Advances in Insect Physiology
Volume 17
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Advances in Insect Physiology
Volume 17
This Page Intentionally Left Blank
Advances in Insect Physiology edited by
M. J. BERRIDGE J. E. TREHERNE and V. B. WIGGLESWORTH Department of Zoology, The University Cambridge, England
Volume 17
1983
ACADEMIC PRESS A Subsidiary of Harcourt Brace Jovanovich, Publishers London New York Paris San Diego San Francisco SBo Paulo Sydney Tokyo Toronto
ACADEMIC PRESS INC. (LONDON) LTD 24/28 Oval Road London NW1 7DX United States Edition published by ACADEMIC PRESS INC. 111 Fifth Avenue New York, New York 10003
Copyright 0 1983 by ACADEMIC PRESS INC. (LONDON) LTD
All Rights Reserved No part of this book may be reproduced in any form by photostat, microfilm, or any other means, without written permission from the publishers
ISBN 0-12-024217-6 ISSN 0065-2806
Printed in Great Britain at The Pitman Press, Bath
Contributors W. Henzel Department of Biology, University of Massachusetts- Boston, Dorchester, Mass, USA
G. J. Goldsworthy Department of Zoology, University of Hull, England
H. Lipke Department of Biology, University of Massachusetts- Boston, Dorchester, Mass, USA
M. Raabe Laboratoire de Neuroendocrinologie des Insectes, P. et M . Curie Universiti, Paris, France
M. Sugumaran Department of Biology, University of Massachusetts- Boston, Dorchester, Mass, USA V. B. Wigglesworth Department of Zoology, University of Cambridge, England
V
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Contents Contributors
V
Mechanisms of Sclerotization in Dipterans H. LIPKE, M. SUGUMARAN and W. HENZEL The Physiology of Insect Tracheoles V. B. WIGGLESWORTH The Endocrine Control of Flight Metabolism in Locusts G. J. GOLDSWORTHY
85 149
The Neurosecretory-Neurohaemal System of Insects; Anatomical, Structural and Physiological Data 205 M. RAABE Subject Index
305
Cumulative List of Authors
314
Cumulative List of Chapter Titles
316
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Mechanisms of Sclerotization in Dipterans Herbert Lipke, Manickam Sugumaran and William Henzel Department of Biology, University of Massachusetts, Boston, Dorchester, Massachusetts, USA
1 Introduction 2 The protomer-matrix transformation 2.1 Criteria for sclerotization 2.2 Kinetics of dimer assembly 2.3 Protein and nucleic acid synthesis 3 Composition and preparation of larval proteins 3.1 The cyclorrhapid integument 3.2 The nematocerid larval cuticle 4 Composition of sclerotized tissue 4.1 The cyclorrhaphid puparial case 4.2 The pupal cuticle of nematocera 4.3 The adult stage 4.4 The egg stage 5 Chemical mechanisms of cross-linking 5.1 Ring substitutions 5.2 p-Sclerotization 5.3 A combined pathway 6 Developments and prospects Acknowledgements References
1 3 3 5 9 14 14 36 38 38 47 49 51 51 53 60 71 73 75 75
1 Introduction
In register with expanding interest in the development of insects, examination of the insect integument currently extends to many processes leading to the synthesis, recycling, deposition and maintenance of skeletal tissue. As a consequence of simultaneous advances in chemistry, microsurgery, microscopy and genetics during the period 1930-70, the influence of the endocrine systems on the control of metamorphosis was established. In the course of these “Advances in Insect Physiology” Volume 17 (edited by M. J. Berridge, J. E. Treherne and Academic Press, London and New York. 1
v. B. Wigglesworth).
2
H. L I P K E e t a / .
early studies, the focus of each investigation was some broad aspect of cuticle development as expressed during pupation, pigmentation or bristle distribution, for example. In the current era, however, and in confirmation of the dictum “everything in the body (cell?) depends on everything else”, students of cuticle biology now assimilate detailed reports on highly specialized systems in the hope that a universal model will obtain. Unfortunately, in the pursuit of this ideal, major inconsistencies in developmental programmes, in enzyme localization, in functional group activity and in protomer composition have become apparent. This search for a generalized mechanism has persisted in spite of the acknowledged heterogeneity of structural components in the cell wall of prokaryotes and plants or in the connective tissue of vertebrates and invertebrates. Indeed, diversity in the biological and chemical aspects of peptidoglycan, lignin, melanin and proteoglycan structure supports a comfortable prosperity, not only among practitioners of these chemical arts, but in the publishing trade as well. The formation and disposal of hardened regions of the exoskeleton require precise integration of virtually all of the major synthetic and catabolic systems of the organism. In species where the trehalose-glucose-glycogen triad provides energy for skeletal development, more than 75% of the carbon can be transferred directly to the integument or consumed in side reactions fuelling deposition of the lamina (Lipke et al., 1965b,c; Ferrus and Kankel, 1981). When mobilization of resources attains this exceptional level, any one of a multiplicity of biochemical systems can be invoked as the key reaction in the formation of the sclerotized entity. Thus peripheral aspects of cuticle development are frequently presented as contributions to the mechanism of sclerotization, per se, when the issue would be better served by greater circumspection. For this reason the present discussion is restricted in two respects, taxonomic and biochemical, With respect to the biochemistry of hardening and bond stabilization, only those reactions contributing to crosslinking and the decline in chemical reactivity will be discussed in depth. Related processes dealing with the characterization of unconjugated phenols, epidermal transformations, gene activations, polymer resorption, endocrine secretion, haemolymph precursors, and wound metabolism will be left to other specialists. In focusing on the Diptera, clear phylogenetic limits are imposed with ample provision for ecological diversity and developmental patterns. Of the 105 species within the chosen taxonomic group, no more than a dozen examples will be taken as representative of the
M E C H A N I S M S OF S C L E R O T I Z A T I O N I N D I P T E R A N S
3
order. Many families of great economic and medical significance have been overlooked by cuticle physiologists on the basis of unavailability or inconvenience. The important families yet to be examined include the Simuliidae, Tabanidae, Gasterophilidae, Tachinidae, Glossinidae, Hippoboscidae and the Tephritidae, to name but a few. At least one species of each of the above families has been reared in the laboratory; lack of material, therefore, cannot be the cause of the neglect. For several reasons, gaps in comparative aspects of cuticle structure should be filled in the near future. Countries formerly dependent on foreign experts have developed indigenous research facilities manned by local personnel. The trend toward integrated pest control requires detailed information on the ecology of each insect-host couple, including the biology of the integument since each life stage is fashioned for a particular environment. On the assumption that safe and efficient pesticides can be developed targeted for those processes without counterparts in benign groups of arthropods or higher forms, unique features of cuticle development are sought as objects for the action of specific inhibitors. The immediate advantages to be derived from these practical exercises are of sufficient consequence to guarantee continued activity in this specialty extending not only to aspects requiring heavy investment in instrumentation but to field practice as well. With these acknowledged limitations concerning the scope of this review, a simplified classification of the Order Diptera is presented in Fig. 1. The phyletics are based solely on the “integumental wisdom” of those investigators choosing to exploit these few groups as experimental subjects. 2 The protomer-matrix transformation
2.1 C R I T E R I A F O R S C L E R O T I Z A T I O N Regardless of the life stage or body region, the changes accompanying crosslinking are easily detected at a superficial level. Although a precise ordering of events has not been realized, the products clearly differ from the reactants in physical and chemical characteristics. A full description of the sequence of reactions requires the delineation of the time-course of each reaction contributing to the completion of the sclerotized matrix. It would appear initially that these objectives would be easily accessible, since crosslinking takes from a few hours to several days to complete in the intact animal, and the process can be accelerated or retarded by manipulation of
4
H. LIPKE e t a / . Order Diptera
Cretaceous)
Suborder
(-108 years)
Vematocera Brachycero
/
+
Tabanomor pha \ A <silo morpha lnfraorder
Lower Oligocene (- 5 x 105 yeors)
\
Cyclorrhapha
' I
Drosophiloidea
Muscdidea
Superfamily
A
'\
Muscidae Family
Subfa mi Iy Callipiorinae
Fig. 1 Simplified phylogeny of the Diptera The Drosophiloidea typify the acalypteratae and the muscoidea represent the calypteratae.
the internal and external environments. In the species examined to date, however, technical difficulties have precluded presentation of a kinetic catalogue using chemical probes. Some of the steps, such as dimerization, for example, call for rapid flow procedure to accommodate time scales in the range of milliseconds or less. Endogenous crosslinkers of natural origin interfere with the monitoring of spin labels by displacement of the probe from the derivatized residues of the primary chain, or by the generation of new signals that interfere with the labelled reactants. When ratios of buried to exposed functional groups are followed during cuticle hardening it has not been possible to distinguish between chemical modification of the side chain of a particular amino acid residue and loss of reactivity due to internalization. The choice is complicated further by the possibility of more than one route to sequestration,
M E C H A N I S M S OF S C L E R O T I Z A T I O N I N DIPTERANS
5
since the transfer of a group to the interior of a globular construct is presently indistinguishable from the compression of fibrous members to form a bundle. A similar ambiguity accompanies the assignment of dehydration indices, since the contributions of each water species can vary with changes in cuticle density, particularly in the categories of non-solvent, immobilized, ice-like and bulk water. Integumental loci subject to crosslinking share properties in common although each manifestation may vary in magnitude and temporal order. Reactions characteristic of puparium formation have counterparts in adult sclerites, the egg shell and in hardened appendages of larval forms. To a degree dependent on the density and hardness of the tissue the criteria for sclerotization include: 1. Decreased solubility of proteins and lipids. 2. Addition of bridges between proteins and between proteins and chitin. 3. Increased molecular weight of the structural polypeptides. 4. Altered packing of protein and polysaccharide. 5. Sequestration of functional groups. 6. Declining response to agents mediating enzymatic or chemical cleavage. 7. Extrusion of water. 8. Reorientation of chitin fibrils. 9. Pigmentation (excluding defensive colorations). 10. Post-translational modification of primary structures. A number of reviews and technical manuals have dealt with various aspects of sclerotization and crosslinking (Horspool, 1969; Rogers, 1978; Andersen, 1979a; Guay and Lamy, 1979; Jungreis, 1979; Silvert and Fristrom, 1980; Miller, 1980; Brunet, 1980; Sherald, 1980; Roberts and Brach, 1981). The breadth of opinions attests to the vigour of this branch of insect physiology and anticipates both lively disagreement and substantial progress in the future.
2.2 K I N E T I C S O F D I M E R A S S E M B L Y The joining of two or more monomeric polypeptides by the introduction of a covalently bound bridging unit calls for chemical definition of the reactants and products, namely the structural proteins, the activated crosslinker, the electron carriers and the fused dimer. Identification of the two proteins participating in the initial stages of matrix formation constitutes the first step, followed
6
H. LIPKE e t a / .
by characterization of the type of residue subject to modification and finally the location of this residue in the primary sequence. If exposure of the susceptible residue is an additional factor in identifying the rate-limiting step, changes in configuration must also be taken into account. The joining of additional protomers to the dimer to form higher n-mers may be very rapid as in an apparent concerted mechanism and may procede by repetition of the dimerization process, although no evidence supports this contention. If indeed the dimer is transient in the route to a multichain complex, the initial bridge would be present in low concentration compared to crosslinks added subsequently and could be overlooked, especially if notably different in chemical structure and stability. In the following discussion it will be shown that sclerotized systems afford a variety of putative crosslinks strongly suggestive of high specificity with respect to participation in different stages of the pathway to oligomers. Although knowledge of the chemical structures is necessary for adequate description of the process, scheduling of the participants is of equal importance to the biosynthetic programme. From the temporal perspective, crosslinking consists of rapid substitution reactions of the order of lo-* to 10-los and slower processes consuming hours or days. The fastest component is associated with the formation of a covalent link between the bridge precursor and the first protein. The interval during which the second structural member is added to the product of the first reaction is also brief, although it may be significantly longer than the initial coupling reaction. The events preceding coupling constitute additional synthetic processes that contribute to sclerotization. In these cases the time scale for synthesis, transport and modification of the polypeptides is much greater than the bridging reaction, per se. Implicit in the elucidation of this slower component is the history of each of the two proteins when viewed in the context of the entire instar. In the simplest case, both proteins are synthesized and transported to the appropriate strata hours or days before crosslinking is initiated. On the other hand dimerization may require monomers of relatively recent origin. These two extremes can be adjusted to accommodate several intermediate conditions to satisfy both the insect and the investigator. The aromatic bridging unit, for example, may be activated by combination with a newly synthesized protein following which an older polypeptide is attacked by the reactive complex. To provide for the large excess of older to newer elements in the Dipteran tanning systems, the initiation step may utilize two pro-
M E C H A N I S M S OF S C L E R O T I Z A T I O N I N D I P T E R A N S
7
teins of recent origin, the resulting dimer serving as the nucleus for subsequent addition of a number of the less reactive older structural polypeptides in concert. In the context of sclerotization, classification of a protein as old or new may be difficult if post-translational modifications immediately precede participation of a preformed polypeptide in dimer formation. The transport of a protein to the reaction site is an additional factor in construction of the time table. An old protein migrating to the reaction locus is modified at least twice, first as a signal for transport to commence, and again on arrival at the site of crosslinking. Proteins such as calliphorin, lucilin, Drosophilin and their counterparts in other families clearly fall within this category (Wolfe et al., 1977; Thomson et al., 1976; Scheller et al., 1980; Roberts and Brach, 1981).
2.2.1 Phylogenetic considerations It is evident that these considerations play a major role in the choice of experimental material. Insects with incomplete metamorphosis synthesize large amounts of cuticle protein during sclerotization, thus an important tool for the study of synthetic mechanisms and reaction kinetics, namely tracer methodology, is compromised. In Dipterans the deposition of structural proteins is by no means uniform either on an anatomical or developmental basis, and tanning procedes with high or low rates of synthesis depending on the tissue and life stage. If the time course and reactants involved in sclerotization are to be described, long lived participants of ready availability are preferred. These requirements speak against the study of crosslinking in the egg, pupa or developing adult and emphasize the advantages derived from the use of the cyclorrhaphan puparium as an experimental subject. In the course of the maggot-puparial transformation, the bulk of the proteins of the ultimate instar are subject to conjugation with companion polypeptides or chitin-protein complexes. In the case of drosophilids, mutants blocked at discrete steps in the pathway to the finished puparial sheath are available. Control of temperature and moisture affords convenient routes to the synchronous development of cultures of the larger Calyptratae. In this group endocrine and nutritional factors are known in sufficient detail to plot the course of ion and precursor flux and a battery of inhibitors are available to block specific steps of matrix formation. In the selection of this model, however, a number of qualifications must be acknowledged. The coloration of the puparium combines two pathways of unknown
8
H . L I P K E eta/.
relationship, first the synthesis of benzenoid chromophor from the arylated amino acid residues of the crosslink and second from the deposition of melanoprotein. The dissociation of these pathways encounters major technical and conceptual difficulties. To some extent, this problem can be circumvented by restricting the search to the early white or amber-pink stages or by organ culture. The assignment of a protein to a given age class, furthermore, constitutes a problem of much greater complexity than originally believed. Whereas gross analysis for chitin and protein suggested that the near constancy of the protein-chitin ratio during pupariation precluded significant turnover, the evidence is no longer convincing with respect to the protein (Fraenkel and Rudall, 1947; Rudall, 1967). That the increase in the dry weight of the puparium is due principally to the dehydration and the deposition of bridging groups and melanin is unquestioned (Hillerton and Vincent, 1979). However, the redistribution of old protein and the synthesis of new polypeptides of unknown function is now well documented. Redistribution increases solubility by loosening non-covalent bonds between two polypeptides. Conditions favourable to dissociation of two chains joined non-covalently would be promoted by participation of one member of the pair in crosslink formation with a third protein (Mitra and Lawton, 1979). The displaced polypeptide may be retained by the integument or may be degraded by epidermal cathepsins (Knowles and Fristrom, 1967; Bautz ef nl., 1973; Deloach and Mayer, 1979; Katsoris et al., 1981). Both de novo synthesis and partial proteolysis, therefore, may be required for sclerotization without causing major changes in protein levels, solvent partition patterns or amino acid composition, and hence may be overlooked by the investigator (Hackman and Goldberg, 1971; Mayer et al., 1979). Inthe Cyclorrhapha the cuticle of the third larval instar is converted to the puparium by modification of both the non-glycosylated proteins and the polypeptide joined to chitin. Some of these proteins are synthesized and deposited in the cuticle early in the last instar and some immediately preceding sclerotization (Snyder et al., 1981). Within the group synthesized at the conclusion of the larval stage, when the maggots have ceased feeding and have migrated from the substrate, additional temporal subdivisions can be detected comprising (a) proteins synthesized during the hours just prior to the retraction of the head and (b) proteins synthesized during formation of the puparium. The destiny of the proteins falling within these two subclasses has not been established. A significant
M E C H A N I S M S O F SCLEROTIZATION I N DIPTERANS
9
portion are added to the puparial case, while others may be directed to the viscera or may initiate particular steps in the deposition of the pupal exoskeleton. In this taxonomic group, completion of the puparium and deposition of the pupal cuticle overlap to the degree that the two processes are not synchronous in head, thorax and abdomen (Roseland and Schneiderman, 1979; Madhaven and Madhaven, 1980; Utsumi and Natori, 1980a). When the entire animal is sacrificed to establish the rate of tissue synthesis, activity of a particular region of the body is confounded at the expense of information on the local economy of protein, nucleic acid and chitin (Pearson, 1974; Kiss et al., 1978; Mayer et al., 1979; White and Lassam, 1979). 2.3
PROTEIN A N D N U C L E I C ACID SYNTHESIS
If deposition of structural proteins was confined solely to that period of the last instar coinciding with food intake, synthesis of protein by the epidermis should undergo a major decline during the post-feeding and white prepuparial stages (Fraenkel and Bhaskran, 1973). By a number of approaches it has been established that such is not the case. Exclusive of fat body, carcass ribosomes originating principally from the epidermis remain more or less constant in number during puparium formation in C. erythrocephala (=C. vicina), thus the potential for polysome formation is retained throughout this interval (Sridhara and Levenbook, 1974). Polysomes prepared from cuticle scrapings purported to consist principally of epidermal cells incorporate [3H]-leucine into ribonucleoprotein when fortified with the pH 5 fraction from rat liver. The synthetic capacity of the polysomes was identical for six-day-old larvae and white (untanned) calliphorid pupae (Fragouli-Fournogeraki et al., 1978). Translation of 5-18s mRNA from 6-day larvae and prepupae on mouse liver ribosomes enabled Fragoulis and Sekeris (1975) to assess gross message synthesis as well as production of dopa decarboxylase, per se. As predicted by differences in ecdysone titres, a four-fold increase in enzyme production was evident in the prepupae judged by titres of specific antibody-precipitable enzyme. In contrast to dopa decarboxylase, total cell-free product including putative structural polypeptides declined only 20% at the commencement of pupation in keeping with the maintenance of appreciable synthetic activity (Table 1). Production of translatable prepuparial mRNA was at the expense of high molecular weight heterogeneous DNAlike RNA. The messenger precursor was recovered from nuclei of
10
H . L I P K E eta/.
TABLE 1 Total cell-free product and immunoprecipitable dopa-decarboxylase from 6-day larval and prepupal 5-18s RNA (from Fragoulis and Sekeris, 1975). The system contained 2.6pg of mRNA from the indicated life stage, mouse liver ribosomal subunits, the pH 5 fraction from rat liver and 3 FCi [14C]leucine. Immunoprecipitation was performed after 30min at 37°C with rabbit anti-dopa decarboxylase
mRNA source
Incorporation of I4C leucine into Total cell-free Enzyme product (A) protein (B)
White prepupae 6-7-day larvae
35000 k 2988 counts/min 250 + 23 48000 k 2111 88f11
B/A 100)
(X
0.70 0.18
epidermal cells at the onset of the maggot wandering stage which falls late in the last instar of C. vicina (Shaaya, 1976a,b). Uniform rates of protein synthesis are expressed by the correspondence of (a) incorporation indices from labelled precursors and (b) the amino acid mole ratios in the polypeptide. With the assumption that precursor pools remain relatively constant, and with normalization of the data to accommodate differences in specific activity of the isotopic building blocks, variations in the time-course of protein synthesis within an instar can be assessed. On the other hand, if the adjusted values for radioactivity are not in accord with amino acid composition, a change in the array of cuticle proteins is indicated during the third instar. When 18 labelled amino acids were administered individually to maggots of Sarcophuga bullata at the commencement of the third instar, major differences in amino acid composition and incorporation rates were evident (Table 2). Discordance of the two parameters was observed within classes of amino acids based on either polarity or nutritional dependence (Sugumaran and Lipke, 1982a). Disparate rates of synthesis could also be inferred from glycopeptide analysis. At least two polypeptides are appended to chitin at the conclusion of pupariation, one present during the larval stage with high levels of glycine in the vicinity of the unions between protein and chitin and a second group added during hardening of the puparium with glutamic acid predominating at these loci (Table 3). The component(s) added late in the process and enriched in glutamic acid could be a protein synthesized and stored prior to polysaccharide modification or could be translated later, at the onset of glycoprotein assembly (Lipke and Strout, 1972; Strout et al., 1976; Kimura et al., 1976).
11
M E C H A N I S M S O F SCLEROTIZATION I N DIPTERANS
TABLE 2 Incorporation of [14C] amino acids into proteins of sarcophagid puparial cases. Each larvae received 1 . s 2 . 0 X lo5dpm of labelled precursor at the commencement of the last larval instar and the puparium harvested following metamorphosis. Portions of the washed and powdered cases were hydrolysed for amino acid analysis and oxidized with HC1O4-H2O2for assessment of total incorporation per puparium (from Sugumaran and Lipke, 1982a) Precursor Tyrosine Lysine Histidine Glutamic acid Proline Aspartic acid Glycine Serine a-Alanine Valine Threonine Isoleucine Leucine Phen ylalanine Arginine Methionine Cysteine Tryptophane
Residue mol percent
percent
3.6 3.6 3.6 12.3 11.8 10.2 10.2 8.6 8.6 8.3 5.9 4.3 3.6 3.1 2.4 nil nil nil
10.0 3.4 8.1 1.2 2.3 1.4 1.4 1.3 3.1 1.9 2.5 1.7 0.9 5.2 8.1 1.4 3.0 5.3
1 4 c
TABLE 3 Mole ratios of amino acids from Sephadex G-10 glycopeptides in formic acid extracts of Sarcophagid cuticle; values expressed as residues00 mg of peptidochitodextrin (from Kimura et a l . , 1976) Fraction Larval LA-I LA-I1 LA-I11 White puparial WA-I WA-I1 WA-I11 Puparial PA-I PA-I1 PA-I11
Glu
Lys
GlY
GlY
0.31 0.23 0.19
0.17 0.28 0.08
0.34 1.47 0.88
0.14 0.44 0.45
1.94 1.77 2.94
0.81 0.62 0.61
12
H. LIPKE e t a / .
2.3.1 Methodology
Without detracting from the value of the maggot-puparial transition as a model system for following crosslink formation, the observations discussed above must be integrated into new proposals on the course of the phenomenon. Regardless of the species under investigation, the appearance of polypeptides with molecular weights in accordance with the dimensions of a dimer or trimer, must be viewed from the position that one or more of the polypeptides are synthesized de novo and may not represent a pre-existing structural member. From the standpoint of methodology, the relevant criteria should include not only classification on the basis of molecular weight, but also (a) the amino acid sequence of the candidates, (b) cross-reactivity with respect to the purported monomer, and (c) the kinetics of assembly of each of the polymeric components of the scheme (Mills et al., 1967; Strout and Lipke, 1974; Willis et al., 1981). 2.3.2 Preparation of larvalproteins
High performance liquid chromatography and two-dimensional polyacrylamide gel electrophoresis are the principal techniques currently favoured for resolution of integument polypeptides. These procedures separate proteins differing very slightly in amino acid composition and molecular weight, but unfortunately problems arise at the preparative rather than the analytical level. With dissociating or non-dissociating gels, thin slabs (0.1 mm) are clearly superior to thick ( l m m ) in resolving power as are analytical scale HPLC supports over larger diameter columns of high capacity. Given the need for unequivocal purification of a polypeptide for sequencing or complement fixation, the current trend is to chemical probes that respond to very low levels of protein recovered from a single narrow band on a thin gel. These new approaches have revealed minor isolates that play important roles in skeletal development. On the other hand, increased sensitivity and resolving power have introduced new uncertainties particularly with respect to microheterogeneity , contamination with non-cuticular protein and spurious variants arising from proteolytic cleavage during sample preparation. The ability of a pure protein to migrate to more than one region of a gel is troublesome on crowded gels and is most frequently caused by substandard ampholytes or protein-protein
MECHANISMS OF SCLEROTIZATION I N DIPTERANS
13
interactions (Cann, 1979). With respect to the presence of contaminating proteins extracted from intersegmental muscles, it may be necessary to establish the cuticular origin of a minor component by immunodiffusion against antibodies to pure muscle proteins (Crossley, 1968). Proteolytic or oxidative changes are minimized by the addition of phenylmethanesulphonyl fluoride and phenylthiourea. Most cuticle proteins exhibit solubility minima below pH 5. In situations calling for minimum exposure to inhibitors, repeated extraction at p H 4-5 under an inert atmosphere in the presence of ascorbic acid can reduce artifacts to acceptable levels. At this acidity cuticle proteins are minimally soluble whereas visceral components pass into solution. A wide choice of equipment and procedures is available for processing both small and large samples. Maggot sheaths can be scraped to release epidermal cells reasonably free of visceral fragments. Providing calliphorin and rel&ed proteins are removed by a preliminary sedimentation, the disrupted epidermal cells are a good source of nuclei, ribosomes and mRNA (Sekeris et al., 1974; Fragouli-Fournogeraki et al., 1978; Sumner-Smith and Phillips, 1979). Fristrom and associates described procedures for the harvest of cuticle proteins applicable to a single Drosophila larva (Fristrom et al., 1978) or for numbers in excess of lo6 larvae (Eugene et al., 1979). Satisfactory preparations have been obtained by judicious operation of conventional blenders and Potter-Elvehjem or Dounce disintegrators, devices capable of considerable flexibility with respect to sample size and solvent volume. The best features of these two apparatuses are combined in the tubular disintegrator designed for Drosophila by Zweidler and Cohen (1971) and modified by Zomer (1978) to accommodate other species. As depicted in Fig. 2, oxygen may be excluded from the system and buffer programmes changed as required. Separation procedures based on attrition of softer organs are suspect with regard to the removal of loosely bound cuticular components along with formed elements from the body cavity. Comparisons of hand versus machine-dissected preparations usually resolve this uncertainty. A genuine need exists for automated methods of separation of individual layers of the cuticle. The practice of retrieval of exuviae for sampling epi- and exocuticular material is difficult in the case of Dipterous larvae inhabiting semi-solid media due to poor visibility, exposure to alkaline pH, and the likelihood of partial autolysis by digestive enzymes secreted into the diet.
14
H. L I P K E e t a / .
I c D E F I Fig. 2 Continuous feed automatic dissection device for dipterous larvae. (From Zweidler and Cohen, 1971; Zomer, 1978) A. Buffer chamber; B. Insect container; C. Mixing chamber on magnetic stirrer; D. Zweidler conical disruptor; E. Second homogenizer; F. Chilled sieving system on magnetic stirrer.
3 Composition and preparation of larval proteins
3.1
THE CYCLORRHAPHID INTEGUMENT
Washed maggot sheaths extracted with 6-8 M urea; pH 8.6; 4-8 M guanidine or 2% NaZB407 containing 0.2-2.0% sodium dodecyl sulphate (SDS) at temperatures ranging from 2°C to 60°C release approximately 50% of the cuticle protein to the solvent. Precise information on the relative solvating ability of the three media has not been forthcoming, usually the proteins can be transferred from one solvent of this group to another without significant loss of individual components. When the soluble proteins are separated by electrophoresis under conditions favouring dissociation it is evident that the bulk of the proteins fall within the molecular mass range between 9 and 30kd with minor components in excess of 50kd. Isoelectric points (PI) range from p H 4 to 6 as assessed with ampholytes poised at these acidities. Unlike the Blattids, the proteins are not conjugated with lipid or carbohydrate and are
M E C H A N I S M S OF S C L E R O T I Z A T I O N I N D I P T E R A N S
Volume eluate ( m i )
'
~
15
1
Fig. 3 Molecular sieve chromatography of proteolytic digests of cuticle from larvae and puparia receiving [7 - I4C] dopamine prior to pupation. Borate extracts of the two cuticles were digested serially with S. aureus V8 protease, subtilisin, Pronase, trypsin carboxypeptidase A and pepsin and chromatographed on Bio-Gel. A, larval; B , amber puparial cuticle. (From Lipke et a / . , 1981)
devoid of pigment if prepared and stored in the presence of reducing agents or copper chelators (Lipke et al., 1965a, 1981; Snyder et al., 1981; Silvert and Fristrom, 1982). Little differences in electrophoretic patterns are manifest when second, early third or first instar sarcophagid proteins are compared. At least one of the genes coding for structural proteins in Drosophila is expressed later than the other four, however (J. W. Fristrom, personal communication). Despite similarities in PI and composition favouring strong noncovalent interactions, shielding of hydrolase-susceptible peptide bonds is not as strongly expressed as in puparial extracts (Lipke and Geoghegan, 1971a). Figure 3 presents the molecular weight distribution of peptides generated from larval and puparial proteins by proteolytic digestion with enzymes specific for linkages of relative abundance in the protein mixture (Lipke et al., 1981). It can be seen from elution pattern A that the bulk of the fragments chromatographed in accord with standards corresponding to amino acids and oligopeptides (0-2-1 kd). When drosophilid proteins were assessed for relatedness by peptide fingerprinting, all the isolates were cleaved by Staphylococcus aureus protease V8 and several afforded fragments of apparent identity (Silvert and Fristrom, 1982). This enzyme cleaves almost exclusively at the C-terminal
16
H. L l P K E
et al.
aspect of aspartyl and glutamyl residues which account for 20-25% of the amino acids in structural proteins of this species (Fristrom et al., 1978). 3.1.1 Unresolved mixtures Amino acid analysis of protein mixtures provided intial stimuli for intraspecific comparisons and selection of reagents for group modification and cleavage, Definitive criteria for homologies, whether mathematical, chemical or immunological are based on properties of purified proteins and can be misleading when applied to unresolved mixtures. Table 4 compares an acalypterate dipteran, D.melanogaster, with three Calypteratae on the basis of amino acid composition. Larval proteins from the drosophilid appear related to the sarcophagid, whereas limited homology has been observed based on N-terminal sequences, total number of proteins or electrophoretic mobility (Mulligan et al., unpublished observations). Conversely, L. cuprina (a calliphorid) and D. melanogaster differ with respect to Asp, Ser, Pro, Ile, Phe and Met content in spite of pronounced immunological crossreactivity (J. W. Fristrom, personal communication). The nematoceran is strikingly similar to C. vicina and L. cuprina, phenylalanine content possibly excepted, whereas habitat and phylogeny are disparate. Comparisons of acidic proteins, furthermore, are suspect without amide titres. This value is 40% for the mixed cuticle proteins from S . bullata. Cuticle extracts comprised of unresolved protein mixtures have provided useful approximations of general properties if interference by companion polypeptides is appreciated. As a group, the proteins soluble in urea are responsible for the 41 8, axial repeat of diffraction patterns (Rudall, 1976). This reflection represents a regularity associated with every four chitobiose residues of a peptidylated chitin chain. Since substantial levels of protein remain attached to the polysaccharide after exposure of the sample to urea, covalently bound protein may be joined to the carbohydrate polymer at a different locus. The patterns could not distinguish between elimination of protein from the 41 8, system or conversion to a less ordered form incapable of generating a reflection at this repeat distance (Rudall and Kenchington, 1973). In this respect, assignment of a given protein to a specified locus in a three-dimensional construct of the cuticle matrix encounters artificial distinctions between structural polypeptides and proteins with enzymic functions. Based on recoveries of soluble enzymes of carbohydrate metabolism as well as
TABLE4 Amino acid composition of extractable larval cuticle proteins of selected Diptera. Values in residues per 1000 ~
Nernatocera" A. aegypti
ASP Thr Ser Glu Pro GIY Ala Val Ile Leu TYr Phe His LYS '4%
Met
80 39 82 117 60 114 132 58 24 46 39 112e 22 42 36
-
Calliphorab vicina
Luciliab cuprina
78.7 48.9 102.8 113.6 80.9 122.3 107.5 76.1 28.1 40.1 37.9 36.8 66.2 42.3 15.0 3.2
80.2 42.6 110.7 109.2 80.6 121.3 115.7 77.1 21.5 43.6 32.4 55.0 52.2 44.2 16.7 2.7
Cyclorrhapha SarcophagaC bullata
140.4 40.2 77.4 141.1 62.1 82.3 87.6 54.1 20.3 49.5 45.8 37.8 63.7 73.4 24.2 -
Drosop hilad rnelanogaster 121 50 66 120 55 99 103 92 51 46 48 28 47 47 26 -
N-termini Ile Val Ala GlY Glu
Asx 51.6% Val 18.4% Gly 14.3% Ala 8.7% Leu 7.0%
Zomer and Lipke (1981). Hackman and Goldberg (1976). Lipke et al. (1981). Fristrom er al. (1978). Includes glucosamine.
18
H. LIPKE eta/.
lysosomal hydrolases, a factor of lo2 probably represents the minimum for the difference in concentration between proteins with a metabolic as opposed to a support function in exoskeleton (Knowles and Fristrom, 1967; Bienz and Diek, 1978; Candy, 1979). However, this classification on the basis of metabolic function is debatable. First, there is virtually no information on the enzymatic activity within the structural proteins as a group, particularly with respect to regulation of ion flux. Second, phenolases, peroxidases and hydroxylases apparently have dual roles in sclerotization, since these enzymes are firmly bound to the matrix, thus constituting structural material while retaining high catalytic activity (Yamazaki, 1969; Locke, 1974; Andersen, 1979b; Barrett and Andersen, 1981; Sugumaron et al., 1982). 3.1.2 Configuration Assessments of secondary and tertiary structure have been attempted in mixtures of cuticle proteins. When prepared from C. vicina by extraction with 7~ urea, eleven components were observed in gels run under dissociating conditions, the polypeptides consisting predominantly of material of M, 14 000-16 000. Perturbation with 20% methyl alcohol or 20% dimethylsulphoxide at p H 6 failed to reveal sequestration of tyrosyl residues by uv difference spectra (Hackman and Goldberg, 1979). The procedure assumes the perturbant did not induce conformational changes in the protein altering the ratio of exposed to internalized groups (Donovan, 1969). Cuticle proteins from cyclorrhaphids are minimally soluble in 20% alcohol at pH5 suggesting this assumption may not obtain for this preparation. The ratio of buried to exposed tyrosyl has also been examined in unresolved samples of proteins from sarcophagid cuticle with the chemical probes, N-acetylimidazole and tetranitromethane (Riordan and Vallee, 1967; Lipke et al., 1981). Borate-SDS extracts containing 21 polypeptides of the same approximate molecular weight distribution as C. vicina were reacted with the acetylating agent for 40min at pH 8-6 and 25°C in the presence and absence of urea. Based on spectrophotometric assessment of the rate of 0-acylation in the two samples, the ratio of buried to exposed functional groups in the native sample was 1: 1. The chitin-bound structural protein on the other hand appeared less ordered, since essentially all phenolic hydroxyl was accessible to modifier (Hennigan and Lipke, unpublished observation). Both chitin-bound and unglycosylated samples were exposed to the borate-SDS extraction medium in the course of sample preparation discounting artifactual
MECHANISMS OF SCLEROTIZATION I N DIPTERANS
19
tertiary structure imposed by previous contact with and removal of detergent. Abundance of helix and p-sheet in urea extracts of Calliphora vomitoria integument varied inversely with the polarity of the solvent selected for measurement of circular dichroism (Hillerton and Vincent, 1979). With C. vicina, addition of less polar solvents was not required for stabilization of either helix or P-sheet (Hackman and Goldberg, 1979). The two investigations differed in selection of solvents and in methods of data processing. Both laboratories agree that cast films show evidence of P-sheet, in confirmation of X-ray analysis of dried material (Fraenkel and Rudall, 1947). The intractability of sclerotized cuticle for the isolation of protomers of larval origin has prevented comparison of primary structures before and after crosslinking. Protein modification as a prerequisite for protomer polymerization remains a strong possibility, particularly when analogies with skeletal material from other phyla are considered. The likelihood of one or more post-translational modifications in larval protomers coincident with pupariation challenges the validity of the assumption that extracts of larval cuticle represent the material ultimately used for puparium fabrication. Without exception, the literature concerning sclerotization accepts post-oxidative coupling of unmodified larval proteins as the rate-limiting step in dimer formation. That this cannot represent the pathway in situ is evident from the absence of dimer and trimer from incubates of sarcophagid larval proteins, dopamine, oxygen and homologous phenolases. Fortification with metal activators, electron carriers, peroxidase, chitin and a wide variety of other stimulants fail to initiate crosslinking in vitro (Strout and Lipke, 1974). In view of widespread success in the resolution of protein mixtures from cuticle and in the development of non-invasive probes of intact organisms, there is little reason to continue experimentation with crude mixtures or to rely too heavily on information culled from these sources. 3.1.3 Drosophilid larval proteins (Superfamily Drosophiloidea) Mass rearing of synchronized cultures coupled with bulk processing enabled Fristrom and associates to prepare litre quantities of third instar larvae from which gram quantities of cuticle were harvested (Fristrom et al., 1978; Silvert and Fristrom, 1982). The urea-soluble proteins were estimated as eight in number, with a later upward revision to include two additional minor components as resolution on the gel was improved (Fig. 4). Five of the proteins coded with a
H. LIPKE e t a / .
20
40
30 20 its
. I I
-
-
-
6 -
I
II
*
+ Fig. 4 Electrophoretic separation and isolation of purified cuticle proteins from Drosophila melanogaster. Upper slab in absence of urea. Lower slab, proteins dissociated with sodium dodecyl sulfate. (From Fristrom, Hill and Watt, 1978)
36 kd DNA segment at chromosomal region 44D, with four of the five falling within 7.9kd of DNA (Snyder e l al., 1981). At least four members of the tetrad cluster were activated for transcription early in the last instar while the fifth component, 8 kd removed from this group was not expressed until later (Chihara et al., 1982).
M E C H A N I S M S O F SCLEROTIZATION I N DIPTERANS
21
Cloning was facilitated by the abundance of poly(A) RNA for structural proteins in late third instar epidermal cells compared to embryonic poly(A) RNA which is virtually devoid of this transcription product. The four major proteins were sequenced and the primary structure confirmed by parallel sequencing of the corresponding cloned genes. CP1 and CP2 are homologous except for residues 1-9 and 12 of CP1, and residues 1-5 and 8 of CP2. CP3 and CP4 are 85% homologous; their interior sequences show homology to CP1 and CP2. The proteins were absorbed quantitatively by deproteinized chitin and could be eluted with urea. None were glycosylated to a significant extent although pupal exoskeleton could be fractionated to resolve polypeptides with 1 to 7% neutral carbohydrate appended. Table 5 presents the amino acid composition of the larval isolates calculated for the sequence. It is evident that Asx and Glx constituted about 25% of the total residues, if Ala, Gly and Pro are included, over half the amino N could be accounted for. The low levels of non-polar components together with the abundance of charged residues imply few if any hydrophobic interactions could be accommodated (Fig. 5). The proteins are compact with few pleated sheets and an even distribution of helical and p reverse turns (Fig. 6). Based on electrophoretic mobility, variants of all of the proteins (except CP4) were readily detected, almost invariably associated with heterozygosity. Gel separations could not detect differences between cuticle proteins from the sibling species D.melanogaster and D.virilis, although haemocyte involvement during pupariation may vary within the genus (Rizki and Rizki, 1980). In support of the information on transcriptional differentials discussed previously, proteins of the third instar were separated into early and lately synthesized proteins. This distinction was not manifest in the first and second instar (Chihara et al., 1982). 3.1.4 Larval proteins from the Superfamily Muscoidea Isoelectric focusing of urea extracts of C. vicina separated twelve proteins with PI 44-5.4 and M, 14-16 kd. N-terminal analysis of the mixed proteins by dansylation indicated Ile, Val, Ala, Gly and Glx in this position (Table 4), mole ratios were not specified (Hackman and Goldberg, 1976). Ampholytes poised from pH4-0-6.0 were exploited for resolution of sarcophagid proteins extracted with borate-SDS buffers instead of urea. A minimum of 20 proteins were identified by staining with Coomassie Blue or precipitation with ammonium sulphate in situ on the gel (Lipke et al., 1981).
i.-n
1
CP1 CP2 CP3 CP4 CPX CP2a # 5 64.68
N
CP1 CP2 CP3 CP4 CPX CP2a X 5 64.68
(D
P
P
V
P L
H A
S P
L V
G R S D V S [ R l E l O V I N I A (N IN E)
(N (
H ( A ) D ( V ) L S R H /A) D /V) L S R N V ElIVIIK E L N E&E (A) D (V) V (K1 (S) L
E N
D E
D
A N A N
V) V
20
CPI CP2 CP3 CP4 CPX CP2a # 5 64.68
I
D
D
v)
A
A
R
H G H G
A I L ) K E m (L ~ N ) G (? E ?
CPI CP2 CP3 CP4 CPX
V
A
N
K
V
CP1 CP2 CP3 CP4
A A
R R
A
(D)
N N
40 I I
H
D
E
G
(F)
D
H H
G G
N N
N [ I
W
V
E
w v
s
V D V
D
D]
N (S) N
R K
A E Q S Y S )
A A
A A
30
H H
T T
s
W W
I I
G
E I
S S S S Y
P P P P E I
s ILI
F F
G G
V
S
v
L
S S
K
G
a
S
(N) G (N) G
50 E G E G E G E G Y V
w
(I) (I)
E E V E A
E E
H H H H P
a 0
s (GI S
(G)
V E V K Y l V E V K Y V V N G K T V
D)
V
E E E E Y
N N N N T
G G G G A
Y Y Y Y D
A A
V V
A A
W W Y Y
L L I I
7n 80 ._ Q P S G A W I P T P P P I P E O P S G A W I P T P P P I P E O P Q S D L L P T P P P I P m O P O S D l l P T P P P l P E E T G Y N P K ? V E A
90 E S H E S H W A N O A H P
A I A I A I A I
100 P P P S
P A P E H P A P E H S K NEnd K E E n d
P P
R R
H H
HEnd HEnd
Fig. 5 Primary structure of larval proteins from D . melanogaster and S. bulluta. (From Snyder et al., 1981; Henzel, Mulligan, Mole and Lipke, unpublished results.) []-no residue; inserted to maximize homology.
23
M E C H A N I S M S O F SCLEROTIZATION I N DIPTERANS
TABLE 5 Amino acid composition of Drosophila cuticle proteins (unpublished results of Dr M. Snvder). Values in mole percent
ASP Thr Ser Glu Pro GlY Ala Val Ile Leu TYr Phe His LYS Arg Asn Gln TrP Acidic Basic Diff
CP1
CP2
CP3
CP4
6.1 1.8 9.6 7.9 12.3 8.8 10.5 7.0 5.3 3.5 1.8 1.8 8.8 0.9 4.4 5.3 1.8 2.7 14.0 14.1 0-1
7.3 1.8 1.0 7.3 10.9 8.2 11.8 7.3 5.5 3.6 1.8 1.8 8.2 0.9 4.5 4.5 1.8 2.7 14.6 13.6 1.o
8.3 2.1 8.3 6.3 9.4 7.3 9.4 10.4 7.3 6.3 3.1 2.1 2.1 5.2 1.0 7.3 3.1 1 14.6 8-3 6-3
7.3 2.1 7.3 9.4 9.4 7.3 9.4 10.4 5.2 6.3 3.1 2.1 3.1 5.2 1.o 6.3 4.2 1.0 16.7 9.3 7.4
s
o
Lipke et al., 1981). Heterogeneity of this magnitude was confirmed by isolation and amino acid analysis of the 20 major components (Table 6). That these represented only a portion of the protein complement was reported by Willis et al. (1981) based on staining patterns characteristic of thin gel slabs (Fig. 7). Both laboratories failed to detect appreciable accumulation of bridged intermediates between the monomeric components and the higher molecular species at the onset of pupariation during the period of declining solubility. The paucity of dimers and trimers favoured a concerted rather than a stepwise assembly mechanism for the sclerotized complex with n-mer half lives on the order of seconds or minutes in vivo. In spite of the presence of phenylthiourea and detergent, the extracts deteriorated on prolonged storage, either in solution or as a lyophilized powder. Isolates declined markedly in solubility and recovery of amino N from hydrolysates was low unless stored at low pressure under vapours of mercaptoethanol. These changes suggested autoxidation of tyrosyl residues, however, phenolase did not constitute one of the proteins of the mixture. In concordance with
24
H. L l P K E e t a / . CP I
H2N I
5
10
80
90 95 100
85
I
CP2
eo
85
90 95 100
I05
110
CP3
40
35
30
60
65
70
75
75
70
COOH 50
55
eo
85
so
95
c P4 95
go 85
eo
65
60
55
50
Fig. 6 Secondary structure of larval proteins from D. melanogaster computed by W. Henzel according to Chou and Fasman (1974). (From Snyder et al., 1981)
the findings in Drosophila, no detectable hexose, pentose or hexosamine was observed in 5 mg (0.30 pmole) of pooled proteins. The assays were sensitive to 0.05 pmole of carbohydrate. Extraction with ether and chloroform did not reduce the dry weight of the lyophilized powder. Intact and hydrolysed proteins were transesterified with BF3-methyl alcohol to release presumptive bound lipids. Gas liquid chromatography failed to reveal fatty acid methyl esters. Sarcophagid isolates were subjected to mapping and the Edman degradation to establish relatedness between proteins and for comparison with Drosophila. It can be seen from Fig. 5 that sarcophagid protein 5-64-68 is essentially homologous with the N-terminal regions of Drosophila CP2a, the six terminal residues are identical and the remaining substitutions are conservative. This finding was supported by comparison of termini of four proteins
TABLE 6 Molecular weights, isoelectric points, composition and yields of cuticle proteins from S. bulluta purified by isoelectric focusing and SDS-Dolvacrvlamide gel electrophoresis (from Lipke et ul., 1981). Amino acid values in residues per mole of protein Protein number
PI Molecular weight ( x lO-’Mr) Relative amount (%y Aspartic acid Glutamic acid Threonine Serine Proline Glycine Alanine Valine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine p-Aianine OH-Lysine Acidic/Basic Polar/Non-Polar Amino Terminus
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
4.63 4.68 4.73 4.80 4.88 4.90 4.97 5.03 5.08 5.13 5.18 5.23 5.33 5.38 5.50 20 17 21 16 22 21 22 24 21 21 21 21 20 19 17 5 29 34 7
6 8 7 3 29 16 1 1
3 8 23 30 46 32 1 2 1 4 31 27 2 9 31 34 15 16 29 27 6 7 8 9 7 6 7 3 3 11 12 7 16 1 1 0 1
n
o
o
o
o
o
o
o
i
1.2 2.2
2.7 1.2
2.7 1.5
3.5 1.9
1.9 1.9
2.1 1.6
1.9 1.4
1.9 1.4
2.0 1.5
2.2 1.4
1.6 1.3
1.3 2.3
2.8 2.1
-
gly
-
gly
-
-
-
-
-
-
asp
-
-
1 25 30 1 1 28 4 38 15
in
o
10 5
22 12 10
5 6 5 3 2
is
0 1
7 6 13 3 39 35 30 35 48 41 42 48 9 9 8 8 12 12 11 8 9 1 0 1 0 1 0 29 25 22 27 17 18 15 18 30 31 28 32 6 6 8 1 1 8 1 1 1 3 1 0 1 1 9 1 0 2 5 8 9 21 17 16 19 24 22 20 22 0 0 1 2 0 0 0 0
11 30 41 6 10 5 25 16
25 9 1 7
5 11 24 1 0
o
n.d.. not determined. a Soluble protein only. Insoluble protein CB-I represents 46% of the total cuticle protein.
2 9 31 29 38 36 9 7 1 12 14 7 1 0 28 27 15 17 25 2s 1 0 1 2 1 9 1 7 7 5 5 14 23 16 15 1 2 0 0
17
18
19
20
CB-I
5.53 5.75 5.88 5.90 17 17 18 19 63
2 3 7 1 1 3 7 5 20 36 38 19 17 22 25 22 6 31 36 42 38 32 29 30 39 12 3 1 9 1 1 1 2 1 1 8 6 1 1 16 17 21 22 23 22 13 23 33 6 1 2 1 4 1 1 4 6 1 2 1 26 49 57 36 28 25 11 35 24 11 8 10 9 8 9 5 13 36 14 16 16 14 13 21 in 25 9 7 7 6 5 6 8 1 0 8 0 9 0 8 7 7 8 1 0 8 8 7 1 2 5 4 2 3 5 6 6 7 4 4 2 1 4 2 4 3 n.d.* 6 48 9 12 8 5 11 17 LO 3 15 21 18 19 20 10 13 10 3 1 0 0 0 2 1 2 1 0 0 0 0 1 0 0 1 0 o o o o o o o o 0
2.7 2.0 tyr
2.1 2.2 -
1.8 1.8 ~
2.3 1.4
1.7 2.0
2.6
-
leu
-
1.5
-
1.5 asp
H. L l P K E eta/.
26
+
-
L-3d L-4d RS
WP
T-lh
T-2h T-4h
T-8h T-16h T-24h
pH 4 - 6
Fig. 7 Dissociated (guanidine) proteins from larval and puparial cuticle of S. bullata. The mixture was resolved by isoelectric focusing. Larval age in days (L), Puparial age in hours post tanning (T). White pupae (WP) Solubility in guanidine in per cent. (From Willis et al., 1981)
representative of both the pH 4 and pH 6 regions of the gel (Table 7). At neither terminal was homology significant within the species (Lipke et al., 1980; Snyder et al., 1981). On the other hand, when relatedness was assessed mathematically, it was evident that the inner regions of many of the proteins probably bore strong resemblances as revealed by peptide mapping (Fig. 8). The degree of relatedness can be calculated from amino acid composition based on SAQ for all possible combinations of the 19 candidates (Marchalonis and Weltman, 1971; Cornish-Bowden, 1980). Since all were similar in molecular weight an average residue number of 220 was used to establish the “strong test” criterion as SAQ = 38.6 as maximum index of similarity. This approach was first exploited by Willis et al. (1981) for D. m e h o g a s t e r and was subsequently confirmed by
TABLE 7 Partial sequences of selected cuticle proteins from S. bullata (Lipke et al., 1980) Protein
PI
N-term
C-term
1 5 NH2-Gly-His-Asx-Ala-Gly
Ile-Val-Ala-COOH
3A
4.4
9A l l H e
1 5 10 5.28 NH2-Asx-Ser-His-Pro-Asx-Asx-Val-His-Ala-Glu
12A He
1 5.30 NH,-Tyr-Tyr-Tyr-Tyr
14A
1 5 10 5-80 NH,-Leu-Gly-His-?-Gly-Gly-His-?-Glu-Ala
CBI
-
NH,- Asp-Val-Ala-His
Ile-Ala-His-Leu-COOH
His-COOH Blocked
H. L I P K E
28
eta/.
E
c
8
(u
L
I
inj,
15
30 Time (mtn )
45
Fig. 8 Peptide mapping of larval proteins from S. bullutu. The polypeptides were digested with trypsin and chromatographed on ultrasphere ODS by the procedure of Fullmer and Wasserman (1979). (From Henzel, Mulligan, Mole and Lipke, unpublished results)
sequencing. When sarcophagid proteins were ranked according to PI, strong relationships were indicated only among entities with close acid-base reactivities and SAQ 4 38.6 (Table 8). Similarly, when calypterate and acalypterate Diptera were compared on the basis of RNA complexity in the egg, only a distant relationship was manifest (Hough-Evans et ul., 1980). In the case of most cuticle proteins, however, questionable relatedness exists between non-terminal areas of D.melunoguster and S. bullutu when SAQ is calculated for proteins of disparate molecular weight. In view of non-identities for termini, at best the values may reflect internal homologies. An unusual component, 12He differed markedly from the other isolates. When this tyrosine-rich protein was separated by focusing
TABLE 8 SAQ of larval cuticle proteins of Surcophugu bullutu (Marcholonis and Weltman, 1971; Lipke et al., 1981). “Strong test” SAQ 5 38.6 in italics ____
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
____
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
156
149 53
287 261 102
213 190 81 102
245 179 73 104 8
240 180 78 121 23 8
259 208 100 125 25 14 5
244 192 77 80 29 21 18 18
178 126 45 87 23 21 20 20 22
136 136 77 168 45 46 33 34 56 22
141 381 355 544 285 325 303 309 367 297 188
179 229 125 121 171 200 209 212 169 129 181 454
169 225 131 152 207 240 254 259 220 161 208 449 12
145 142 71 148 191 204 211 231 164 149 201 417 103 100
154 124 65 149 193 189 179 207 139 140 184 433 141 161 28
131 45 31 136 96 99 99 118 103 52 71 330 132 148 122 102
138 215 113 140 82 96 79 88 97 71 60 199 185 216 194 166 90
150 36 26 161 132 128 134 147 123 77 111 375 141 138 62 75 47 167
H. L I P K E e t a / .
30
TABLE 9 Composition of tyrosine-rich larval proteins of S.bullutu (from Lipke and Henzel, 1981) 1st Isoelectric point Molecular wt kd 5 0.4 Relative amount (%)" Aspartic acidb Threonine Serine Glutamic acid Proline G1ycine Alanine Valine Isoleucine Leucine Tyrosine (%) Phenylalanine Lysine Histidine Arginine Acidiclbasic Polarmon-polar
12He
4.51 5.30 23 26 1 1 12 25 6 7 21 14 24 20 8 9 31 26 9 15 11 9 2 4 1 6 72 (35) 48 (20) 27 4 15 4 7 1 4.0 2.1 0.5 0.6
Percentage of total protein in extract. Values in molesimole of protein rounded to nearest whole number. Amide N not determined.
on a granulated bed (Bio-Gel P-100) similarity was noted to an aromatic polypeptide recovered from a polyacrylamide disc gel and previously designated 1st (Lipke and Henzel, 1981). The two focusing methods were not strictly comparable due to the different sieving and binding properties of the ampholyte supports. Mobility can also be effected by losses in amide N which are difficult to control at low pH, especially in cuticle proteins with a high degree of amidation of acidic amino acid residues. Proteins 1st and 12He contained exceptionally high titres of tyrosine; in the case of 12He, a oligotyrosyl peptide was located at the N-terminus. The yield of PTH-tyrosine was acceptable for the first four cycles of the Edman degradation, when continued beyond this point, unidentified products were released (Table 9). In view of the ubiquity of aromatic proteins in cyclorrhaphid haemolymph and the sharing of immuno determinants with cuticle proteins, protein 12He may represent a component transferred to the larval integument toward the end of the third instar (Scheller et al., 1980). In the event that protein-
MECHANISMS
OF SCLEROTIZATION I N DIPTERANS
31
TABLE 10 Radioactivity of S. bullata cuticle proteins from larvae administered [7-I4C] dopamine (from Lipke et al., 1981) 14c
Protein number
Per cent of Total durna
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 CB-Ib Control 12CProteins 1-20+ [7-W] dovamine ~
Cuticle only.
1.2 1.0 4.8 1.8 3.0 5.5 1.8 8.4 2.8 6.9 1.3 7.5 2.2 2.4 3.7 1.6 1.2 1.8 3.4 3.6 21.1
dumhmole 34 46 34 38 34 51 56 44 62 58 48 72 51 40 41 30 29 37 29 38 79 0.8
~
Polypeptide only
bound tyrosyls are substrates for phenolase or peroxidase in the course of crosslinking, the polytyrosyl sequence would be among the more vulnerable of the sites for modification. In addition to the well-defined aromatic amino acid complement of 12He, a second entity derived from aromatic precursors was associated with the borate-soluble larval proteins. At the conclusion of the post-feeding stage, cuticle proteins acquired radioactivity administered as [714C] dopamine earlier in the third instar (Lipke et al., 1981). The dopamine derivative was probably incorporated during a preparatory stage to crosslinking. The label was distributed more or less uniformly among the integumental polypeptides, in keeping with the concerted mechanism revealed by electrophoretic analysis (Table 10). Non-radioactive larval proteins did not bind dopamine and administration of labelled catecholamine during pupariation did
32
H. LIPKE e t a / .
not generate 14C02 from C7 or C8, thus non-specific binding or recycling did not account for the prevalence of isotope among the polypeptides. Affinity for borylcellulose, a bidentate ligand of o-diphenols, was undetectable (Sugumaran and Lipke, 1982b). These properties indicate that dopamine was converted to an aryl ether or an open chain metabolite in the course of addition to soluble components in anticipation of puparial metamorphosis.
3.1.5 Glycosylated components In contrast to the multiplicity of different proteins in urea or borate extracts of cuticle (Tables 6, 7) the chitin-bound insoluble fraction from S. bullata, fraction CB-I, was composed of a single protein with the N-terminal sequence: HN-Asp-Val-Ala-His-Tyr- and with the C-terminal blocked (Sugumaran et al., 1981, 1982). Brief exposure to chymotrypsin cleaved a 63000 dalton entity from the mucoprotein complex. Fraction CB-I was composed of significantly more proline and serine and less aspartic acid than the boratesoluble components (Table 6). Serial digestion with pronase, subtilisin-BPN and chitinase released a peptidochitodextrin with mole ratios: Gly (5):Glx (4):Asx (4):Thr (1):Val (1):Arg (1):glcNac (20). These values were fully in accord with the excess of glycine over glutamic acid in limited digests of larval glycopeptides previously reported (Kimura et al., 1976). In addition to the predominant protein species, and unlike the soluble protein mixture, phenolase activity characterized the chitin-protein complex. Whereas the cuticular phenolase of C. vicina was released into neutral buffers, the sarcophagid enzyme remained insoluble after prolonged extraction with borate-SDS. The ability of the phenolase to withstand mild proteolysis with thermolysin, pronase, trypsin, or chymotrypsin to affect release from the insoluble matrix was confirmed (Yamazaki, 1977; Barrett and Andersen, 1981). Oxidative activity was expressed with or without solubilization, in either instance thiols, copper chelators and withdrawal of oxygen inhibited quinone production from dopamine. The acellular strata of S . bullata include peroxidase in the enzyme complement and warrant detailed cataloguing of transferases and ion pumps to augment compositional studies (Quesada et al., 1978; Gupta and Hall, 1979; Sugumaran et al., 1981, 1982). The chitin-bound protein was dissimilar to the soluble faction in configuration. Whereas a globular shape was indicated by a 1:l ratio of buried to exposed tyrosyl groups in the
M E C H A N I S M S OF S C L E R O T I Z A T I O N I N D I P T E R A N S
33
borate-soluble fraction, all the phenolic residues were external in the residue (Hennigan and Lipke, unpublished observations). The scheduling of the reactions forming the chitin and the protein chains destined for joining has not been addressed directly. The heavy predominance of older chitin and protein synthesized during the third instar as opposed to new materials synthesized just prior to pupariation makes the kinetics of peptidoglycan deposition difficult to follow by tracer technique. Based on the small amount of protein added to the polysaccharide during tanning, however, relatively few polysaccharide chains would be required to accommodate the new glutamic acid-rich proteoaminoglycan. If the usual pattern of glycoprotein synthesis obtains, protein synthesis precedes saccharide addition. Protein synthesis was unaffected by polyoxin D, an inhibitor of chitin assembly in calliphorid peritrophic membranes, suggesting the polypeptide is supplied to the new chitin-protein complex well before the glycan was synthesized (Ishaaya and Casida, 1974; Peters, 1976; Becker, 1980). On the other hand there is no reason to rule out delayed peptidylation of chitin deposited before sclerotization or the storage and editing of preformed glutamic acid-rich polypeptide until conjugation was initiated. The localization of enzymes oxidizing phenols in cuticle well before visual evidence of pupariation or sclerotization is manifest is coincident with the presence of benzenoid species of low molecular weight (Lipke and Henzel, 1981; Lipke et al., 1981; Sugumaran and Lipke, 1982a). As discussed in Section 3.1.4, at this time labelled dopamine was incorporated into soluble proteins and extensively metabolized. Accordingly, the insoluble chitin-protein complex from larvae also contained label derived from tyrosine or dopamine in a variety of metabolites. Indeed, based on specific radioactivity, the residual Faction CBI exceeded the soluble in isotope uptake (Table 10). Tyrosine was converted to four distinct materials when administered early in the third instar (Sugumaran et al., 1981, 1982). Each metabolite was easily separated from tyrosine and companion substances on a cation exchange resin developed with gradients composed of volatile buffers (Lipke and Henzel, 1981). The best defined products were bi- and tertyrosine, the former present at levels of 2 nmole/mg during the post-feeding stage when peroxidase activity was high and at lower levels thereafter (Fig. 9). The polypeptide including this material in the primary structure was cleaved from the insoluble matrix by 0.1111 NaOH at 100°C for 5 min, yielding the two multi-functional aromatic amino acids on
34
H. L I P K E eta/. I
I
white yellow pink
red
dark brown
PUPARIAL STAGES
Fig. 9 Dityrosine and orthodiphenols during pupariation in S. bulluta. Following removal of soluble components by extraction with borate buffer, p H 9.2, odiphenols were determined in the residue according to Arnow (1937). Dityrosine was assessed by fluorimetry following isolation by chromatography. (From Sugumaran et ul., 1981, 1982.)
acid hydrolysis. The alkali-soluble protein and the original chitinprotein complex released peptides of clearly defined primary structure on extensive treatment with proteolytic enzymes (Fig. 10). Distinction between the two position isomers N-terminal to bityrosine remains to be established. Bi- and tertyrosine was identified in residual larval skins from Tabanus bivattatus but could not be detected in Aedes aegypti. In S. bullata labelled on the schedule described above, the sum of the radioactivity in the two tyrosine conjugates was equal to that of tyrosine. Generally, recovery of bityrosine was five-fold greater than tertyrosine. Bityrosine is refractory to phenolase and is not involved in the pigmentation of insoluble chitin-protein complexes that followed partial proteolytic digestion. In these preparations from S. bulluta, phenolase was active while still bound to the matrix, hence darkening on treatment with proteolytic enzymes was due to release of oxidized products to the buffer or the removal of blocking groups separating the oxidase from substrate(s). The endogenous materials subject to oxidation consisted of two types of substrates. The first group contained monotyrosyl peptides cleaved from the matrix or exteriorized from a sequestered tertiary structure. The peptides were prepared by proteolytic digestion of extracted skins with the phenolase previously inactivated by boiling, and conformed in
M E C H A N I S M S O F SCLEROTIZATION I N DIPTERANS
35
f: Hz H,N -ASP
- NH-CH-CO-P
RO
- S E R-COOH
Fig. 10 Primary structure of Peptide SH-3 from 5’. bullata larval cuticle. Sequence determined by the subtractive Edman procedure and carboxypeptidases A, Y and proline-specific endopeptidase. (From Sugumaran et al., 1981, 1982).
structure and properties to conventional tyrosine polypeptides susceptible to phenolase. The second object of phenolase action was a tightly bound o-diphenol that was alkali-labile unless complexed with molybdate-nitrite, this property also serving as a convenient route to quantitation (Arnow, 1937; White et al., 1979). It can be seen from Fig. 9 that the o-diphenol occurred at levels in excess of bityrosine and that turnover followed a different time course. Following prolonged treatment with chemical or enzymatic oxidizing agents, dark polymeric material accumulated in the reaction medium or was deposited within the chitin-protein complex. This pigment bears an obscure relationship to the pathway of sclerotization as observed in vivo and may be formed by a side reaction of little biological significance from the standpoint of crosslinking. The aromatic precursor of the pigment is of undeniable importance and is probably identical with the benzenoid species liberated by hydrolysis under reducing conditions with mercaptoethanesulphonic acid (Sugumaran and Lipke, 1982a). This reduction product did not chromatograph with dopamine although this catecholamine could serve as a precursor to the aromatic moiety. Two products were generated by the thio acid. In addition to the aromatic species, a non-benzenoid derivative of dopamine formed an acidic mercaptan or thioether clearly distinguishable from the positive charged metabolites discussed above. Whether the disappearance of aromatic character preceeded exposure to mercaptoethanesulphonic acid or is artefactual is not known. There was no question that the insoluble cuticle oxidases and substrates initiated crosslinking first in the matrix and then in the soluble components. During the course of
36
H. LIPKE eta/.
the darkening reaction initiated by the action of thermolysin on the protein-bound diphenol, the aspartyl N-terminus of the structural protein was completely blocked by coupling with quinone and was unreactive to Edman’s phenylthioisocyanate reagent. Masking or non-reactivity was not due to aspartyl removal by thermolysin since titre was unchanged during protease treatment if oxygen was excluded from the system. The attack on the N-terminal aspartyl residue clearly differentiated tanning of the chitin-protein as observed in covalent aggregates of sclerotized puparial proteins. In the latter example, lysyl and histidyl residues located in positions other than the N-termini were modified (Sugumaran and Lipke, 1982a). The enzymic reactions discussed above have an unusual counterpart in a purely chemical transformation produced by the action of 6~ HCI on borate-extracted larval cuticle at room temperature. Under conditions precluding enzymic activity (pH