTIDES FROM NORMALH U M A N LIGHTCHAINS" Yield of peptides Mole/mole
Light chaiii BeliceJones protein, Type I< (oxidized) Belice-Jones protein, Type L (oxidized) Oxidized riormsl light chairis "
K
O i
Corrected yield h
K
-
10 (nrbitmry)
-
0 5
0 45
0 20
-
0 63
A
-
10 (arbitrary) 0 40
Rec;ilculated from Milstein ( 1965).
Peptides of the A chain are more difficult to identify in unfractionated normal light chains because they occur in lower concentrations. Using a radioactive technique, two peptides that account for the last twenty-three residues of abnormal h chains have been identified in expected yields in fingerprints of normal pooled light chains ( Milstein, 1966d). These results indicate that C-terminal halves of normal light chains consist essentially of a mixed population of C-terminal halves of K and h chains. A similar study on peptides of the N-terminal half will be more difficult, because each variant sequence must be present in very small amount. However, sequences in the N-terminal half which occur in a high proportion of Bence-Jones proteins may be detectable. An Nterminal tryptic peptide has been observed in fingerprints of normal light chains (Putnam and Easley, 19651, although it is not possible to say whether it represented a mixture or a pure peptide. The study of the N-terminal sequence provides another approach to this problem. The N-terminal residues of human pooled light chains are Asp and Glu, which correspond to the two residues found in monoclonal K chains. The N-
51
STRUCTURE AND ACTIVITY O F IMMUNOGLOBULINS
terminal residue of monoclonal chains is either blocked or it is Tyr (see Table XVI), which is an elusive residue in N-terminal analysis. Stepwise degradation of normal human light chains gave Ile in the second position and either Val or Gln in third (Hood et al., 1966). This agrees very well with the residues more commonly found in the Nterminal sequence of the majority of K chains. In many cases a methionine is the fourth residue of Type K Bence-Jones proteins. The N-terminal peptide of cyanogen bromide cleavage has been prepared from such chains, and it should be possible to isolate the same peptide from normal pooled light chains. This has, in fact, been done ( Milstein, unpublished), but the amino acid composition of the peptide was not as straightforward as the one obtained from Bence-Jones proteins (Table XVIII ) ; however, TABLE XVIII THE N-TERMINAL PEPTIDEFHOM CNBr CI.EAVAC:E OF HUMANL m m CHAINS Light chain
Abnormal Ker ( I )"
B-tJ ( 1 ) Normal Poolctl 'ight chains (2)
'IS"
Ile
Val
Glri
HOSer
1 0 1 0
0 x5 -
10
1 1 1 1
0 75 0 5
1 1
o x
0 4
0.7
0.5
Minor components: Ser 0.2; Gly 0.2; Thr 0.1; Xla 0.1 'I
Key to references: ( I ) Milstein ( 1 9 6 6 ~ ) ;( 2 ) Milstein (unpublishecl).
this is not surprising because the isolation procedure may not have separated all the variants. The analysis nevertheless indicates that the most common N-terminal sequence shown in Table XI11 is present in normal light chains. The C-terminal residues of normal human IgG reported by Beiser et al. (1966) present a problem. The presence of Arg in very good yields in the C-terminal position, as shown by hydrozinolysis, is difficult to reconcile with analyses of myeloma proteins. The reason for the discrepancy remains obscure. An explanation for the pattern of electrophoretically separable bands of normal light chains can be given on the basis of differences in amino acid sequences. The electrophoretic mobility of a protein is a function of physical (size and shape) and chemical (charge) properties. If the former remains constant, the latter at appropriate pH values will vary stepwise by a minimum unit (one charge). Variation of charge may occur at several places in the N-terminal half, but mobility will be determined
52
SYDNEY COHEN AND CESAR MILSTEIN
by net charge and will be independent of the number and nature of the variations. The electrophoretic pattern of bands shows a characteristic random distribution, which suggests that the population is derived from a very large number of variants, which depart from a given or ancestral sequence. The same conclusion is suggested by the amino acid sequences of different Bence-Jones proteins. TABLE XIX PTH-AMINO ACIDS IDENTIFIED AT AMINO-TERMINALEND OF IhlMUNOGLOBULIN LIGHTCHAINS" Position in peptide chainb
Light chain source
1 Rabbit 7-globuliii
Ala
(Ile,Asp, Glu) Mouse and human
Asp Glu
2
Val (Leu)
Ile
3 Val Leu (Gln/Glu) Val Gln
4 Val (Glii/Glu) Val Leu Met
5
(;In
6 Glu (Thr,Ala)
Thr
Gln
From Doolittle ( 1966). numbered from amino-terminal end. PTH-amino acids shown in parentheses occurred in small amounts. a
' Positions
As regards other species, structural studies are more difficult because no preliminary information can be obtained from myeloma proteins. Doolittle ( 1966) has studied, by Edman degradation, the N-terminal sequence of rabbit y-globulin which can be compared with the corresponding sequence of mouse and human K chains (Table XIX). Since the y chains of rabbit have a blocked N-terminus, the results are considered to represent the N-terminal sequence of pooled light chains. 3. Sequence Studies on Heavy Chains
Elucidation of the primary sequences of light chains encouraged the more difficult task of studying the detailed structure of heavy chains; such studies are complicated by the size of the chain and by the number of classes and types. Nevertheless, progress in this area has been rapid and it seems IikeIy that sequences of myeloma and of large sections of normal heavy chains, will soon be available. Cyanogen bromide cleavage has been very useful in preliminary studies of heavy chains, and the arrangement of five fragments obtained from a human yzI, protein has been proposed (Press et al., 1966a)b; Piggot and Press, 1967) (Fig. 7 ) .The five fractions were separable by Sephadex filtration. The smallest was identified as the C-terminal peptide
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
53
and could be isolated and analyzed in several species and in types of human monoclonal IgG (Table XX). Normal IgG heavy chains from rabbit have also been split with cyanogen bromide and fractionated by Sephadex filtration (Press ct al., 1966a). A larger number of fractions was obtained, but a major fragment seemed to include practically thc whole of Fd (about 200 residues). However, there were indications that Fd was heterogeneous as far as methionine content was concerned and that a significant proportion ( 2 0 3 0 % ) of the population contained methionine in position 36 or 38 giving rise to an N-terminal peptide linked by a disulfide bridge (or bridges) to the rest of the Fd fragment. Papain
cleavage
Fd - f ragmen?
Fc - f ragrnent
FIG. 7. Cyanogen bromide fraginentc of a y:,, heavy chain ( D a w ) . The interfragment bridges and the moIecolar weights of fragments are indicated. The sequence of Fragment 2b is shown in the text. (From Piggot and Press, 1967.)
n. The C-Terminal Half. It has seemed likely that the C-terminal half of the heavy chain is invariant, since the pioneering work of Porter (1959) revealed that this part of the molecule readily crystallizes. This is being confirmed by finserprint studies of Fc fragments (Frangione and Franklin, 1965) and by sequence analysis of myeloma proteins and normal heavy chains. The similarity of C-terminal sequences of IgG in several species is remarkable (Table XX) , The C-terminal octadecapeptide of human yna.and yJb are identical but y Z c differs by either one or two residues. Larger differences between y types are to be expected in other sections of Fc fragments. The variation within y Z c may be related to Gm(b) and Gm(g) specificities, but more proteins are needed to establish a statistically significant correlation. Normal human IgG contains the C-terminal sequence of yna and Y.~, in very good yields as would be expected since these two types constitute the major part of normal chains ( see Table IV )
.
TABLE XX C-TERMINAL SEQUENCES OF HEAVYCHAINS ~
Chain Pooled human IgG Human yra myeloma Hiiman p b myeloma Human yzC H chain disease Gm (b+) Human yZe myeloma Gm(b+) Hiiman yZC myeloma Gm(b- g + ) Horse IgG(T) Horse IgG Rabbit IgG a
Sequence (.\let) His-GI u--lla-Leu-H is-.~sn-His-Tyr-Thr-Gln-Lys-Ser-Leu-Rr-Leii-Ser-Pro-~lS (AIet His-Gl~i-,Ua-Leu-His-;\sn-His-Tyr-Thr-Gln-Lys-~er-Leii-Ser-Le~i-Ser-Pr~-Gly (11et His-Glu-.~la-Leri-His-Asn-His-Tyr-Thr-Gln-Lys-Ser-Leu-Ser-Leri-Ser-Pro-Gly (Met ) H is-Gl~i-AIn-Leu-His-.~sn-d rg-Phe-Thr-Gln-Lys-Ser-Len-Ser-Leu-Ser-Pro-Gly (.\Iet ) His-GIu-Ala-Leii-His-.~sn-.-l,.g-Phe-Thr-Gln-L~s-Ser-Leu-Ser-~ii-Ser-Pro-Gly ( l l e t ~His-Glii-.~la-Leii-His-;\sn-.I rg-Tyr-Thr-Gln-Lys-Ser-Leli-Ser-Leu-Ser-Pro-Gly
(Net )His-G1ii-.lla-T‘al-Gl1c-~lsn-His-Tyr-Thr-Gln-Lys-il sn-T’al-Ser-His-Ser-Pro-Gly (Met)His-Glri-~~la-Leu-His-hsn-His-l’yr-Thr-Gln-I,~s-Ser-T’al-Ser-l,ys-Ser-Pro-Gl~~ (Met )His-Glu-Ala-Leri-His-.~sn-His-Tyr-Thr-Gln-Lys-Ser- Zle- Ser-.4 rg-Ser-Pro-Gly
Key to references: ( 1 ) Prahl ( 1966; and unpublished). ( 2 ) Press et d.(1966b). ( 3 ) Weir et al. ( 1966). ( 4 ) Givol and Porter (1965).
~~
Referenceo
STRUCTURE AND ACTIVITY O F IMMUNOGLOBULINS
55
Sequence stiidieq of rabbit Fc have been initiated by Hill ct al. ( 19666a,b). Twenty-seven tryptic peptides derived from the Fc fragment have been isolated; their total composition accounts, within experimental error, for the composition of the whole Fc. Chymatryptic digestion and cleavage with cyanogen bromide have provided evidence for the overlap of most of these peptides and a partial sequence of the Fc fragment of rabbit y chains has been proposed (Table XXI). Although two types of rabbit Fc have been suspected from studies of allotypes (see Section II,C,2), sequence studies suggest that this fragment is homogeneous. Rabbit IgG is known to be heterogeneous on papain digestion (Goodman, 1965), so that the seqiience may account only for a major fraction of Fc. However, when whole heavy chains were siibjected to tryptic digestion, no apparent discrepancy emerged. In fact only three extra peptides (one probably the N-terminus and two small peptides) were obtained in good yields. A number of unidentified peptides appeared in very low yields but their origin is unknown. A definite conclnyion about the homogeneity of rabbit Fc cannot be reached at present especially as the need to establish overlaps leaves considerable uncertainty about the partial sequence depicted in Table XXI. The results, nevertheless, suggest that more than haIf of the heavy chain (the C-terminal half) can be accounted for by a sequence which may be common to most or all of the rabbit y-chain population. A carbohydrate moiety is covalently bound to the F c fragments of IgG in several species (reviewed by Press and Porter, 19sS). It has been tentatively placed on residue 150 numbered from the C-terminus (see Table XXI) of rabbit y chain. However, in some IgG molecules the existence of more than one site of attachment of carbohydrate has been suspected (see Fleischman, 1966). In horse IgG( T ) , two-thirds of the carbohydrate is attached to the Fd fragment (Weir and Porter, 1966). The characteristics and distribution of the carbohydrate moiety might vary in different Ig molecules, even of the same type. Clamp et al. ( 1966) studied the composition of ten carbohydrate-containing peptides from a human IgA myeloma protein and found extensive heterogeneity. This very interesting observation raises the question of the specificity of structure and biological function of the carbohydrate in pure molecular species of immunoglobulin. 1). The N-Terminal Half. It is widely believed that the N-terminal half of the normal heavy chain of a given type is heterogeneous. This belief arises because there is little doubt that ( a ) antibody specificity resides at least in part in this half of the molecule and ( b ) antibody specificity is associated with variations in amino acid sequence (see
TABLE XXI A TENTATIVE SEQUENCE OF RABBIT FcaSb CXBr 2 40
u1
1
230
H,X-Cys-Pro-Pro(Pro,Glu) Leu(Pro,(;ly,(;ly,Vnl,Ser,Leu,Phe) Ile(Pro.Phe,Pro,Lys,Pro,Lys)~sp-Thr-Leu-?tIet-
0)
210
190
200
(.lsp?,Thr,,Ser3,C;lu3,Pro,,C~I~,i\lrt,C~s,Val~,Ile,,Leu,Phe,L~~) l(A~I)~,Thr~,Ser,C;l~i~. 180
160 (Ile-Asp-~~~p-C~lu-(C;I~i,Val~Arg) Thr-~lln-Arg-Pro-Pro-Le~i-~~rg-~l~~-
I
150
CHO
I 140 GIii-GIii-Phe-rlbl)-Ser-Thr-I le-~lrg-Val-Val-Ser-Thr-I,e~i-Pro-lle-.ila-Hiz-(;I~i-_ly,-Try-Leu-Ar~-Gly130
120
110
Lys-C;lu-Phe-Lps-Cys-L~z-Vrtl-Hiz-.l~p-Lys-Ala-Leu-Pro-Ala-Pro-Ile-C~lu-L~s-Thr-Ile-Ser-Lys-.llrtCSBr 100
1
90
Irg-Gly-G lu-Pro-Leu4 lu-Pro-Lys-Val-TS r-Thy-lkt-(: Iy-Pro-I’ru-Alrg-C;lu-C: lu-Leu-Ser-Ser-.irg-Ser-
CNBr
80 1 70 Val-Ser-Leu-Thr-Cys-Me~Ile-Bsp-Gly-Phe-Tyr-Pr~-Ser-.4sp-Ile-Ser-Gly-Val-Try-Glu-Lys60 50 hsp-Gly-Lys-Ala-Glu-Asp-Asp-Tyr-Lys-Thr-Thr-Pro-~4la-Val-Leu-Asp-Ser-Asp-Gly-Ser-Try-Phe-
40 30 20 Leu-Tyr-Ser-Lys-Leu-Ser-Val-Pro-Thr-Ser-Glu-Try-Gln-Arg-~ly-Asp-Val-Phe-Thr-Cys-Ser-ValCSBr
1 10 1 ~~et-EEis-Glu-Ala-Leu-His-Asn-His-Tyr-Thr-Glu-Lys-~r-Il~Ser-Arg-Ser-Pro-Gly-COOH a Vertical bars indicate positions where overlaps have not been established. The four peptide bonds cleaved by CNBr are indicated by the arrows. The numbering of residues is arbitrary and, in contrast to convention, residue 1 is at the COOH-terminal position. From Hill et a2. (1966a,b). There are differences between the sequence given by Hill et al. in 1966a and in 1966b. Where the discrepancies occur this table gives the data of the latter publication.
58
SYDNEY COHEN AND CESAR MILSTEIN
Section V ) . In fact, there is at present very little direct evidence for such variability. Heterogeneity in the N-terminal sequences of heavy chains of normal rabbits has been shown by Wilkinson et al. (1966) (see Table XXIII). Study of the N-terminal sequences has been greatly facilitated by the observation that the N-terminus of a myeloma protein (Porter and Press, 1965) was blocked by pyrolidone-carboxylic acid. This could arise under mild conditions from Gln residues in the N-terminal position; however, the authors found no evidence that the conversion was an artifact and favor the idea that it represents a more specific process (Press et nl., 1966b). The N-terminal sequences of rabbit heavy chains (Civol, unpublished) and of the cyanogen bromide fragment of a human pathological myeloma ( Daw ) heavy chain have been established and are compared TABLE XXII C O h l P A l ~ I S O N OF N - T E H M I N A L
CNHr FHACWENT 01%'A
* From Piggot
SEQUENCES O F RABBIT H E 4 V Y C H A I N S A N D OF TH1.:
H U h I A N PATIlOLOGlCAL
MYELOMA( b w ) HEAVYC H A I N " '
and Press ( 1967). residues, assuming a deletion in the second residue of labbit.
' It'ilics-identical
in Table XXII. As in the case of light chains of different species, there seems to be a significant degree of homology between the N-terminal sections of rabbit and human heavy chains. Apart from variations in the N-terminal peptide of rabbit (Table XXIII), very little is known about the variability of the N-terminal half of the heavy chain. Analysis of peptides isolated from antibody molecules by the use of hapten analogs (affinity labeling) indicates very extensive heterogeneity ( Singer and Doolittle, 1966); it is not clear, however, whether the tyrosines labeled by this method are in different parts of the molecule, in different types of chains, or, in fact, occupy equivalent positions in different sequences of a single chain type. Direct information
STRUCTURE AND ACTIVITY OF IhiMUNOCLOBULINS
TABLE XXIII THE ~ - ? ' E H b l I N A L
SEQUENCE OF I I E A V Y C H A I N S " '
59
'
Sormal mhbi t Glp-Ser-\.':tl-G In Glp-Ser-Leu-Glu Glp-Gln Longest sequence isolated :
Glp-Ser-Val-Glu-Crlu-Ser-C~ly-GI~-Arg " The numbers refer to approximatt. proportion of each sequence in the mixture. The pattern of rabbit peptitles \ w s essentially unchanged when a single hoinozygous r a l h i t and purified nntil,otly to hiiman serum allmmin were investigated. Glp = pyrralidone-carboxylic acid. " From Wilkinson et al. (1966).
will be obtained by sequence studies on Ftl fragments of myeloma proteins. The problem can also be approached by asking how much of the N-terminal half of the molecule is invariant in each type; two observations are of interest in this connection. First, some allotypic Gm characters are located in the F d fragment, and sequences determining their specificity should be essentially invariant (see Table VIII ). Second, light chains are attached to the Fd fragment and each class of human heavy chain appears to have characteristic sequences around the specific cysteine residue:
I
I& (ria) l'ro-I~eu-.~l,z-(Ser,Cys,I'ro)
I
(YLb)
Ser-Cys-A\sp-L>s
I IgM
Pro-Leu-Val-Ser-Cys-GIx-.2sx-Ser (Asp,Thr,Ser,Pro)
Those from IgG were obtained in expected yields from normal heavy chains (Pink and Milstein, 1967). Unfortunately the precise position of this particular cysteine is not yet known. It seems probahle, therefore, that the F d fragment resembles the light chain in having invariant sections and parts which vary within each type. This resemblance has been the subject of considerable speculation and some authors have suggested the possibility that the variable portions of light and heavy chains are controlled by a specific cistron (Burnet, 1966). The N-terminal sequence of the Y _ . ~heavy , chain of protein Daw can he compared with the N-terminal sequences of several human h chains (Table XV) and with a peptide isolated from the h chain of protein Daw ( Piggot and Press, 1967):
60 Heavy Daw
SYDNEY COHEN AND CESAR MILSTEW
Glp-Val-Thr-Le
ti- Arg-Glu-Ser
Humau
G1p-&--Ala Val-Leu-Thr-Gln-Pro
Daw
Asp Glp,Ser,Val
The N-terminal sequences of normal rabbit light (Doolittle, 1966) and heavy (Wilkinson et al., 1966) chains can also be compared: I:ahl)it~,light
Ilabbit, heavy
Ma (Ile, Asp Glu) 611)
Val (Leu)
Val (Leu Glu, Gln)
Ser (Gh)
Val (Leu)
Val Gln (Glii, Glu)
Glu
Glu
G 111 (Thr, Ala)
Ser
The results indicate that the light and heavy chain N-terminal sequences are quite different, This is in agreement with the fact that antibody specificity may be associated with isolated heavy chains but not usually with light chains (see Section V ) . The number of disulfide bridges between heavy chains is still a matter of controversy (see Fleischman, 1966). Smyth and Utsumi (unpublished) have isolated two carboxymethylated peptides from the rabbit Fc fragment after partial reduction under mild conditions and carboxymethylation with i ~ d o a c e t a t e - ~ These ~ c . peptides were rich in proline, carried 60%of the total radioactivity, and had identical sequences; one contained a blocked N-terminal carboxymethyl cysteine as an artifact of the isolation procedure. The carboxymethylated residue was believed to be the N-terminal cysteine of Fc (see Table XXI). However, a peptide also rich in Pro but containing two carboxymethyl cysteines has been isolated from mildly reduced and carboxymethylated myeloma proteins and human IgG proteins (Pink and Milstein, 1967). Utsumi and Karush (1964) have proposed that there are, in fact, two disulfide bridges joining the two chains in a nonsymmetrical bridge. Disulfide interchange is invoked to explain the low yield of carboxymethyI cysteine after mild reduction. The proximity of two cysteines in one peptide makes such a model attractive, but at the same time emphasizes the technical difficulties involved. Piggot and Press (1967) present evidence to indicate the position of some of the S-S bonds. They isolated a single, symmetrical disulfide-bridged peptide linking the two halves of Fc (interheavy-chain disulfide bridge ) and present in the N-terminal cyanogen bromide fragment of Fc (Fig. 7 ) . The authors point out that their results do not exclude the presence of other interchain bridges. The positions of intrachain bridges that seem to emerge from these studies also merit consideration. In light chains the “variable” and common sections are
61
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
bridged separately. If heavy chains followed the same pattern, Fragments 2b and 4 (Fig. 7 ) should be joined, but, in fact, both seem to form bridges with Fragment 2a. Of course, since no evidence about variable and common sections of heavy chains is available, it is still premature to draw further conclusions. The results do, nevertheless, emphasize the differences between the two chains. Elucidation of these problems requires more structural information and this may so011 be available.
4 . The Evolutionary Pattern of Immunoglobulin Chains There is convincing evidence from structural studies of hemoglobin chains and some proteolytic enzymes that related proteins in higher organisms may derive from a common ancestor gene (see Dixon, 1966; Epstein and Motulsky, 1966). The homology of myoglobin and hemoglobin chains is made even more striking by the close similarity of their tertiary structure. Similarities between the two types of light chains are sufficiently great to suggest that both are derived from a common ancestor. In the same way, similarities in the C-terminal peptides point to the common origin of heavy chains of different types. Although the evidence is tenuous for the p and y chains (and by extension perhaps to all classes of heavy chains), there are indications that these may also have evolved from a common ancestor; this has been the interpretation given by Singer and Doolittle (1966) for the common alIotypic specificities of the two chains of the rabbit ( Todd, 1963; Stemke and Fischer, 1965). Fingerprint similarities may also support this possibility (Lamm and Small, 1966; van Dalen et nl., 1967). In addition, IgM seems to precede IgG, phyIogenetically (Good and Papermaster, 1964; Marchalonis and Edelman, 196s) so that y and (1 chains may have evolved from a precursor p-type chain. Some authors have gone further in trying to understand evolutionary relationships between different chains. A similarity between C-terminal sequences of heavy and light chains has been suggested by comparing known stretches (Singer and Doolittle, 1966) and also the whole of the Fc fragment (Hill et al., 1966a,b) with light chains. The positions of the cysteine residues are also in agreement with this suggestion. If residues are numbered from C-terminal ends of human K chains and rabbit y chains, the cysteines occiir at the following positions:
I
p-l
K
Chain? (human)
191
134
. . . Cys
. . . Cys
195
Iinbhit Fc fragment
-
80 .
170 126(?)
. . . (Cys).
. Cys. .
..
Cys
.
Cvs
80
1
...
20 Cys
1
. . . Cyvl
2’2
. . . Cys .
.. .
..
C entl C end
62
SYDNEY COIIEN AND CESAR MILSTEIN
The similarity would be even more striking if it were found that heavyand light-chain disulfide bridges involve homologous residues. Doolittle et QZ. (1966) have reported the presence of cysteine as the carboxyterminal residue of a pathological, human, macroglobulin, heavy chain, which agrees very well with the hypothesis of a common origin of light and heavy chains. Another striking observation which points to a common origin of light and heavy chains is the position and sequence around the first Cys of a yZl,protein (Daw) and light chains. Daw heavy chain ( Piggot and Press, 1967), first Cys at position 22. Sequence: Thr-Leu-Thc-Cys-Thr
X chains (Table XVI), first Cys at position 22. Sequence:
K
chains (Table X V ) , first Cys at position 23. Sequence:
It seems that even in the N-terminal sections there may be significant similarities between heavy and light chains. The y chain (but not the p chain) is about twice the molecular weight of light chains, and the former could have arisen from a gene doubling of the latter (see Fig. 8 ) . However, the light chain includes a highly variable N-terminal half, whereas in heavy chains it is very unlikely that there are two “variable”
/,’
IS t Precursor gene
2nd ~
-~~~ + Precursor
gene
doubling
Ancestral heovy chain
gene
doubling
Frc. 8. A possible evolutionary pattern of I g chains.
K
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
63
stretches. Singer and Doolittle (1967) go even further and suggest that the light chain itself may have evolved from an ancestral half-molecule. This postulate is based mainly on the symmetry of the S-S bridges (see also Putnam et a?., 1966). This has also been suggested by studies of the Fc fragment. Hill et nl. (1966a,b) have observed that there is homology between the two halves of the Fc fragment. Comparison of residues 1-57 and 106-161 (numbering from C-terminus) shows 29%of the residues to be identical provided three gaps are left to obtain maximum alignment, This poses some interesting questions concerning the possible evolutionary pathway of the chains which may have important genetic implications (discussed by Singer and Doolittle, 1967). V.
Antibody Combining Site
The isolation of monovalent Fa11 fragments from IgG antibody molecules (Porter, 1959) proves directly that each 4-chain unit carries two combining sites. Nonprecipitating antibodies found in certain antisera are sometimes assumed to be univalent. However, Klinman et al. (1964) have shown by equilibrium dialysis that 7 S units of a nonprccipitating equine antibody each contained two combining sites of high affinity. The nonprecipitability of such divalent, high-affinity antibodies may be ascribed to structural features that prevent the opening up of Fab portions of the molecule to cross-link with antigen ( Fig. 3 ) . The available evidence indicates that IgM antibodies carry five or six combining sites per molecule. This has been shown by equilibrium dialysis experiments using purified rabbit anti-p-azobenzenearsonate IgM (Onoue et nl., 1965) and by precipitation tests with IgM antibody and isotopically labeled bovine serum albumin ( BSA) (Lindqvist and Bauer, 1966). In addition, the number of binding sites determined by equilibrium dialysis remained unchanged after dissociation of antihapten IgM to 7 S units by reduction and alkylation at pH 8 (Onoue et al., 1965). These results suggest that 7 S IgM subunits, which may retain full combining activity (Hill and Cebra, 1965) are, in fact, monovalent; whether this indicates the presence of only a single combining site or the availability for stereochemical reasons of only one of the two sites present on the 4-chain unit, has not been determined. Kaplan and Kabat (1966) obtained evidence that the combining sites of IgM antibodies are, in some instances, significantly smaller than those of IgG antibodies of similar specificity. The variable portions of heavy and light chains are present in the active Fab fragments which contain the antibody-combining sites; it is generally assumed that this variability in primary structure is related
64
SYDNEY COHEN AND CESAR MILSTEIN
directly to differences in combining specificity, The same conclusion is suggested by the finding that characteristic variations in total amino acid composition of specific antibodies are confined to active fragments and are located in the N-terminal portions of both heavy and light chains (Koshland, 1966), but whether such overall differences can be related directly to combining specificity is doubtful. The best evidence indicating that specificity is dependent on primary structure comes from experiments in which Fab fragments of various rabbit antibodies, completely reduced in 6 M guanidine-HC1, were shown to regain a significant degree of combining affinity after removal of denaturing and reducing agents (Buckley et al., 1963; Haber, 1964; Noelken and Tanford, 1964; Whitney and Tanford, 1965a,b). A similar recovery of activity has also been observed with whole IgG antibody (rabbit anti-BSA) which was reacted with polyalanyl residues before complete reduction in 8 M guanidine ( Freedman and Sela, 1966). The inference from these experiments, namely that amino acid sequence is the sole determinant of threedimensional structure and, hence, of specificity at the combining site, is supported by the fact that molecules denatured in the presence of guanidine have many properties of truly random coils (Tanford et al., 1966). Since available data suggest that the N-terminal regions of both heavy and light chains are highly variable, it might be anticipated that both polypeptide chains would be involved in forming the combining site. It has proved extremely difficult in practice to establish the relative importance of the two chains in this respect mainly because the conditions required for their separation lead to disruption of steric structure and considerable loss of combining affinity (see Porter, 1966). Nevertheless, isolated heavy chains have in several instances shown a degree of combining specificity which cannot be accounted for by contamination with light chain (Fleischman et al., 1963; Utsumi and Karush, 1964; Haber and Richards, 1966; Porter and Weir, 1966). In addition, catabolic studies have shown that Fd fragments, but not light chains isolated from specific antibody, bind to circulating antigen in vivo (Spiegelberg and Weigle, 1966). Antigen binding by the isolated light chain is uncommon but has been reported (Goodman and Donch, 1965; Mangalo et al., 1966). The light chain does, however, lead to enhancement of binding activity when recombined with specific heavy chain. Light chains derived from the original antibody are more effective than those from either nonspecific immunoglobulin, unrelated antibody, or even from antibody of the same specificity derived from a different pool or having different binding affinity (Metzger and Mannik, 1964; Roholt et al., 1965; Franek ct al., 1965; Hong and Nisonoff, 1966; Porter and Weir, 1966; Lamm et
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
65
nl., 1966). There is 110 evidence that the number of combining sites is altered by recombination, but combining affinity is greatly euhanced by association of homologous heavy and light chains (Haber and Richards, 1966). With the technique of affinity labeling, a specific hapten has been covalently bound to tyrosine residues in the vicinity of the combining site. The label is distributed in a constant molar ratio of about 2 : l between heavy and light chains of various specific antibodies; this suggests that both chains are involved in the combining site (reviewed by Singer and Doolittle, 1966). It is evident that no definite conclusion can be reached at present about the location of the combining site. Some heavy chains may carry all the information required to form the specific site. The remarkable heterogeneity of the light chain suggests its role in combining specificity but whether it has a relatively nonspecific modulating effect or participates directly at the site is a problem which remains unsettled. If combining specificity is determined by both heavy and light chains, then it is possible that a relatively restricted number of chains could generate a far larger number of distinct antibody specificities. The actual number generated might, however, be limited by the ability of various forms of heavy and light chains to associate. Studies on homogeneous chains from monoclonal proteins show that some recombination does occur between unrelated chain pairs and the ability to associate is apparently independent of the electrophoretic mobility and isotypic or allotypic specificity of the chains. However, there is a definite preference for autologous recombination which is revealed most strikingly when studied under conditions involving competition with homologous chains (Grey and Mannik, 1965; Gordon and Cohen, 1966; Mannik, 1967). Such specific interactions which have also been observed with the chains of antihapten antibodies (Roholt et nl., 1967) must be determined by regions within the variable portions of heavy and light chains which may not be the same as those areas which generate combining specificity. Studies on the chemical basis of combining specificity would be facilitated if monoclonal proteins with defined antibody activities were available for chemical analysis. Several apparently homogeneous IgG and IgM proteins with some form of combining affinity have been described (Table XXIV), but it has not as yet proved possible to stimulate the production of monoclonal immunoglobulins with well-defined combining specificity. Even if such proteins were available it seems likely that sequence data in the variable regions of heavy and light chains would have to be interpreted in terms of four properties which may or may not be related. namely ( 1 ) the Combining site, ( 2 ) regions modulating the
66
SYDNEY COHEN AND CESAR MILSTEIN
combining site, ( 3 ) the specific heavy-light chain association site, ( 4 ) idiotypic specificity of the antibody preparation. Preliminary attempts have been made to investigate the configuration of antibody-combining sites by means of physical methods. Antidinitrophenyl antibody can be differentiated from inert IgG by optical rotatory dispersion measurements in the ultraviolet region; this suggested the presence of a distinguishing configuration in the specific antibody apparently unrelated to allotypy and charge (Steiner and Lowey, 1966). TABLE XXIV MONOCIOV A L PHOI'EINSWITH SOME FORMOF COMBININGAFFINITY
Stored eryi Iirocyi es ICry Lhrocyt e " I-ant,igen' ' Htreptolysiii
niid p Ilpoproteiii Group A streptococcd carbohydrate (Y
Electron-spin resonance studies of the interaction between anti-DNP and a spin-labeled hapten (dinitrophenyl nitroxide), suggest that the combining sites have a high degree of structural rigidity (Stryer and Griffith, 1965). Chlorine-% nuclear, magnetic, resonance studies of the interaction between anti-DNP and a mercury-containing hapten [2,4dinitro-4- ( chloromercuri ) -diphenylamine] have shown that the Hg atom of the bound hapten is exposed to C1 ions of the solvent (Haugland et aE., 1967). Interpretation is complicated by the inany parameters which influence such physical measurements. However, in view of the inherent difficulty of interpreting sequence data in terms of biological activity, the use of physical methods and especially of X-ray crystallography (PoIiak and Dintzis, 1966) and electron microscopy, is of great potential interest in studies of antibody structure.
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
VI.
67
Synthesis and Assembly of Peptide Chains
Protein synthesis takes place on polysomes, and the size of the polysome appears to be related to the length of messenger and the polypeptide chain which is being synthesized (iVarner et ul., 1963; Staehelin ct al,, 1964; Kiho and Rich, 1965). Since the genetic markers of heavy and light chains are apparently unlinked, the chains should be synthesized an polysomes of different sizes. Several attempts to characterize the polysomes of lymphoid cells have been unsatisfactory (Stenzel et ul., 1964; Scharff and Uhr, 1965; Norton et al., 1965; Manner et al., 196s) because degradation to single ribosomes readily occiirs, probably through the action of nucleases. More recently, intact polysomes synthesizing immunoglobulin chains have been prepared by disrupting lymphoid cells in the presence of excess HeLa cell cytoplasm (Scharff and Uhr, 1965; Shapiro et al., 1966b) or under conditions of strict temperature control (Askonas and Williamson, 1966b; Williamson and Askonas, 1967). The study of mouse plasma cell tumors has shown that radioactively labeled, heavy and light chains, characterized either by electrophoresis (Shapiro et al., 1966b) or by immune precipitation (Askonas and Williamson, 1966b; Williamson and Askonas, 1967) are synthesized on polysomes of different sizes. Heavy chains are found only in association with larger polysomes (about 300 S ) and pulse-labeling experiments indicate that they are made within 60 seconds. Light chains appear to be synthesized within 30 seconds on smaller polysomes (about 180 S ) and can also be identified in free form in the cytoplasm (Askonas and Williamson, 1966;~;Shapiro et nl., 1966a; Nezlin and Kulpina, 1966). Labeled light chains can be demonstrated in association with heavy chains on the larger polysomes. However, in a chase experiment, labeled light chain was still present on the larger polysomes when all nascent heavy and light chains had heen removed (Shapiro et al., 1966b). Since free heavy chains are not detectable in the cytoplasm even after short labeling times, these findings suggest that assembly of the whole molecule occurs by attachment of free released light chains onto the polysome-bound heavy chains. Heavy and light chains also appear to be synthesized on separate polysomal sites in the normal lymphoid tissues of rabbits and rats (Becker and Rich, 1966) by processes essentially similar to those in other mammalian cells (Tawde et nl., 1966). Protein synthesis occurred on two sets of polysomes which, by analogy with those synthesizing hemoglobin in rabbit reticulocytes, were estimated to be of appropriate size for synthesizing peptide chains with molecular weights (25,000 and 55,000-60,000) corresponding to light and heavy chains, respectively.
68
SYDNEY COHEN AND CESAR MILSTEIN
In contrast to the remarkable heterogeneity of peptide chains isolated from the total immunoglobulin population, several observations indicate that individual cells synthesize only a restricted number of variants of each polypeptide chain. Immunofluorescent techniques using specific antisera have shown that heavy and light chains occur in the same cell. However, individual human or rabbit lymphoid cells produce only one type of light chain (Bernier and Cebra, 1964, 1965; Burtin, 1965), one class of heavy chain (Burtin and Buffe, 1963; Mellors and Korngold, 1963; Chiappino and Pernis, 1964; Bernier and Cebra, 1965; Cebra et al., 1966) including the d chain of IgD (Pernis et al., 1966), and probably only one type of the y chain. Moreover, in heterozygous animals, chains carrying allelic forms of allotypic specificity are always found in different cells (Pernis et al., 1965; Weiler, 1965; Cebra et al., 1966). This remarkable degree of specialization of immunoglobulin-producing cells is borne out by the properties of monoclonal proteins which characteristically are class- and type-specific and carry a single allelic form of alIotypic specificity (Table VI). In this connection it is of interest that the heterogeneity of myeloma proteins as judged by electrophoresis, arises at least in part from changes in charge properties which occur after secretion and can be induced in vitro by incubation of newly synthesized molecules with serum (Awdeh et al., 1966). Additional differences between the electrophoretic mobilities of intra- and extracellular Ig appear to arise during secretion and are attributable to changes located on the Fc portion of the heavy chain (Fleischman, 1963; Notani et al., 1966). Possible exceptions to the specialization of lymphoid cells have been recorded by Pernis and Chiappino (1964) who observed K and chains within individual cells of germinal centers, and Nossal et nl. (1964) who provided indirect evidence for the transient production of y and p chains by the same cell. It is possible, therefore, that at least in the differentiated cell, only two genes are active for the production of heavy and light chains and each chain represents predominantly the product of only one of two allelic cistrons. A similar degree of allelic exclusion has been observed in female animals with respect to genes located on the X chromosome, e.g., in heterozygous females only one allele controlling synthesis of glucose6-phosphate dehydrogenase is active ( Davidson et al., 1963; Beutler, 1964). However, in the case of autosomal genes controlling hemoglobin synthesis, both parental alleles are commonly expressed in each cell (Beutler, 1964). Immunoglobulin synthesis constitutes the only known example of complete or partial inactivation of one or other of a pair of
STRUClTJJX AND ACTIVITY OF IMMUNOGLOBULINS
69
autosomes. Whether this degree of differentiation precedes or follows antigenic stimulation is unknown. However, normal rabbit lymphocytes from A5, A6 heterozygotes show a summation of lymphoblast transformation with antisera to A5 and A6; this suggests that even primitive cells may be differentiated before antigenic stimulation to respond in terms of one or other chromosome of the pair (Gel1 and Sell, 1965). VII.
Genetic Implications of Immunoglobulin Structure
The combination of genetic experiments and protein sequence studies in bacteria and viruses has provided information having far-reaching consequences for understanding the molecular basis of protein synthesis. Unfortunately, what appear at first sight to be elementary genetic experiments on the nature of the immune response have sometimes proved to be of a complex nature (Green et al., 1966; McDevitt and Sela, 1965; Lennox, 1966). For this reason the increasing amount of protein sequence data has been used to the limit (and sometimes beyond it) in an attempt to understand some aspects of the genetic control of immunoglobulin synthesis. As in the case of mammalian hemoglobin and haptoglobin this approach has yielded some extremely informative results. Although many more sequences of the various chain types are needed, the available data indicate some puzzling contradictions. A good deal of space has been devoted to the evidence suggesting that both light and heavy chains are made up of a C-terminal section having a sequence defined by the type of chain, and an N-terminal portion specific to the clone from which the chain is derived. No two proteins from different clones (myelomas) have so far proved to be identical, and no two C-terminal sections have shown significant differences that could not be ascribed to an isotypic or allotypic distinction. This concept is derived mainly from studies on light chains, but recent results suggest that the same observations are applicable at least to the major types of y chains. In the following discussion we shall assume that regions of restricted and high variability are, in fact, characteristic of all types of immunoglobulin chains. Such an arrangement ideally fulfills the biological function of antibodies which must be unique in their specific recognition of an indefinite number of antigens and yet be able to maintain several properties characteristic of all antibodies of the same class or type. However, a structure which is in part repeated in all antibodies of a given type and, in part, is individually defined leads to an apparent contradiction regarding the number of genes controlling separate halves of the chains.
70
SYDNEY COHEN AND CESAR MILSTEIN
A. NUMBER OF GENESCONTROLLING C-TERMINAL STRETCHES There is convincing evidence that the minimum number of structural genes involved in the synthesis of immunoglobulins is of the same order as the number of classes and types of chains, i.e., in humans so far approximately twelve structural genes appear to be involved in the synthesis of C-terminal sections of immunoglobulins. That this is so is indicated by the following facts: 1. C-terminal halves of light chains of the same type and the known Fc fragments of heavy chains of a given type are remarkably homogeneous. It is difficult to imagine that an apparently identical sequence of over 100 residues (above 250 in Fc fragments) is under the control of many genes. 2. Population and family studies indicate that allotypes are segregated as Mendelian genes. It is difficult to visualize how homozygosity could be maintained for a factor controlled by, say, more than 100 different genes. One could postulate that, in the case of InV allotypes, mutation involves transfer ribonucleic acid ( RNA), and, therefore, affects the products of a triplet repeated in an equivalent position in many genes. But Gm(a) specificity, for example, appears to involve at least two different residues, and mutation in this case would have to involve at least two transfer RNA’s at the same time. These arguments taken together render most unlikely any hypothesis that necessitates a large number of genes coding for the C-terminal stretch of a given chain type,
B. NUMBEROF GENESCONTROLLING N-TERMINAL STRETCHES There is as yet no evidence that any two proteins derived from different clones are identical. All individual differences in chains of the same type and allotype are apparently located in the N-terminal sections. However, available data suggest that variability is restricted in several respects: ( a ) chain length is essentially constant, though variations in size due to insertions or deletions have been observed; ( b ) certain positions are highly variable and others highly conservative; ( c ) some positions involve one of two residues; and ( d ) the majority of the variations can be ascribed to a one-step mutation process involving both transitions and transversions (Table XXV; see also Gray et al., 1967). Even apparent exceptions may be ascribed to a one-step process when more data are collected; e.g., residue 91, Table XXV in which two steps would be required to go from Asp to Thr but single base substitutions would account for consecutive changes from Asp to Asn to Thr ( Milstein,
71
STRUCTURE AXD ACTIVITY OF IhlMUXOGLOBULINS
1966d). It must be emphasized that restrictions listed above are based on the study of a far from adequate number of proteins. Some proteins appear to have larger differences but these have not been studied in detail ( Milstein, 1966a; Baglioni and Cioli, 1966). From a statistical point of view it appears that, despite restrictions, the number of variants is theoretically very large. If variation can occur at 30 places (more than 30 have already been observed), and the number of variants at each place is restricted to only 2, then the total number of possible chains, assuming all theoretical combinations, is 2 or more than a million. If the average number of possible variants in each position is increased to 3 and the total number of variant sites to 50, then the theoretical number of variants becomes 3-"'-an astronomical figure. The chances of finding two identical proteins under these conditions is almost negligible. If, on the other hand, the number of variants is of a lower order of magnitude, one can ask how many proteins need to be studied before two are found to be identical. This can be calculated by assuming a population of infinite size containing n variants. The probability, Pr, of drawing r samples without observing a repeat is given by )'I,
Figure 9 shows a graphic representation of this equation for different values of n. If the number of variant sequences of a single type does not exceed 1000, then the probability of finding two identical sequences is large after studying 50 proteins (P,,, = 0.29). If after studying 77 proteins ( Pi, = 0.05), no two identical sequences were found it would be highly likely that the total number of variants is much larger than 1000. If the study is restricted to fingerprints rather than full sequences then the probability of finding complete identity is considerably increased because ( a ) core peptides are not detected and ( b ) variations such as Thr/Ser, Asp/Glu, Asn/Gln, and Leu/Ile are not likely to be detected. This approach has been initiated by Raglioni and Cioli (1966) who found no identical fingerprints after analyzing 25 K chains and 20 x chains, which indicates that the minimum number of variants is likely to be at least 250 K chains and above 200 chains (see Fig. 9 ) . Unfortunately no similar study has, to our knowledge, been carried out in inbred mice. In humans the possibility that some-or many-of the observed differences are due to allelic variation cannot be excluded. However, if this were not the case and each variant structure was under the genetic control of a separate gene, then a minimum approaching 500 genes for each type of light chain would be necessary.
TABLE XXV CODONSOF
THE
OBSERVED SUBSTITUTIONS IN HUMANK CHAINS"'~
1
(:APu; GAPy
39
GGX; AAPu
83
GUX; UUPy; AUPy
2
AUPu;GUX
46
SUPY
84
GCX; GGX
53
AAPu; AAPy
85
GUX; ACX
AUG; g:U;GUX
55
GCX; GAPu
90
GCX; GUX; AUPy
56
ACX; GCX; AGPy
60
lJCx. GAPy AGPy '
4
19
65 66
;E;
1
CAPu; AUG
I
ucx
92
GAPy; Gr\Pu;
=I
93
BCX; AAPy;
GAPy; GAPu
UUPU
CUPy
100
31
AUPy; AAPy; A1Pu
27
CAI’u; C U S
ccs;
C.1Pu;
I C;Gx
U
w
I]!,
” The numbers indicate the positions in the chain as shown in Table XV. In boxes are known variants that cannot be derived single base changes. Pu is either A or G; Py, U or C; X, any of the four. ‘’ From hlorgan et al. ( 1966).
74
SYDNEY COHEN AND CESAR MILSTEIN
I t is obvious that the minimum number of genes apparently required to code for N-terminaI stretches is of a different order of magnitude from that involved in coding for the C-terminal stretches. This contradiction involves a fundamental problem of immunology, namely, the origin of antibody variation ( and presumably of combining specificity ), and several mechanisms have been invoked to explain it. As discussed above,
r
FIG. 9. Probability ( P r ) curves indicating the number of proteins ( r ) which must be analyzed, out of a pool containing a total of n sequences, in order to obtain two identical sequences. (The collaboration of J. K. Moffat is gratefully acknowledged. )
conservation of invariant C-terminal sections and the Mendelian inheritance of allotypic specificity, indicate that the C-terminal end of each chain type must be controlled by a limited number of genes. The Nterminal sections must be controlled by a large number of genes which could have arisen during the course of evolution by a gene doubling process with selective preservation of variants in the germ line or be generated by somatic mutation of a limited number of genes. There are, therefore, two major problems in relation to the genetic control of immunoglobulin synthesis; namely: ( 1 ) Are the C- and N-
STRUCTURE AND ACTIVITY OF IMMUNOGLOBULINS
75
terminal sections of a given peptide chain controlled by two separate genes or by a single gene? ( 2 ) Is the variability of the genes (or specific parts of genes) controlling N-terminal sections of chains generated by mutation and selection during the course of evolution or by somatic mutation during the lifetime of the individual? No conclusive answer can be given to either of these questions at the present time, but it seems worth while discussing some of their implications in relation to the known structural features of immunoglobulin chains. The postulate that each variant chain is controlled by two structural genes visualizes that one is common to all proteins of the same type and the other is selected from a large set of genes which give rise to individual specificities. Fusion of the two halves could occur at the level of deoxyribonucleic acid (Dreyer and Bennett, 1965), messenger RNA, or at the protein level (Cioli and Baglion, 1966; Burch and Burwell, 1965) The general restrictions in chain variability, which were listed above, are qualitatively similar to those observed in several species for sequences of a given protein likely to have arisen by selection after random mutation [e.g., cytochrome c (Margoliash and Schejter, 1966) and insulin (Smith, 1966)l. Moreover, there does not appear to be a restriction of the type of base substitution since both transitions and transversions are observed. Proteins, Roy, Ag, B-J, and Ker are extremely similar in their sequences, but in 19 positions have 2 or more variants. Two of these cannot be accounted for by a one-step mutation; of the others, 8 are transitions and 15 are transversions. Such findings have suggested to several authors that genes controlling N-terminal sectiom have arisen by a mutational process followed by selection of suitable variants and are carried in the germ line. However, there are several difficulties in regard to such a theory involving two genes per chain. If the two polypeptides were synthesized separately and then joined, the two halves should behave as independent chains during amino acid incorporation; experiments with the pulse-labeling technique have, as yet, provided no direct evidence for this. In vitro studies of lymphoid cells have shown that polysomes involved in Ig synthesis are of appropriate size for heavy and light chains and not smaller units. Gin specificities located on different halves of monoclonal y.,, chains are invariably paired (see Section 11,C); if the specificities of the N-terminal half [Gm( z) and Gm( f ) ] are, in fact, localized in the “variable” region of the chain, then any mechanism involving gene fusion must be excluded. Finally, if the rabbit allotypes present on Fd and controlled by the “a” locus are associated with amino acid substitutions in the variable 9
76
SYDNEY W H E N AND (SESAR MILSTF.IN
region, then the explanation of their Mendelian inheritance becomes problematical. A major difficulty involving an independent set of genes for the Nterminal half stems from the evolutionary pattern of the system. If the N-terminal halves of the chains of different animal species are derived from a common ancestor set, then “conservative residues” present in the sncestors should have been selectively preserved. However, such residues are apparently not always common to digerent species (Table YIX) and may diger in the K and chains of a single species (Table XV ) despite evidence for their common ancestry. A further evolutionary difficulty concerns the specific attachment of a large set of N-terminal halves to a small group of C-terminal halves. The single evolutionary ancestor of K and C-termini must be capable of recognizing every N-terminal half. A doubling of the ancestor C-terminal half to produce the second type of chain would presumably have had the best chance of survival if it could still recognize the pre-existing set of N-terminal halves. The N-terminal halves should, therefore, be common to the two types of chains, and this is not the case (see Section IV,C,l,b). A more detailed discussion of evolutionary dif€iculties which complicate the two-gene model of immunoglobulin chain synthesis is given by Singer and Doolittle ( 1967). The available genetic and structural data, therefore, provide no positive support for the two gene model of chain synthesis, and certain structural features appear to be incompatible with this theory. An alternative postulate is that each variant chain is controlled by a single gene. If only a limited number of genes control C-terminal sections, then variability of the N-terminal stretch must arise by somatic hypermutation. Earlier hypotheses ( Burnet, 1959; Lederberg, 1959) did not restrict somatic mutation to a given part of the gene, but subsequent theories have attempted to account for variability confined to one-half of the chain. Sequence data can be used to test some of these hypothetical mechanisms. Because of their simplicity, crossing over mechanisms have been favored by some workers (see, e.g., Watson, 1965). One can ask, for instance, if the observed variants could have arisen from two different sets of nucleic acid sequences by multiple crossing over with strict preservation of phase. In other words, is it possible to construct two nucleotide sequences from which all amino acids in each position can be coded? This has been tested by Milstein (1966a). The more general test is to show that all substitutions involve only two bases in each position. In Table XV, however, there are 13 places in K chains which include more than two residues; among these at least three different bases are
STRUCl’URE AND ACTIVITY OF IMMUNOGLOBULINS
77
required either in the first codon position (residues 4, 83, and 96) or in the second codon position (residues 91, 96, and 100) (see ‘Table XXV). In h chains there are ten places (Table XV) with three or more variants, and five (3, 13, 15, 24, and 30) require more than two different bases in one of the codon positions. The possible occurrence of crossing over between more than two strands becomes very difficult to test in this way and is impossible if four or more strands are postulated since there are only four bases to code for any amino acid. As the number of postulated strands is increased, special assumptions must be made to explain the invariance of the C-terminal section and the Mendelian behavior of allotypes. Smithies (1965) has proposed that variations could arise by somatic rearrangements of genes controlling the polypeptide chains. Inverted duplications were suggested and these should give rise to variations clustered at the site of the inverted loop; in fact, residue differences occur along the length of the chain, cf. Roy, Ag, B-J, and Ker which have single residue differences in positions 2, 19, 30, 31, 46, 53, 56, 65, 67, 77, 91, 93, 96, and 100 (Tables XI11 to XV). Another hypothesis which can be tested by sequence data was proposed by Potter et al. (1965) and is based on a specialized messenger translation mechanism. Unusual triplets present in specific places in the gene could be translated differently depending upon small changes in the amino acid activating enzyme or in transfer RNA. This has been discussed by several authors (Milstein, 1966a; Titani et al., 1966; Gray et al., 1967; Singer and Doolittle, 1967). The fact that the chains showing a general restriction in size, may nevertheless, vary in length, is probably the strongest single argument against this hypothesis. However, it could be argued that there are, for instance, two genes of different sizes with identical C-terminal sections. Even then a variation such as that observed in residue 2 of human K chains, where an “invariant residue” (Ile) was eventually found to be replaced, seems difficult to reconcile with the prediction that “the final protein will vary by single amino acids at key points.” Other difficulties have been discussed by the authors mentioned above. Selective mutation could be achieved if a specific stretch of DNA at the beginning of the invariable part of the gene and common to all immunoglobulin genes acted as a recognition site for initiation of the mutation process. Brenner and Milstein (1966) have discussed a mutation mechanism which postulates a defective repair enzyme becoming operative after stretches of DNA coding for the N-terminal half of the chain have been split off. This model generates a far larger number of
78
SYDNF,Y COHEN AND CESAR MILSTEIN
variants than appears necessary (Dreyer and Bennett, 1965). One prediction of this hypothesis is that there should be an identical nucleotide sequence near the beginning of the C-terminal section of aZZ chains. In K and h chains, residues 110-114 are identical (Milstein, 1966e), suggesting an identical nucleotide sequence. A nucleotide sequence ( see Table XXV), Py, CCX, GCX, CCX, A, seems, in fact, to be common to K chains of human and mouse and h chains of human, starting at the end of residue 109 (Table XI). However, our knowledge of the nature and extent of the variable sections of chains is fragmentary and a stringent test of the above postulate requires further sequence data. It seems, therefore, that the available sequence data provide no positive proof for various hypothetical mechanisms whereby variability of certain sections of immunoglobbulin chains could be generated from a small number of genes. We are left with the fundamental question of whether or not each variable region is controlled by genes carried in the germ line. If this is so then a postulate that each variant chain is controlled by two genes seems unavoidable but, as outlined above, certain structural features seem to be incompatible with such a theory. A more critical assessment of its validity may come from an understanding of the evolntionary pattern of immunoglobulins and from studies on the inheritance of well-characterized variants. At present, the occurrence of somatic hypermutation seems more likely, but such a process may not necessarily be recognizable on the basis of sequence studies alone. VIII.
Comments
Since immunoglobulin structure was last reviewed in this series, the 4-chain model proposed by Porter has been amply confirmed and found to apply even to the most primitive vertebrates examined. Understanding of the general configuration of antibodies has been extended by electron microscopy which shows that, when cross-linked to antigen, the Fab portions of IgG antibodies are extended and the molecule appears as a Y-shaped strand. Although certain overall structural problems remainnotably in regard to the configuration of seromucous IgA and the apparent monovalency of the 7 S subunits of IgM, considerable progress has been made in defining the general chemical and biological properties of distinct immunoglobulin classes and types. Even more striking has been the accumulation of detailed sequence data which was obtained, in the first instance, from the study of monoclonal proteins and is now being derived increasingly from normal immunoglobulin chains; this work provides further evidence for monoclonal proteins being individnal species of normal immunoglobulin. It seems likely that these studies will
STRUCTURE AND ACITVITY OF IhIAfUiVOGLOBULIXS
79
before long lead to a detailed understanding of the chemical differences that distinguish classes, types, and allotypic variants of immunoglobulins and will also delineate evolutionary relationships between various chains. Amino acid sequence studies have revealed the surprising fact that light chains ( a n d probably also heavy chains) have C-terminal sections with sequences defined by isotypic and allotypic specificities alone, whereas N-terminal stretches appear to be specific for their clone of origin and no two chains have so far proved to have identical structures. The number of genes required to code for N-terminal stretches is, therefore, of a far greater magnitude than that involved in coding for C-terminal stretches. The fundamental problem of whether or not each variable region is controlled by a gene carried in the germ line or generated by somatic hypermutation cannot as yet be answered conclusively. It seems reasonable to assume that the remarkable degree of structural variation of immunoglobulin chains is related to combining specificity, especially as the successful refolding of antibody molecules in the absence of antigen has provided strong evidence that such specificity is dependent upon covalent structure. However, peptide chains also show individual antigenic ( idiotypic ) specificity related to, but not necessarily corresponding with combining specificity, and also considerable individual specificity in regard to interchain association. Even if monoclonal proteins with defined antibody activity become available for analysis, the relationship between combining specificity and primary structure of heavy and light chains may prove to be extremely complex. Understanding the structural basis of combining specificity may ultimately depend upon physical rather than chemical methods of analysis.
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SYDNEY COHEN AND CESAR MILSTEIS
Benacerraf, B., Ovary, Z., Bloch, K. J., and Franklin, E. C. (1963). J. Exptl. Med. 117, 937. Bennett, J. C., Hood, L., Dreyer, W. J., and Potter, M. (1965). J. Mol. B i d . 12, 81. Berken, A., and Benacerraf, B. (1966). J. Exptl. Med. 123, 119. Bernier, G. M., and Cebra, J. J. (1964). Science 144, 1590. Bernier, G . M., and Cebra, J. J. (1965). J. Immunol. 95, 246. Bernier, G. M., Tominaga, K., Easley, C. W., and Pntnam, F. W. (1965). Biochemistry 4, 2072. Beutler, E. (1964). Cold Spring Harbor Symp. Quant. Biol. 29, 261. Binaghi, R. A., and Benacerraf, B. J. ( 1964).J. Immunol. 92, 920. Binaghi, R. A,, Benacerraf, B. J., Bloch, K. J., and Kourilsky, F. M. (1964). J. Immunol. 92, 927. Binaghi, R. A., Oettgen, H. F., and Benacerraf, B. J. (1966). Intern. Arch. Allerg!! Appl. Immunol. 29, 105. Bloch, K. J., Kourilsky, F. M., Ovary, Z., and Benacerraf, B. J. (1963a). J. Exptl. Med. 117, 965. Bloch, K. J., Ovary, Z., Kourilsky, F. M., and Bennccrraf, B. J. (196311). Proc. SOC. Exptl. Biol. Med. 114, 79. Bornstein, P., and Ondin, J. (1964). J. Exptl. Med. 120, 655. Boyer, S. H., Hathaway, P., hscasio, F., Orton, C., and Bordley, J. (1966). Science 153, 1539. Brenner, S., and Milstein, C. (1966). Nature 211, 242. Buckley, C. E., Whitney, P. L., and Tnnford, C. ( 1963). Proc. Nail. Acad. Sci. U.S. 50, 827. Burch, P. R. J., and Burwell, R. G. (1965). Quart. Rec. Biol. 40, 252. Burnet, F. M. (1959). In “The Clonal Selection Theory of Acquired Immunity.” Vanderbilt Univ. Press, Nashville, Tennessee; Cainbridge Univ. Press, London and New York. Burnet, F. M. (1966). Nature 210, 1308. Burtin, P., and Buffe, D. (1965). Immunopathol., Intern. Symp. 4th, Monte Carlo, 1965, p. 273. Burtin, P., and Buffe, D. (1963). Proc. SOC. Exptl. Biol. Med. 114, 171. Cahnmann, H. J., Arnon, R., and Sela, M. (1965). J. Biol. Chem. 240, 2763. Cahnmann, H. J., Arnon, R., and Sela, M. (1966). J. Biol. Chem. 241, 3247. Cebra, J. J., and Robbins, J. B. (1966). J. Immunol. 97, 12. Cebra, J. J., and Small, P. A. (1967). Biochemistry 6, 503. Cebra, J. J,, Colberg, J. E., and Dray, S. (1966). J. Exptl. Med. 123, 547. Chaplin, H., Cohen, S., and Press, E. M. (1965). Biochem. J. 95, 256. Chiappino, G., and Pernis, B. ( 1964). Puthol. Microbiol. 27, 8. Choules, G. L., and Singer, S. J. ( 1966). Immunochemistry 3, 21. Cioli, D., and Baglioni, C. (1966). J. Mol. Biol. 15, 385. Clamp, J. R., Dawson, G., and Hough, L. (1966). Biochem. J. 100, 35C. Clem, L. W., and Small, P. A. ( 1966). Federation Proc. 25, 437. Clem, L. W., and Small, P. A. (1967). J. exptl. Med. 125, 893. Cohen, S. ( 1966). Proc. Roy. SOC. (London) B166, 114. Cohen, S., and Dresser, A. M. (1965). Immunopathol., Intern. Symp., 4th, Monte Carlo, 1965, p. 243. Cohen, S., and Freeman, T. (1960). Biochem. J. 76, 475. Cohen, S., and Gordon, S. (1965). Biochem. J. 97, 460.
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Genetics of Immunoglobulins in the Mouse MICHAEL POTTER AND ROSE LIEBERMAN laboratory of Biology. National Cancer Institute. and the loboratory o f Clinical Investigations. National lnrfilute of Allergy and Infectious Diseases. Notional Institutes o f Health. Bethesda. Maryland
I . Introduction . . . . . . . . . . . . . . . . Structural Chanicteristics of Immnnoglol~ulinsin hlice . . . . . . . . . . . . A . General . B . Su1)unit Structure . . . . . . . . . . . C . Chemical Identification of Uiffercnt Im~iiuiioglol~ulin Chains . D . Localization of Allotypic Determinants on Different Imnirlno. . . . . . . . . . . . glohlins E . hlyeloma Proteins Ct)utiolled by Cenes Other than Those Found . . . . . . . . . . . in BALB/c . 111. Preparation and Testing of Homologous Antiscra . . . . . A . General . . . . . . . . . . . . . R . Preparation of Allotype Antisern with Normal Iiiimunoglol~ulins . C . Preparation of Allotype Antisera with Myeloma Imtiiunoglol~i~lins . D . Testing of Homologous Antisera . . . . . . . IV . Distribution and Localization of Ilcavy-Chain Determinants . . A. General . . . . . . . . . . . . . B . yG Heavy-Chain Determinants . . . . . . . . C. y H Heavy-Chain Determinants . . . . . . . . D . yA Heavy-Chain Determinants . . . . . . . . E . y F Heavy-Chain Determinants . . . . . . . . F . Unassigned Immunoglobulin Determinants . . . . . G . Failure to Produce Homologous Antisera in Some Donor-Recipient . . . . . . . . . . . Combinations V . Comparison of the Results 0l)tained with the Inhibition of Precipitation . . . . . . . . . . . . . hfethocl . VI . Linkage of &nes Cotitrolling IIcnvy-Chain Ileterminuits . . . A . Ceiwtic Studies . . . . . . . . . . . B . Structural Similarities I)rt\vwu (: ;inti f 1 1’;ipaiii Fc Fragrntwts of HALH/c hlicc . . . . . . . . . . . VII . Distrilxltion of IIcavy-Chain Dcterminants i n 1nl)rctl nntl Wild hlicc . A . Inbrcd Strains . . . . . . . . . . . B . Wild hlice . . . . . . . . . . . . C . Possible Evolution of Heavy-Chain Linkage Groups . . . VIII . hlyeloma-Specific Homologous Antisera . . . . . . . IX . Hemolytic Complement Component ( IIc’ or hluB‘) . . . . X . Concluding Rcmarks . . . . . . . . . . . References . . . . . . . . . . . . . 91 11.
92 95 95 97 9‘3
101 101 103 103 10:3
104 104 109 109 110 113 117 119 122 126
126 127 127 131 1S1 131
13’3 137 139 140 141 143
92
MICHAEL POTTER A N D ROSE L I E B E R M A N
I.
Introduction
The mouse is a highly advantageous species in which to study the qenes controlling immunoglobulin structure. The wild and domesticated forms constitute two different populations in which different selective factors affecting survival are at work. In wild mice, iminunoglobulin genes from individuals living under natural conditions can be examined. With domesticated, inbred strains developed by extensive consecutive brother-sister matings, it is possible to use classical genetic methods for establishing the segregation and linkage of genes controlling immunoglobulin structure. Hopefully, this will lead to a quantitative evaluation of the number of genes involved in immunoglobulin formation and ultimately provide insight into the mechanism of variation in immunoglobulin structure. Another important advantage in mice, very relevant to genetics, is that the polypeptide chain products of immunoglobulin genes may be recovered in sufficient quantity and purity to permit detailed chemical and amino acid sequence analysis (Hood et nl., 1966; Perham et al., 1966; Gray et al., 1967). It has become possible by the establishment of the colinear relationship between the gene and polypeptide chain ( Yanofsky, 1963; Yanofsky et nl., 1964) and the genetic code (Nirenberg and Leder, 1964) in other systems, to anticipate that immunoglobulin genetic structure may be established through amino acid sequence analysis of immunoglobulin polypeptide chains. However, normal serum immunoglobulins are a heterogeneous group of closely related proteins, and it is very difficult, if at all possible, to isolate homogeneous molecular forms which are espntial for this type of analysis. This problem is resolved by the homogeneous myeloma immunoglobnlins secreted by plasma cell tumors, and the mouse is one species in which plasma cell tumors can be readily obtained. In mice, plasma cell tumors very rarely occur spontaneously (RaskNieIsen and Gormsen, 1951; T. B. Dunn, 1954, 1957), but in one highly inbred strain, the BALBIc strain, they can be induced with high frequency by solid plastic materials such as Lucite disks or shavings ( Merwin and Algire, 1959; Merwin and Redmon, 1963), immunological adjuvants (Potter and Robertson, 1960; Lieberman et al., 1962), and mineral oils (Potter and Boyce, 1962). Plasma cell tumors can be propagated as transplant lines in syngeneic hosts where they grow to relatively enormous size in each new recipient. The cells of a tumor acti\lely synthesize and secrete specific homogeneous forms of immunoglobulins
GENEHCS OF IhfMUPiOGLOBULINS IN THE MOUSE
93
(Potter et al., 1957; Potter and Fahey, 1960). Each tumor consists of one type of plasma cell that is differentiated from others in the type of immunoglobulin synthesized. Further, tliese neoplastic cells are fixed, in a way that, when they continue to divide mitotically, the succeeding progeny produce the same type of immunoglobulin. For these reasons, one homogeneous farm of iminm~oglobulinappears in high concentration in the serum of tumor-bearing mice and permits both the separation of the immunoglobulin from the underlying normal immunoglobulins and its immunochemical characterization. There are five classes of immunoglobulins in mice: yM, yA, y F ( y l ) , 7G( y2a), and yH( 7211) (Faliey et a/., 1964; Potter et al., 1965). Each c l x s of immunoglobulin possibly has a specialized function in the immune response, although this has not been demonstrated for all forms as yet. For example, the yM-immunoglobulin is the most efficient immunoglobulin for binding complement. Eorsos and Rapp ( 1965) have shown that the presence of a single antisheep red blood cell yM molecule on a sheep red blood cell surface is sufficient to bind complement and lyse the red blood cell. YM-Immunoglobulins are 18 S molecules usually made in a pentameric form (Miller and Metzger, 1965). The large size and its multivalence endows the yM molecule with special physicochemical properties that are pertinent to some types of antigen-.antibody reactions. The yA-immunoglobulin class is of particular interest in the mouse, because the chief type of immunoglobulin-producing plasmocytoma induced by plastic ( Merwin and Redmon, 1963) or oil (Potter and Lieberman, 1967) produces yA myeloma protein. Further Mandel and Asofsky ( 1967) have shown that mesenteric node and intestinal tissue contain immunocytes that produce chiefly yA-immunoglobulins. These findings suggest that large numbers of the immunocyte population in the peritoneum (where the induced plasmocytomas arise ) are differentiated to produce yA-type immunoglobulin. Serum levels do not reflect the relative large numbers of yA cells in peritoneal tissues, as the yA leiiels in NIH-WS mice are low (0.4 mg./ml.) as compared to y F (2.5 mg./ml.) or yG plus yH (4.0 mg./ml.) (Fahey and Sell, 1965). One explanation for the low serum levels of yA is that these serum yA-immunoglobulins are concentrated in tissues or secretions. This is supported by the findings in the rabbit and in man that yA-immunoglobulins are transported across epithelial cells (e.g., salivary gland; mammary gland) and secreted into saliva, milk, nasal secretions, etc. (Tomasi et al., 1965; South et al., 1966). It is believed that during passage across the epithelial cell,
94
MICHAEL POTTER AND ROSE LIEBERMAN
the yA-immunoglobulin acquires an additional polypeptide called the “transport piece” (Cebra and Small, 1967). Thus far transport piece in the mouse has not yet been found. The 7 S mouse yF-immunoglobulins are able to bind skin (Barth and Fahey, 1965) and, thus, could be concentrated in specific tissue sites. The 7 S yG- and yH-immunoglobulins do not have this skin-binding property. The special function of these two immunoglobulin types in the immune response has not as yet been fully elucidated. In the humoral immune response in the mouse, five different classes of antibody molecules are produced (Fahey et al., 1964). The identification of genes that control the structure of these different immunoglobulin types are one of the primary genetic problems. As a direct outgrowth of the knowledge of genetic polymorphism of serum proteins and immunoglobulins in rabbits (Oudin, 1960; Dray and Young, 1959), it was discovered that genetic polymorphism of mouse immunoglobulins also existed. Kelus and Moor-Jankowski (1961) showed that mice of me inbred strain could be immunized with the immunoglobulins from a genetically different strain. The recipient mice formed precipitating antibodies that identified a specific, immunoglobulin, antigenic determinant in the donor strain. The genetic significance of this finding was immediately appreciated by observing that these determinants were present in some and absent in other inbred strains. Dubiski and Cinader (1963) identified a second immunoglobulin determinant, and Dray et al. ( 1963), utilizing reciprocal homologous antisera, demonstrated the allelism of two genes controlling different, immunoglobulin, antigenic determinants. When immunizations were extended to other donor-recipient inbred strain combinations, an elaborate system of antigenic determinants was identified (Lieberman and Dray, 1964). Based on the distribution of these determinants, five basic immunoglobulin allotypes were found among thirty-eight inbred strains. Herzenberg et al., (1965) extended this work and identified additional determinants. The assignment of immunoglobulin antigenic determinants to specific immunoglobulin chain types began with Mishell and Fahey’s (1964) observation that one of the homologous antisera identified a determinant on a yG ( y2a ) -immunoglobulin; Herzenberg ( 1964 ) found a second on a yA-immunoglobulin, and Lieberman et al. (1965) found a third on a yH-imrniunoglobulin. Herzenberg ( 1964 ) demonstrated that the genes controlling yG and yA were linked, and Lieberman et al. (1965) demonstrated the close linkage of the yG and yH genes. From these basic observations, a complex immunogenetic system in the mouse has evolved. The important general finding of genetic significance is that specific
GENETICS OF IMMUNOGLOBULINS I N THE MOUSE
95
homologous antisera can be used to identify the products of genes that control immunoglobulin structure in the mouse. Highly purified, myeloma immunoglobulins or their subunits can be used as the products of the immunoglobulin genes, and this constitutes a highly specific system. The identification reaction for this system can be an agar gel precipitin reaction which is easily accomplished with small quantities of material. It becomes possible with these antisera to test many different immunoglobulins for the presence of specific determinants. The potential of the mouse allotype system is far from being realized; for example, no polymorphisms of light chains have yet been found. Because of the rapid development of the mouse allotype system, we shall present the current data available pertaining to this system. We shall not make any attempt to describe all of the genetic aspects of the immune response; for example, discuss genes that regulate the recognition of antigenicity ( McDevitt and Sela, 1965). A brief mention of the distribution of the allotypes of Hcl (Herzenberg et al., 1963) or MuB' (Cinader and Dubiski, 1963) seriim protein among inbred strains is included. The Hcl component is not an immunoglobulin in the strict sense, but is related to the complement system and, therefore, participates in some immunological phenomena. Further, some inbred strains do not make any Hc'; these are the Hco strains. Some of these Hcn strains will form precipitating antibody when immunized with serum of mice that do produce Hc'. In preparing homologous antiserum to the immunoglobulin allotypes, antibody to the Hcl component is sometimes obtained and a brief description of the Hc' component is included here for purposes of distinguishing between the immunoglobulin allotypes and the Hc' component. 11.
'4.
Structural Characteristics of Immunoglobulins in Mice
GENERAL
The immunoglobulins of most vertebrate species, including the mouse, are constructed according to a common, 4-polypeptide-chain plan established by the noiv classic investigations of R. R. Porter and his associates (Porter, 1959, 1963; Fleischman et al., 1962, 1963; and Edelman and Poulik, 1961) . Essentially, the 4-polypeptide-chain unit consists of 2 identical light chains (25,000 mol. w. each) and 2 identical heavy chains (50,000 mol. w. each). The light chain is believed to interact with only about one-half of the heavy chain chiefly by hydrogen bonds, but also by a covalent disulfide bone ( L H disulfide). Amino acid sequence studies (Milstein, 1965; Perhani et nl., 1966) have shown the contributing cysteine from the light chain is actually the carboxyl
TABLE I IMMUNOGLOBULIN NOMENCLATURE IN MICE AND CLASSIFICATION OF
REPRESENTATIVE MYELOMAPROTEINS RZolecular forms of immunoglobulins in mice
Characteristics Classesa Chain types Light Heavy Forms0 Predominant sedimentation coefficient Representative myeloma proteins BALB/C
C3H
hL/N BL4LB/c-2(2/2 homozygotes)'
Y
Yz 4
YF
YG
IgM
IgA
IgGrl
IgGy2a
YH IgGy2b K
h
K
P
LY
(ma5 18 s
(K2a2)pb 7, 9, 11, 13 s
RIOPC 104
Adj PC-6.4 RIOPC 209 RlOPC 241 MOPC 153 X-5647 MOPC AL-3 lLIOPC 320
K
K
9 K292
Y
7
K2y2
x272
7s
7s
7s
MOPC 21 MOPC 31 MOPC 70A
Adj PC-5 RlOPC 173 LPC 1
RlOPC 141 MOPC 172 MOPC 195
X-5563 MOPC 300
MOPC 352
"Classes are distinguished by the differences in heavy chains. A class may contain two forms, i.e., two types of molecules possessing similar type heavy chains, but containing a different type of light chain ( K or h ) ; this prevails in man, but has not yet been demonstrated in mice. ' p = polymer. ' See Table 11; Section 1,E.
GENETICS OF IMMUNOGLOBULINS IN THE MOUSE
97
terminal residue. The remaining portions of the heavy chains are joined together again chiefly by hydrogen bonds and by a covalent bond, the H-H disulfide (Hong and Nisonoff, 1965). While most functional immunoglobulin molecules are constructed according to this 4-chain plan, some forms, notably the yM and yA classes, function as polymers of these 4-chain units. A second structural relationship between immunoglobulins within a class is based on the sharing of light-chain subunits, In the mouse, the yA-, yF-, YG-, and yH-immunoglobulins thus far appear to utilize the ronimon type of light-chain subunit, the K chain (Potter et al., 1965), whereas the yM-immunoglobulin contains the &-type ( McIntire et al., 1965) light-chain subunit (Table I ) . The mouse differs quite strikingly from man. In man, there are two light-chain types, the K and A. Either one of these light chains can combine with the same type of heavy chain. Six different heavy-chain types have been identified in man and taking into consideration the ability of either light-chain type to act as a subunit, there are at least twelve molecular forms of immunoglobulins in man (Kunkel et al., 1964; Terry et al., 1965).
R.
SUBUNIT STRUCTURE
There are two main types of subunits of immunoglobulins, the genetic subunits and the chemically derived proteolytic subunits. The light and heavy polypeptide chains are genetic subunits and are obtained by reducing the interchain disulfide bonds with reagents such as mercaptoethanol or dithiothreitol and dissociating the chains in urea, guanidine, propionic acid, etc. (Fleischman et a]., 1962; Small et al., 1963; Potter et al., 1965) by gel filtration. Dissociated heavy chains often become insoluble and lose their antigenicity and are not usable as antigens in agar-gel diffusion methods. The 4-chain immunoglobulin molecule is cleaved proteolytically by several enzymes (including papain which is chiefly used) into several types of subunits. The most common!y obtained are the papain F a b and Fc fragments (Porter, 1959). Papain splits the 4-chain unit in a vulnerable region about midway in the heavy-chain polypeptide into two similar Fab fragments and an F c fragment. Each Fab fragment contains an entire light chain and about one-half of a heavy chain. The Fc fragment ronsists of tbe two other halves of the heavy chains joined together by the H-H disulfide bond. The sedimentation and viscosity studies of Noelken et 01. (1965) show that the F c and Fab fragments each behave as compact globular proteins whereas the 4-chain protein is considerably extended. This has prompted Noelken et a]. to represent schematically
98
MICHAEL POTTER AND ROSE LIEBERMAK
the immunoglobulin molecule as consisting of three globular units, two Fab units, and one Fc unit. It is generally surmised that the Fab fragment is the region containing the interactive sites between the light and heavy chains. Further, Porter (1959) has shown the Fab fragments from rabbit antibody contain the antigen-combining sites.
4 Chain monomer
Papain
/
t
Reduction olkylotion
Llght chain
i
?l
COOH‘ICrOH
COOH Heavy chain
FIG. 1. A schematic iiiotlel of a 4-chain imnirinoglobulin molecule based upon the model described by Porter (1963), Fleischman et al. (1963), and Noelken et al. (1965) and the types of fragments or subunits obtained By papain digestion or reduction and alkylation. Coils not intended to connote a-helix.
The various fragments of the 4-chain immunoglobulin molecule are identified schematically in Fig. 1 which is a composite of the Porter (1963) and Noelken et aZ. (1965) schemes. In contrast to the isolated chains, the Fc and Fab fragments retain their solubility and antigenicity. Of particular relevance to the present study is the fact that most of the polymorphic, immunoglobulin, antigenic determinants are found on the papain Fc fragments. Thus, the structural basis for the antigenic poly-
GENETICS OF IMMUNOGLOBULINS IN THE MOUSE
99
morphism is probably accounted for by the amino acid sequence variations existing in the Fc region of the heavy chains. However, this is by no means the only possibility. The carbohydrate side chains are located on the Fc fragments in the rabbit (Fleischman et al., 1963), and presumably, also in the mouse and these could contribute to the antigenicity. The genes controlling carbohydrate structure would be independent of those controlling peptide structure. C. CHEMICAL IDENTIFICATION OF DIFFERENT IMMUNOGLOBULIN CHAINS The chemical identification of immunoglobulins is based upon the analysis of the polypeptide chains of a large variety of myeloma proteins (Table I ) . Initially, myeloma proteins of mice were classified by heterologous rabbit antisera (Fahey et at., 1964) and, then, further characterized by tryptic peptide maps of the heavy chains or the papain Fc fragments (Potter et al., 1965, 1966). When chains of the same type were compared by the tryptic peptide map technique, each chain derived from a different tumor source was found to differ from each other by several tryptic peptides while at the same time resembling one another by possessing a large number of common tryptic peptides. Although the genetic basis for the structural variation existing in polypeptide chains of the same type is not understood, it is chemically accounted for by changes in amino acid sequence (Hood et al., 1966; Perham et at., 1966, Gray et al., 1967). In the K-type light chains, it has been shown that amino acid variations are predominantly found scattered in the amino terminal half of the chain; the carboxyl terminal parts of K chains are similar in sequence. In the heavy chains, the same general picture probably prevails, and although this is not yet supported by amino acid sequence data, the tryptic peptide map analysis of Fc and Fab fragments indicate the heavy chain is divided into a common region (Fc) and a variable ( F d ) region (Fig. 1). Papain Fc fragments have been isolated from the yA-, yF-, yG- and yH-immunoglobulin classes in mice. Although it can be demonstrated that the heavy chain of each immunoglobulin class contains “distinguishing” tryptic peptides, the papain Fc fragments isolated from different myeloma proteins of the same class all produce the same tryptic peptide map. In the BALB/c mouse, we have found tryptic peptide maps of the Fc fragment for 2 yA myeloma proteins to be identical (Mushinski, unpublished operations) as have the maps for 6 yF, 5 yG, and 5 yH myeloma proteins. Further, the papain Fc fragments from myeloma proteins
FIG.2. Tryptic peptide maps of four types of BALB/c papain F c fragments upon which immunoglobulin determinants have been identified. Upper left and right are the G and H Fc fragments, arrows pointing right indicate common C and H tryptic peptides. Lower left is the y F F c fragment; arrow to left indicates common C and F tryptic peptides. Lower right is the S-carboxmethylated yA Fc fragment.
GENETICS O F IMMUNOGLOBULINS I N THE MOUSE
101
in the same class, have the same relative mobility in agar-gel electrophoresis which also helps in classifying the proteins (Potter, 196%). Tryptic p q t i d e maps of papain Fc fragments are, therefore, an effective means for classifying immunoglobulins (Fig. 2 ) .
I>. LOCALIZATION OF ALLOTYPICDETERMINANTS ON DIFFERENT IMMUNOGLOBULINS
Myeloma proteins from BALB/c inchding yM, yA, y F , YG, and yH types and free K- and A-type light chains (Bence-Jones proteins) were tested with a large number of homologous allotype antisera that precipitated with immunoglobulins in normal BALB/c serum. These specific antisera identified antigenic determinants on the yA, y F , yG, and yH myeloma proteins, specifically on their heavy chains in the Fc region (Dray ct al., 1965; Lieberman et a]., 1965, Lieberman and Potter 1966a; Potter et al., 1966). Allotypic antigenic determinants in BALR/c can now be assigned to specific heavy chains i.e., CY, 4, 7, and 7 (Table I ) . Thu$ far, no allotypes havc been found on light chains in the mouse. BY GENESOTHER E. MYELOMAPROTEINSCONTROLLED THAN THOSE FOUND IN BALB/c
To enable u s to compare immunoglobulin genomes in different strains of mice, it is important to assign any antigenic determinants found to a specific chain. We have only a few myeloma proteins from mice of other genotypes than BALB/c (Table I ) because plasma cell tumors are very rarely found in other strains or cannot be induced by the usual means (Merwin and Redmon, 1963; Potter, 1967a). However, it is possible to introduce genes controlling determinants not found in the BALB/c into the tumor-susceptible BALB/ c genotype by repeated back-crossing and by selecting progeny that carry the specific determinant. After several generations, it becomes possible to induce plasma cell tumors in the hybrids. For example, the unassigned immunoglobulin 2 determinant derivcd from the C57BL has been backcrossed into BALB/c for twelve consecutive generations (Tablr I1 ). At the sixth backcross generation, we mited backcross mice with each other and selected mice homozygous for the 2 determinant. Of forty such hybrids injected with mineral oil, sixteen have already developed plasma cell tumors. Tumors producing yA, ylF and yH myeloma proteins have been found. In other crosses, the genes controlling the 3 determinant from the DRA/2 and the 4 de-
102
MICHAEL POTTER AND ROSE LLEBERMAN
TABLE I1 SCHEMEFOR PRODUCING NEW PLASMACELL TUMOR-SUSCEPTIBLE TYPESOF MICE BY BAC~CROSSING DIFFERENT IMMUNOGLOBULIN GENESINTO BALB/c MICEORIGINOF BALB/c-2 Plasma cell tunior susceptible BALB/c
Plasma c e ll tumor resistant X
2/20
C57BLb
G1/G1 a C
G1/G1 C
(CX B)F,
I G1/G1 C
1
G1/GIC
G1/G1 C
tCbcl
X
G1/'+Cbc2
X
,.
Cbcnd
G1jz Cbcn
- - - - - .- .discard G1/l Cbcn
Continuous backc r o s s line, plasma cell tumor susceptible Cbcn parents of new plasma cell tumor susceptible s tra in 2/2
Defines antigenic determinant, see Table I V Symbols C = BALB/c and B = C57BL bc = backcross hc = number
terminant from AL have been backcrossed over six generations into BALB/c. Plasma cell tumors can be induced in some FI hybrids of BALB/c mice and other strains. Goldstein et al. (1966) have reported tumors in (NZB X BALB/c)F, hybrids, and we have observed them in several different hybrids (Potter, 1967a). Of particular interest, is the finding (Warner et al., 1966) that in neoplastic plasma cells originating in a host that is heterozygous for two different heavy-chain linkage groups, only one of the alleles appears to be used for immunoglobulin synthesis; the other is permanently repressed. It is possible, then, to recover the products of immunoglobulin genes not found in the BALB/c genome by backcrossing and hybridization to
GENETICS OF IMMUNOGLOBULINS I N THE MOUSE
103
BALB/c and inducing plasma cell tumors in the tumor-susceptible hybrids. Ill.
Preparation and Testing of Homologous Antisera
A. GENERAL In this section, the immunological methods used will be described. These differ from general immunological methods in only a few detailed respects but, nonetheless, it is essential to the discussion to describe the methods used as these reveal some characteristics of the allotype system, First, the immunization procedures with mice are of some interest. The mouse has not been as widely studied for its ability to produce precipitating antibody as have other species, and, especially, its ability to produce precipitating homologous antibody. Second, the methods for identifying polymorphic antigenic determinants vary in different laboratories, and this point requires some discussion. OF ALLOTYPEANTISERAWITH B. PREPARATION NORMAL IMMUNOGLOBULINS Washed “immune bacterial agglutinates” carrying the donor’s immunoglobulin are used uniformly by this laboratory to prepare homologous precipitins to immunoglobulin determinants. Whole serum has been used but usually evokes a poorer and delayed antibody response. Agglutinins to Proteris mirabilis are prepared in a selected inbred strain of mouse by immunization with 3 to 4, 0.5 ml. intraperitoneal injections, each containing approximately lo9 heat-killed organisms given at 3 to 4 day intervals. Immune agglutinates are prepared by mixing 0.1 ml. of anti- Proteus mirabilis antiserum with 0.1 ml. of packed P. mirabilis organisms. The mixture is allowed to stand at room temperature for 15 to 20 minutes, and the suspension containing the clumped organisms is centrifuged at 2000 R.P.M. for 10 minutes. The supernatant is discarded, and 0.85%saline is added to the packed organisms and mixed. The washing of the clumped organisms (agglutinates) is repeated three times. These immune agglutinates carry predominantly the 7 S (yF, YG, and yH ) immunoglolmlins of the immunized donor. Selected strains of mice are injected subcutaneously in several lymph node draining areas with a 0.1 ml. of the mixture of equal parts of complete Freund’s adjuvant and immune agglutinate (carrying the donor’s immunoglobulins ) . Three similar injections, one in incomplete Freund’s adjuvant and then two more without adjuvant are given at weekly intervals. Mice are bled f3-7 weeks after the first immunization injection, and the antisera are tested
104
MICHAEL POTTER AND ROSE LIEBERMAN
in Ouchterlony plates for precipitation with the donor’s serum. If antibody is not found, two additional subcutaneous injections of immune agglutinates without adjuvant are given. Thereafter, boosters are given whenever antibody begins to disappear. Whole-serum (Dubiski and Cinader, 1963; Herzenberg et at., 1965) precipitated 7-globulin ( Wunderlich and Herzenberg, 1963) , immune precipitates ( Gengozian and Doria, 1964), and allogeneic homologous anti-red-blood-cell ( RBC ) agglutinates ( Herzenberg 1964, Herzenberg et al., 1965) have also been used to produce homologous allotype antisera, but many of these fail to consistently evoke strong precipitating antisera. All of these antigen preparations contain mixed immunoglobulins in varying proportions. C. PREPARATION OF ALLOTYPE ANTISERAWITH MYELOMA IMMUNOGLOBULINS
Myeloma proteins have also been used to prepare homologous antisera and are especially useful since defined antigens can be used to immunize recipients. Recipient mice of selected strains are injected directly with myeloma . are proteins. Subcutaneous injections varying from 50 to 150 ~ g each given using the same regimen as is employed for bacterial agglutinates. Myeloma yG proteins evoke a strong antibody response in selected inbred strains and frequently require as little as 200 pg. for a total immunizing dose (Potter et al., 1966). This is also true of yA myeloma proteins but to a lesser degree. Myeloma yH proteins are poor immunogens and frequently require many injections, often 900 pg. of protein is injected before antibody is elicited. This is also true for y F myeloma proteins but with these, an even more intense immunization is rarely successful in inducing anti-yF antisera.
D. TESTING OF HOMOLOGOUS ANTISERA
1. Identification of Determinants Before describing the criteria for defining a determinant, it is necessary to describe some important characteristics of the homologous antisera that are produced by immunizations with immune agglutinates or myeloma proteins. Some antisera are highly specific and contain antibody to one determinant; the specificity of these antisera cannot be altered or partially changed by absorption. Further the specificity of the antiserum does not change during progressive immunization of the
GENETICS OF IMMUNOGLOBULINS IN THE MOUSE
105
mouse from which it is derived. Antisera with these characteristics can be used to define determinants. The majority of homologous antisera produced by immunization with immunoglobulins are polyspecific. These antisera may identify more than one determinant on a single immunoglobulin or may recognize determinants on two different immunoglobulins. Characteristically, the specificity of these antisera can be partially changed by absorption. In addition the specificity of this type of antiserum often changes with progressive immunization. A determinant is defined by the distribution of the antigenic determinant in question among a group of genetically different inbred homozygous strains. Monospecific antisera present no difficulties in assigning determinants, whereas polyspecific antisera that identify more than one determinant require absorption before the specificity can be determined and the determinants assigned.
2. Oitchterlonj Aiztilysis A diagram of a typical micro-Ouchterlony plate illustrating the antigens used in establishing the identity of an immunoglobulin determinant in this laboratory is given in Fig. 3. Wherever an antiserum identifies a determinant in the BALB/c and other strains presumably having thc same determinant, the antiserum is further tested in Ouchterlony plates to determine the specific immunoglobulin molecule as well as the region of that molecule bearing that determinant (Fig. 4 ) . Sevcml myeloma proteins of each class (Table I ) indicated in Fig. 4 iire roiitinely testcd for precipitation with the antiserum in question. Wherever a determinant has been identified that is not present in RALB/c or related strains and until a myeloma protein carrying the determinant can be obtained from the moiise strain in question, the determinant cannot be assigned to n specific immunoglobulin class. In those instances, immunoelectrophoresis can be used for identification. Characteristic precipitin arcs produced by the homologous antiserum reacting with its reference antigen are used to establish that the antiserum precipitates proteins in serum that are immunoglobulins. These arcs have the same shape and electrophoretic distribution as the arcs produced by similar systems where both the specificity of the antigen and the antibody are known (Lieberman and Dray, 1964; Potter and Lieberman, 1967). The specificity of some antisera can bc increased by iin absorption
19
24
31
36
FIG. 3. Model of a n Ouchterlony plate showing the procedure used to identify an immunoglobulin determinant. The homologous antiserum ( ) and the antigen (donors serum or myeloma protein) ( * ) are placed in alternate wells of the center row as indicated in each diagram. The precipitin bands between these alternate wells serve as reference bands. The top and bottom six wells of each diagram are filled with sera from the inbred strains of inice indicated. The inbred strain is considered to have the same determinant as the donor strain if the prcc:pitin hands produced by the donor strain coalesces with that produced by the strain in question. The distribution of the precipitin bands among the thirty-eight inbred strains is noted and identifies the specific determinant found in these strains and the donor strain.
+
GENETICS OF IMMUNOGLOBULINS IN THE MOUSE
107
PC-6A
@O PC-6A
000 MOW141
MOPC.21
FIG. 4. Model of an Ouchterlony plate showing the procedure used to identify the immunoglobulin carrying the specific determinant. Specific myeloma proteins from BALB/c are placed in the wells indicated. The homologous antiserum ( + ) and the antigen (donors serum or ntyelon>aprotein ) are placed in wells as indicated. Wherever a precipitin line occurs with a specific myeloma protein that coalesces with the precipitin line produced with the reference antigen, that immunoglobulin molecule is considered to carry the specific determinant that has been identified. The homologous antiseruni is further tested with the heavy-chain fragments of specific myeloma immunoglobulins to complete the identification of that determinant.
procedure. The absorbent is placed first in the antibody well and then the antibody is added; those antibody molecules that are not trapped in the immune precipitate diffuse toward the antigen well. In principle, the antiserum is absorbed with a specific antigen. This absorption procedure has been found to be particularly effective when the antigenic determinants are located on different immunoglobulin molecules. When two determinants are identified by an antiserum, one giving a strong reaction and the other a weak one; the weaker can often be removed by absorption. There are many advantages to using direct visual methods for
108
MICHAEL POTTER AND ROSE LIEBERMAN
studying antigen-antibody interactions in agar gels. One can often establish by the number of bands if there are antibodies to more than one antigenic determinant. 3. lnhibition of Precipitation hlethod
Immunoglobulin antigenic determinants have also been identified by the inhibition of precipitation method. In this highly sensitive procedure, antisera that fail to precipitate in agar gels can be used (Herzenberg et al., 1965). The sensitivity of the precipitin-inhibition method depends upon the ability of dilutions of antisera ranging from lo-' to to quantitatively precipitate small quantities of antigens of high specific activity. Iodine-125, 10,000 cpm, is introduced into 0.01 pg. quantities of r-globulins; the equivalence ratio of an homologous antiserum (or reference antibody Abr) is determined by reacting dilutions of the antiserum TABLE I11 SCHEMEOF PRINCIPLES OF PRECIPITIN-INHIBITION METHOD" lZ6IRadioactivity in 10,000 y Reaction
+ +
I *Agr Abr I1 *Agr 0 111 *Agr Agt Abr (a) Agt = Agr (b) Agt - Agr ( c ) Agt f Agr
+
+
Precipit2ate
1 /+ 4
l*Agr 0
- Abr]
Supernatant 0 *Agr
+
+ -9
+
[Agt - Abr] [Agt - A h ] [*Agr - Abr]
+ [*Agr + Abr]
*A@, 100% of I1 *Agr, partial of I1 Ayt, 0% of I1
Key to symbols and abbreviations: * = '"I substituted; Agr = reference antigen; Agt = test antigen; Ahr = antiserum that precipitates Agr; I / = run at approxiniateIy equivalence ratios; II/= same as I/ for Agr; III/ Agt is introduced at varying concentrations ( a ) is complete resemblance, ( b ) is partial resemhlance, and ( c ) is no resemblance to Agr.
with 0.01 pg. quantities of ""I reference antigen (Agr). After 3 hours at 37"C., the Agr-Abr suspension is centrifuged at 10,000 g for 10 minutes and the precipitate is removed; the amount of precipitation is determined by measuring the radioactivity remaining in the supernatant. An antiserum may identify a number of antigenic determinants in the reference antigen. The identification of specific determinants is established by introducing another unlabeled test antigen (Agt) into the Agr-Abr reaction at slightly below equivalence. These molecules may compete with the labeled lraIAgr molecules for the available Abr molecules to
GENETICS OF IMMIJNOGLOBULINS I N THE MOUSE
109
form precipitates. Three possible results are observed; no inhibition of precipitation, complete inhibition of precipitation, or partial inhibition of precipitation (Table 111). Antigenic determinants are identified in cases where the test antigen removes a part of the antibody molecules [Table I11 ( IIIa)]. Here it is postulated Agr and Agt share common determinants and that the Agr possess additional determinants not shared by Agt. IV.
Distribution and Localization of Heavy-Chain Determinants
A. GENERAL Immunoglobulin determinants have been identified by two kinds of homologous antisera in our laboratory. Homologous antisera were prepared with either mixtures of normal immunoglobulins (immune agglutinates-see Section III,B immunization methods) or with myeloma immunoglobulins derived chiefly from BALB/ c. Homologous antisera prepared with normal immunoglobulins have identified many determinants not present in BALB/c mice, and, hence, we are unable to assign them to any specific immunoglobulin class. TABLE IV IMMUNOGLOBULIN ANTIGENICDETERMINANTS OF MICE IDENTIFIED BY AGAR-GELPRECIPITINREACTIONS Strains
-
1 , 6, 7, 8
9, 1 1
12, 13, 14
1, 6, 7, 8
9, 11 9
12, 13, 14 -
9, 11
-
3
-
4, 10 4, 10 5
-
8
6, 7, 8 6, 7, 8 7, 8
-
13
-
9, 11
14
10 2
BALB/c, BDP, BRSUNT, CBA, C3H/He, C57BR, C57L, C58, MA, PL, ST, STR, 129 DD C57BL, C57BL/6, C57BL/10, NBL, HR, LP, SJL, Shl, STll/l DBA/l, DBAIL, I, RF, RIII, STOLI, SWIl, YBR A/He, A/d, AKIt, AL BL CE, UE, N H
At the present time, we have identified fourteen immunoglobulin heavy-chain determinants in inbred strains and their distribution among thirty-nine inbred strains is given in Table IV. It has been possible to assign nine of these to yG-, yH- or YA-, immunoglobulins; five have not been assigned. The yF heavy-chain determinants present a special
110
MICHAEL POTTER A M ) ROSE LIEBERMAN
problem and have not been included in TabIe IV but are described in Section IV,E. The notation system for immunoglobulin determinants used in our laboratory will be the one employed here. Essentially, when we have assigned a determinant to a specific chain, we have used a letter designation. For determinants assigned to the y heavy-chain, we have used G ; for the 7 chain; H; for the (Y chain, A (Table I ) ; and for the 4 chain, F. Consecutive numbers represent determinants as they are identified and are not speciated for G, H, A, or F. Thus a number may be assigned to two different letters, as is the case for the determinant 8 which is found on both the G and F immunoglobulins in BALB/c. (The symbols for the immunoglobulin genes G should be written Ig-G; for the sake of brevity, we have omitted the Ig prefix.)
B. YG HEAVY-CHAIN DETERMINANTS Four yG heavy-chain determinants, 1, 6, 7, and 8 have been identified among the inbred strains. As may be seen in Table IV, these are widely distributed among the thirty-nine inbred strains tested. The donor-recipient combinations used to prepare the homologous antisera that identify the four yG determinants are given in Table V. We shall TABLE V HOh~fOLOcoUs DONOR-RECIPIENT COMUINATIONS USED TO PREPARE ANTISERATHATIDENTIFY G DETERMINANTS Determinants on immunoglobulins of donor strain
Determinants on recipient strain
G Determinants 1, 6, 7 , 1, 6, 7, x I , 6, 7, X 1, 6, 7 , 1, 6, 7, 8
G Strain
Determinants
C58 DD BALB/C R.1
6, 7, X
ST
Y
7, 8
x
Determiiiaiits irlentified
Strsiii
.iL SM 3H DU.\/l
Lt F
7,8
NH
CRiBL
8
DBil/B
C57BL/10
1, 6,7 , 8
BALB/c $3"
LP
G1 (:
1
(:1
(;I GI
{ C:
I
+CX + Gti + (:7 +
~~8
G8 G1
+ G6 + G6 + '27 G I + (:6 + (;7 + C;8 G1
" Imiiirinogen was a BALB/c yG-type Inyeloma protein; in d l others, ilninune agglutinates were used.
GENETICS OF IMMUNOGLOBULINS I N TRE MOUSE
111
describe the determinants in some detail and show how the homologous antisera are prepared and how the determinants are identified. The G1 determinant is found in only fourteen of the thirty-nine inbred strains and has the narrowest distribution among the G determinants. Antisera specific for this determinant are very diacult to prepare since most recipients immunized with immunoglobulins carrying the G1 determinant make antisera simultaneously against the other G determinants that are more widely distributed. Antisera harvested in the early phases of some immunizations have proven to be the best source for this specific type of antiserum. For example, we have succeeded in preparing antisera specific for the G1 determinant in the early phases of some immunization of strain AL with C58, SM with DD, and NH with BALB/c immunoglobulins. These same combinations more frequently produce polyspecific antisera in later phases of immunization. Fw each donorrecipient combination, immune agglutinates carrying the donor’s immunoglobulins were used as immunogens. Anti-G1 antisera can also be prepared by absorption of specific isoantisera, e.g., AL anti-C58 antiserum absorbed with either a specific BALB/c myeloma protein ( yH MOPC 141 Fc fragment) or with a normal serum from a specific strain; namely, C57BL/6 (Fig. 5 ) . Homologous antisera identifying G6, G7, and G8 determinants are more easily prepared. The G6 determinant is identified by an antiserum prepared by immunizing strain DBA/1 with immune agglutinates from strain MA. This antiserum, like all the others described in this section, precipitated normal BALB/c serum and when tested with the different BALB/ c myeloma proteins, precipitated only the YG forms and the Fc fragments of the yG myeloma proteins. Normal sera from nineteen different strains were precipitated by this same antiserum (Table IV), and these strains are considered to have the same determinant, designated G6, found in BALB/c. I n another combination involving a recipient that lacks G6; namely, in RF immunized with immune agglutinates from a strain ST which has the G6 determinant, isoantisera are sometimes produced which precipitate the normal sera of the same nineteen inbred strains. More frequently, antisera from this same combination will precipitate the sera of the same nineteen strains and also the sera of three additional strains. Such an antiserum defines the G7 determinant and is found in twenty-two of thirty-nine strains (Table IV). It has not been possible to absorb an R F anti-ST antiserum (Table V ) with normal serum from a strain having only the G6 determinant to obtain an antiserum that identifies only the three additional strains. It, therefore, must be assumed that an isoantiserum such as the one described identifies both the G7 and the G6 determinants (Table V ) .
FIG. 5 . Identification of G1 and H9 determinants. Ouchterlony plates showing precipitin reactions of an homologous antiseruni 2684 ( + ) prepared in an AL mouse immunized with C58 immune agglutinates. The antigen wells contain the following: ( * ) C58 serum; ( - ) BALB/c myeloma yG Fc fragment; ( # ) BALB/c myeloma yH Fc; ( = ) BALB/c myeloma y F Fc (MOPC 21 ); and the sera from different inbred strains in wells numbered 1-38 (see Fig. 3 and Table I V ) . Precipitin reactions of antiserum 2584 after alxorption with BALB/c yH Fc (MOPC 141) and with C57BL/6 serum are also shown. The unalisorliet1 antiserrm identifies inimunoglobulins in all strains (except A/He, AKR, AL, and B L ) and on yC and yH Fc fragments. Thus this serum identifies G1, H9, and H11 determinants. Absorption with MOPC 141, yH Fc, fragment removes the antibodies identifying yH determinants and the resulting antiserum identify only the G1 determinants. C57BL/6 normal serum also removes the yH reactivity and the strong G1-specific antibodies remain.
GENETICS OF IMhfUNOC,LOBl.ILINS IN THE MOIISE
113
In another combination involving two strains, C57BL/6 and NH, neither of which carries the G6 determinant, immunization of C57BL/6 with NH immune agglutinates also produces an isoantiserum that identifies the G7 determinant in the same group of twenty-two mice (Tables IV and V ) . In more advanced phases of this immunization, the serum of eight additional strains are precipitated; this antiserum defines the G8 determinant (Table IV). Thus, R F anti-ST identifies both G6 and G7 determinants; C57BL/6 anti-NH identifies both G7 and G8 determinants (Tables IV and V ) . Antiserum identifying the G8 determinant may sometimes be shown to also identify the G7 determinant by absorption. For example SM antiBALB/c antiserum (Table V ) absorbed with DBA/2 carrying the 8 determinant will be specific for the G7 determinant; absorption with AL or CE serum will remove all the antibody reactions. On the other hand, SJL anti-DBA/2 antiserum (Table V ) only identifies the G8 determinant and not the G7 determinant but also identifies the unassigned 3 determinant (this can be demonstrated by absorption with BALB/c serum). When strain L P mice lacking G1, G6, G7, and G8 determinants are iminunizvd with BALB/c yG myc>loma immunoglobulins which carry these four determinants, antisera of different specificities are produced. Some antisera recognize only G1 G6, others G1, G6, and G7, and still others G1 G6, G?, and G8.
+
+
DETERMINANTS C. yH HEAVY-CHAIN
No homologous antisera prepared thus far with mixtures of normal immunoglobulins ( immune agglutinates ) recognize determinants exclusively on the yH BALB/c myeloma immunoglobulins. All these antisera have been prepared in either A/He or AL mice with immune agglutinates from inice with the G 1 determinant, and will identify both the heavy-chain H9 and/or H11 and G1 determinants (Tables IV and VI). Figure 5 shows an example of an antiserum identifying determinants on two different immunoglobulins. An AL mouse 2684 immunized with C58 immune agglutinates identified determinants H9 and G1. It is possible to remove the antibodies identifying G determinants from these antisera by absorption with BALB/ c yG myeloma proteins to make antisera specific for the H9 and/or H11 determinants alone (Dray et a?., 1965). The two determinants, H9 and H11, have a wide distribution among the inbred strains (Table IV). The H9 determinant was previously reported to be present in the RL strain and was based up011 precipitation of a BL pooled serum, obtained from another laboratory in 1963, with an anti-H9 antiserum. Subsequently, we found that sera from many
FIG. 6. Identification of determinants by nntisern collected at different stages of in~munizntionfrom an individual mouse. Ouchterlony plates of precipitin renctions of two homologous antisern placed in wells indicated ( ). Wells indicated ( a ) contain donor sera; wells numbered 1 3 8 contain sera of inbred strains (see Fig. 3 and Table I V ) . Antiserum N227 prepared in a C57BL/6 mouse immunized
+
115
GENETICS OF IhlhfUNOGLOBULINS IN THE MOUSE
TABLE VI L)ONOR-RECIPIENT COAIUINATIONS USEDFOR ANTISERA
THATIDENTlFY H, A,
BALB/r i.:L. BALB/c i.a. BALB/c. i.a. BAL13/[*i.a. lJAl,l3/c* ?l€ n1.p.”
A/He A/He A/He A/He A/He
BAIA13/c y L i ni.p.* BALB/c ?A n1.p. BALU/c yA m.p. HAIB/c yA 1n.p. C57BL/G i.n. DBA/2 Lit. AL i.n. NH i.a. BL La.
AND
PHEPAHATION OF €iohro~o::ous UNASSIGNED IXIhlUNOGLOUULlN DETERMINANTS
None h’oiie BALB/c. y U m p . ly the G6 antl C7 tleterminmits; 3177 A/He inlti-C58 antiserum identified the H9, H11, and C;l rletenninants on day 330, and on day 342 identified the H11 and C1 determinants.
116
MICHAEL POTTER AND ROSE LIEBERMAN
FIG. 7. Identification of the H9 determinant by an antiserum 3146 preparcd in an AL mouse immunized with a BALB/c yH myeloma protein MOPC 195. Wells indicated ( ) have homologous antiserum, wells indicated ( * ) contain normal BALB/c serum, wells numbered 1-38 (except 4 ) contain serum of inbred strains (see Fig. 3), BALB/c myeloma proteins yG (MOPC 173), and yH (MOPC 195, MOPC 141, and MOPC 172) are in the wells indicated on the figure. This antiserum did not precipitate yF myeloma proteins (not shown),
+
GENETICS OF IMMUNOGLOBULINS IN THE MOUSE
117
Immunization of strain A/He or AL mice with BALB/c yH myeloma proteins ( MOPC-141, MOPC-195) produce antisera which are specific for heavy-chain yH determinants alone and eliminate the problem of having mixtures of antibodies to determinants on two different immunoglobulins. In general, it is difficult to prepare such antisera. The precipitin reactions of an antiserum prepared in an AL mouse immunized with MOPC-195 with the sera of thirty-eight inbred strains is shown (Fig. 7 ) . This antiserum 3146 identifies the H9 determinant; the reactions with other yH myeloma proteins and with a yG myeloma protein may also be seen. This antiserum did not precipitate any y F myeloma proteins. Homologous antisera that identify the H9 and H11 determinants, and absorbed with normal BALB/c serum, possess a curious, and as yet unexplained, capacity to precipitate specifically BALB/c yH myeloma immunoglobulins and normal ( y H ) immunoglobulins in the serum of 8 strains carrying the unassigned 3 determinant (Table IV). This finding suggests a similarity between BALB/c yH myeloma proteins and the normal yH proteins in genetically different strains that is not found in normal BALB/c yH proteins. D. yA HEAVY-CHAIN DETERMINANTS In assigning the y A-immunoglobulin determinants, several facts concerning the characteristics of yA-immunoglobulins must be known. First, in comparison to the other immunoglobulins, yA levels in the serum are apparently quite low ( Fahey and Sell, 1965); second, yA-immunoglobulins in some strains including BALB/c, AL, AKR, BRSUNT, CBA, BDP, and PL loose their antigenicity during storage and during repeated freezing and thawing. Reference samples must, therefore, be continually evaluated. Finally, the age of the mouse from which the reference sera are obtained is important. Adult mice of sufficient age, usually 2 months old or older must be used, since the concentration in a serum of young mice is so low as to often be undetectable. Particularly in strain AKR, detectable serum levels of 7A-immunoglobulins are only found in older mice. The immunizations used to produce specific yA antisera are given in Table VI. The assignment of the yA-immunoglobulin antigenic determinants is given in Table IV. In our previous assignment of yA determinants we separated strains PL, CBA, BRSUNT, and BL from strains BALB/c, BDP, C3H, C57BR, C57L, C58, MA, ST, STR, and 129. The PL, CBA, and BRSUNT strains differed from the others by lacking a determinant we called the A14
11s
MICHAEL POTTER AND ROSE LIEBERMAN
FIG, 8. Identification of the A12 determinant by an antiserum 2942 prepared in on AL mouse immunized with a BALB/c yA myeloma protein (Adj. PC-GA). Wells marked ( ) contain the antiserum, wells marked ( " ) contain normal BALB/c serum, and wells numbered 1-38 (except 4) contain sera from inbred strains (see Fig. 3); well 4 contains myeloma protein Adj. PC-GA. This antiserum (2942) did not precipitate yG, yH, or yM myeloma proteins of BALB/c (not shown).
+
GENETICS OF IMMUNOGLOBULINS I N THE MOUSE
119
determinant. This separation, however, does not exist when fresh serum is used. The sera of all these strains except BL are precipitated by an antiserum, e.g., 2942 prepared in an AL mouse immunized with myeloma Adj. PC-6A protein that identifies the A12 determinant (Fig. 8). The A14 determinant has been reassigned and is found in strains CE, DE, and NH and strains carrying the A12 determinant (Table IV). Fresh sera obtained on the day of testing from ten separate BL mice varying in ages from 4 to 12 months old failed to precipitate with antisera identifying the A12, A13, or A14 determinants. This was in contrast to previous results obtained with a pool of sera collected from several BL mice in 1963 and repeatedly frozen and thawed in the ensuing 3 years. This pool of sera continued to precipitate with the antisera identifying the A12 and A13 determinants. Until we can resolve this enigma we cannot assign the A12 or A13 determinants to the BL strain. The identification of any new yA determinants will be on the basis of reactions with fresh serum antigens. It is of considerable interest that the distribution of G1 and A12 determinants is the same among the inbred strains as is the G6 and A13 and the G8 and H9 determinants. Some plasma cell tumors of BALB/c mice produce a 3.9s yA protein that appears in the urine. This type of protein contains one K-type light chain and one chain (Potter and Kuff, 1964) and is called a ?A halfmer. We have prepared a number of homologous antisera to the halfmers Adj. PC-6C and MOPC 47A; none of these precipitate the normal sera from thirty-eight inbred strains but do precipitate the BALB/c halfmers MOPC 116, MOPC 88, MOPC 4G, MOPC 47A, and Adj. PC-6C irrespective of the halfmer immunogen used to prepare the homologous antisera. None of these halfmers are precipitated by antisera that identify the A19, A13, or A14 determinants. Only antisera prepared with Adj. PC-6A react with Adj. PC-6C halfmer and this may be attributed to a common myeloma protein specificity (see Section VIII). The two myelomas, Adj. PC-6A and Adj. PC-GC, were originally derived from the same mouse (Potter and Kuff, 1964). These results are described in detail ( Lieberman, Potter, and Mushinski, in preparation). Urine concentrates from hyperimmunized mice tested with the antihalfmer antisera give no precipitin reactions which may indicate that yA halfmers are proteins that are not liberated from normal cells. (Y
E. y F HEAVY-CHAIN DETERMINANTS The YF-immunoglobulins in mice have not as yet been shown to pos;ess unique polymorphisnis. Tryptic peptide maps of Fc fragments of
120
MICHAEL POTTER AND ROSE LIEBERMAN
y F myeloma proteins differ markedly from the YG and yH Fc pattern
(Fig. 2 ) (Potter, 1967). Composite maps show that a few peptides are shared, but for the most part, the peptides are nonoverlapping. This is in direct contrast to similarities in structure observed between the yG and yH Fc fragments (Potter et al., 1966). W e have previously reported that some antisera prepared with normal immunoglobulins ( immune agglutinates ) precipitated strongly with myeloma yG proteins and very weakly with myeloma y F proteins (Dray et al., 1965). At that time, we used the whole yF myeloma protein to test these antisera and did not attempt to determine if this precipitation would occur with the Fc fragments of the myeloma y F proteins. In agargel electrophoresis ( p H 8.2) the y F Fc fragments migrate far more TABLE VII DONOR-RECIPIENT COMBINATIONS USED IN PREPARATION OF HOhfOLOGOUS ANTISERATHATPHECIPITATE yF MOPC 21 Fc FRAGMENT A N D IDENTIFY F8 AND G8 DETERMINANTS Donor
net errniriants identified
Deterniiiiaiits
YG 1, 6, 7, 8 7, 8 1, 6, 7, 8 6, 798
8 8
I'
rF
Strain
8 BALB/c 8 NH 8 BDP 8 AKR 8 DBA/2 8 BALBlcyF m.p.n (MOPC 21)
Recipient *trail1
SM C57RL/6 SJL C57BLj6 SJL LP
YG
1, 6, 7 , 8 7,8 1, 6, 7, 8 6,7,8 8 8
yF
8 8 8 8 8 8
imp. = myeloina protein.
anodally than BALB/c yG or yH fragments (Potter, 1967), and when finally separated by this method, they can be considered to be essentially free of contamination. We have prepared papain Fc fragments from two y F myeloma proteins, MOPC 21 and MOPC 31C. These two fragments have similar electrophoretic distributions in agar gel and similar peptide maps. We have re-examined some of these antisera, and other antisera, and tested them against specific isolated y F Fc fragments (Table VII; Fig. 9 ) . Only the MOPC-21 Fc fragment was precipitated by these antisera. The data obtained indicate that the myeloma MOPC-21 y F and yG proteins have a common determinant. The determinants, designated G8 and F8, show the same distribution pattern among the thirty-
121
GENETICS OF IMMUNOGLOBULINS IN THE MOUSE
nine inbred strains (Table IV) and are identified on F c fragments of myeloma yG and y F proteins, respectively. Antisera to y F determinants are difficult to prepare by direct immunization with myeloma y F proteins. Thus far, we have only succeeded in immunizing one strain, LP, with a y F myeloma protein MOPC-21. As seen in Table VII, this antiserum behaves similarly to the antisera prepared with normal immunoglobulins and identifies determinant 8. Although the same distribution among the thirty-nine inbred strains is obtained for both G8 and F8, nevertheless, the great difficulty in obtaining
FIG.9. Precipitation of G8 and F8 determinants on Fc fragments of BALB/c yG (MOPC 173) and y F (MOPC 21) myeloma proteins, respectively. Ouchterlony plate showing the precipitin reactions of an homologous antiserum 313 ( ) (prepared in an SJL mouse immunized with BDP immune agglutinates) with the Fc fragments of several BALB/c myeloma proteins yG ( MOPC 173), yH (MOPC 141), y F (MOPC 21) and (MOPC 31C). Precipitin lines are formed with (MOPC 21) y F Fc and yG Fc and show partial identity; spurring may also be seen.
+
antibody to F8, and the ease in obtaining antibody to G8 determinant in the LP strain, suggests that some differences exist between these two determinants. (This is also shown by the spurring of the precipitin lines in Fig. 9.) An unexplained finding was that the antisera (Table VII) that identified the G8 determinant on MOPC 21 Fc fragment did not precipitate MOPC 31 Fc fragment. The tryptic peptides of Fc fragments of MOPC 21 and MOPC 31 are similar. This suggests genes controlling y F heavy chains are more complex and require further study. Possibly
122
MICHAEL POTTER AND ROSE LIEBERMAN
there are differences in MOPC 21 and MOPC 31 not revealed by tryptic peptide maps. Table VII lists the donor-recipient combinations in which the antisera were prepared that identified the G8 and F8 determinants. In general, these findings are supported by results obtained with heterologous rabbit, antimouse, immunoglobulin antisera ( Fahey et nl., 1964) where it has been observed that the yG and y F proteins share common antigenic determinants. Polymorphism in yF proteins in different inbred strains is demonstrable by a variation in electrophoretic mobility of y F papain Fc fragments derived from different strains (Herzenberg et al., 1967, Potter and Lieberman 1967). The BALB/c y F Fc fragments migrate more anodally at p H 8.2 than do those from C57BL. A y F producing tumor derived from the BALB/c-2 backcross (BALB/c mice in which the unassigned 2 determinant has been introduced) yields a y F Fc fragment of different mobility from any so far obtained from yF producing tumors of BALB/c origin. Herzenberg, using this electrophoretic characteristic, has evidence that'the y F gene is linked to the other genes in the heavy-chain linkage group. Preliminary studies have not shown that the genetic change which causes the electrophoretic variation also confers a special homologous antigenicity. This is another example of a genetic polymorphism to which we have been unable to produce an identifying homologous antiserum.
F. UNASSIGNED IMMUNOGLOBULIN DETERMINANTS Five determinants, 2, 3, 4, 5, and 10, have been found in sera of inbred strains that have not been assigned to specific immunoglobulins (Table 11). The donor-recipient combinations in which these antisera were prepared are given (Table VI ) . All of these five determinants are presumed to be 7 S immunoglobulin determinants since in immunoelectrophoretic plates the precipitin arcs have electrophoretic mobilities characteristics of 7 S immunoglobulins (YF, Y G or YH). Determinants G1, 2, 3, 4, and 5 have been shown to be controlled by five allelic chromosomal regions ( Lieberman and Dray, 1964). Homologous antisera to the 2, 3, and 4 determinants are the easiest to prepare in recipient strains having the G1 determinant. The 2 determinant differs from all other determinants inasmuch as antisera to this determinant alone can be prepared in mice having the 3, 4, and 5 determinants (Table IV). Antisera to the 3 determinant alone can also be made in mice having
GENETICS OF IMMUNOGLOBULINS IN THE MOUSE
123
the 5 determinant, e.g., DE anti-RF. However, in mice having the 4 determinant, all mice do not behave the same way when immunized with immunoglobulins carrying the 3 determinant. For example, AKR mice having the 4 determinant immunized with SWR carrying the 3 determinant will produce antisera that identify the 3 determinant alone. Another combination presumably of the same genotypes, namely, AL anti-DBA/ 2, will produce antisera that will identify the 3 and H9 determinants ( Table IV) . Recipient strains having the 2 determinants usually recognize the 3 and G8 determinants when immunized with immunoglobulins carrying the 3 determinant. Three types of antisera that identify the unassigned determinant 4 have been prepared. One type, e.g., BALB/c anti-BL (Table VI) identifies the 4 determinant. A second type identifies two unassigned determinants, 4 and 10. The 10 determinant is found in strains having the 4 determinant and also in one strain, DD, among the fourteen strains carrying the G1 determinant (Fig. 10; Table IV). The third type of antiserum in addition to the 4 determinant recognized other determinants that have been assigned to specific immunoglobulins. This is exemplified hy the N217 antiserum prepared in a C57BL mouse immunized with AL immunoglobulins ( immune agglutinates ) which identify the G8, G6, and 4 determinants. This can only be demonstrated by appropriate absorptions (Fig. 11). The unabsorbed antiserum identified determinant G8 which has a very wide distribution among the strains tested. When this antiserum was absorbed with normal serum from the DBA/2 strain, which contains determinants G8, H9, H11, and unassigned 3, the ability to recognize the G6 determinant remains. Hence the unabsorbed N217 antiserum identifies at least two determinants, G8 and G6. When this same antiserum is absorbed with normal CE serum, which contains G7, G8, H9, H11, and unassigned 5, only the ability to recognize 4 remains. Variation in recognition of antigenic determinants by an individual mouse at different stages of immunization is illustrated by mouse N227, a C57BL/6 immunized with AKR immunoglobulins, which recognized only the 4 determinant on day 48 following immunization and recognized several determinants, 4 and G8 and possibly G6 and G7 on day 166 (Fig. 6). An antiserum that identifies only the unassigned 5 determinant is very difficult to prepare. For example, BALB/c immunized with NH immunoglobulins rarely elicits precipitating antibody. The C57BL/6 antiNH combination, however, produces very strong precipitating antibody which identifies the 5 and G8 and G7 determinants. These antibodies appear to be equally strong for all the determinants, and it is difficult to
124
MICHAEL POTTER AND ROSE LIEBERMAN
FIG. 10. Identification of determinants 10 and 4. Onchterlony plate of precipitin reaction of antiserum 3484 ( + ) prepared in a BALB/c mouse immunized with BL immune agglutinates. Wells indicated ( " ) contain BL serum; wells numbered 1-38 contain sera from inbred strains (see Fig. 3 and Table IV).
GENETICS OF IMMUNOGLOBULINS IN THE hlOUSE
125
FIG.11. Identification of G8, and C6 and 4 determinants by a single antiserum. Ouchterlony plates showing precipitin reactions with homologous antiserum N217 ( + ) prepared in a C57BL/6 mouse immunized with AL immune agglutinates. The antigen wells contain the following: ( * ) AL serum; (-) BALB/c myeloma yG Fc (MOPC 173); ( # ) BALB/c myeloma yH Fc fragment (MOPC 141); ( = ) BALB/c myeloma y F Fc (MOPC 21); and the sera from different inbred strains in wells numbered 1-38 (Fig. 3 and Table IV). Precipitin reactions of antiserum N217 after absorption with DBA/2 show identification of G6 and 4 determinants; after absorption with CE serum, antiserum identifies 4 determinant alone.
126
MICHAEL POTTER AND ROSE LIEBERMAN
remove any by absorption without completely removing all determinants. Sometimes, however, specific antisera are obtainable from heterozygotes made from crosses of mice of two completely different genotypes (Table IV). For example, we were able to prepare an antiserum specific for the 5 determinant in a heterozygote of a cross of C57BL/6 and BALB/c immunized with NH immunoglobulin, The use of selected hybrids as recipients has considerable potential for the production of new types of homologous antisera.
G. FAILURE TO PRODUCE HOMOLOGOUS ANTISERAIN SOME DONOR-RECIPIENT COMBINATIONS Different donor-recipient combinations presumably of the same genotype do not necessarily recognize the same determinants, or for that matter, produce comparable strengths of antibody. For example, the BALB/c makes excellent antibody to the 2 determinant present in C57BL/6, whereas PL mice fail to recognize the 2 determinant in C57BL/6 mice. As far as we know, both these mice, BALB/c and PL, have the same immunoglobulin genotype. The MA mice presumably having the same genotype as BALB/c require much greater amounts of C57BL/6 immunogen to recognize the 2 determinant. Even then, only about half the immunized group will produce antibody. On the other hand, the MA mice are immunologically competent and have no trouble producing excellent antibody to the 4 determinant present in A L mice. These findings and others (Lieberman and Potter, 1966a) suggest genetic variations in the ability to recognize antigens and play an important role in the production of homologous antisera. V.
Comparison of the Results Obtained with the Inhibition of Precipitation
Herzenberg (1964) and Warner et al. (1966) have described sixteen immunoglobulin antigenic determinants in mice using both direct precipitation and the precipitin-inhibition methods (Table 111). The antigenic determinants described by Herzenberg and his group have been assigned to genetic loci designated Ig-1, the locus controlling the y heavy chain; Ig-2, the locus controlling the cr heavy chain; and Ig-3, the locus controlling the 7 heavy chain. The determinants are recorded as numbers, and for each locus, the first determinant identified is numbered 1 followed by consecutive numbers as determinants are identified. Thus Ig-1.1 denotes the determinant controlled by the y heavy chain gene, and the Ig-3.1 denotes the 1 determinant controlled by the heavy-chain gene. The 1 determinants of Ig-1.1 and Ig-3.1 are unrelated, In both
127
GENETICS OF IMMUNOGLOBULINS IN THE MOUSE
laboratories, the determinants identified and their distribution among tlic inbred strains, have been done for some of the same donor-recipient combinations, and it is possible to compare the findings. There appear to be seven determinants identified in both laboratories (Tab!e VIII ) . Thwe of the four yG determinants appear to be similar. Determinant G6 has only been identified b y our laboratory. Unessigned immunoglobulin CO&WAl3ISON OF
TABLE VIII ANTICENICDETERMINANTS DESCHIBED IN T H E
(:I)
(:I (;6 ( k i (;S ~
-.
-,A
YH
>(; Inimriiitrglol~riliiis~~
(1)) 1g-1 .10 -
Ig-1 . 1
hfOLJSE
lT~~:~srig~~etl
l n i n i i i i i : ) g l ~ ~ l ) ~ i i i i iIsn, i~iir ~ i r r o g I~ ~ I~ i i l iInimriiiogl~~l~uliiis~ i i s~ ~ (:I)
€19
HI 1 .-
Ig-1 . 2 Ig-1 9 1g-1 . (i
-
Ig-1 . 7 rg-1 .s
-~
-
. .-
(11)
(:t)
,112
~
-
-413
Ig-:il IK-3 2 lg-3 ::
,114
(1,)
(1))
2
Ig-1 4h Ig-1 :ih Ig-1 . 3 Ig-1 . I I h
~
-
)
4
.-
-
-
-
-
Ig-2. I Ig-2 .: