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Analysis of the cuticular proteins of Hyalophora cecropia with two dimensional electrophoresis
Insect Biochem. Vol. 17, No. 3, pp. 457-468, 1987 Printed in Great Britain. All rights reserved
0020-1790/87 $3.00+0.00 Copyright © 1987 PergamonJournals Ltd
ANALYSIS OF THE CUTICULAR PROTEINS OF H Y A L O P H O R A CECROPIA WITH TWO DIMENSIONAL
ELECTROPHORESIS DIANA L. COX* and JUDITH H. WILLIS Department of Entomology, University of Illinois, Urbana, IL 61801, U.S.A.
(Received 2 January 1986; revised and accepted 25 June 1986)
Abstract--The soluble cuticular proteins of defined anatomical regions from different metamorphic stages of the giant silkmoth, Hyalophora cecropia, were characterized by two dimensional electrophoresis. As urea concentrations in 2D gels were increased, some of the cuticular proteins from the larval dorsal abdomen decreased in mobility relative to the molecular weight standards. This decrease was also influenced by the pH and ionic strength of the resolving gel. Clustering of proteins into groups, whose members showed similar behavior under different electrophoretic conditions, was indicative of membership in multigene families. By such criteria, common families were found in cuticles with similar mechanical properties from different metamorphic stages, yet there was evidence that different members of a single family were independently regulated.
Key Word Index: Cuticle, cuticular proteins, metamorphosis, 2D gels, protein hydrophobicity
INTRODUCTION Proteins extracted from different cuticular regions within a single metamorphic stage yield quite different electrophoretic patterns. We have been able to correlate the isoelectric points of these proteins with the different mechanical and functional properties of the regions (Cox and Willis, 1985). Indeed, there is an increasing body of evidence that the particular proteins found in cuticles are more related to the functions and properties of the cuticle than they are to the metamorphic stage of the insects (Andersen and Weis-Fogh, 1964; Andersen, 1971; Shawky and Vincent, 1978; Bordereau and Andersen, 1978; Willis et al., 1981; Chihara et al., 1982; Roter et al., 1985; Cox and Willis, 1985; Wolfgang and Riddiford, 1986). The hydrophobicities of the amino acids from hydrolyzed cuticle also have been correlated with different cuticular properties (Andersen, 1979; Vincent, 1980; Hillerton and Vincent, 1983). Yet, to date, such analyses have failed to consider the role of individual proteins or the possibility of different hydrophobicities among the cuticular proteins. In this paper, the conditions for best resolution of cuticular proteins in isoelectrofocusing and SDS gels are described. Using 2D gels with various concentrations of urea and made with buffers of different pHs and ionic strengths, we present evidence consistent with differences in hydrophobicities among individual proteins extracted from the larval dorsal abdomen of the giant silkmoth, Hyalophora cecropia. Using the optimal conditions established, we have *Present address: Department of Molecular Biology, Vanderbilt University, P.O. Box 1820, Station B, Nashville, TN 37235, U.S.A. 457
examined the cuticular proteins from several anatomical regions in each metamorphic stage. We found that there is even greater similarity between the cuticular proteins of regions having similar properties than was apparent from isoelectrofocusing. MATERIALS AND METHODS
Sample preparation Cuticular proteins from H. cecropia were prepared as described in Cox and Willis (1985). In brief, proteins were extracted from scrupulously cleaned cuticles using buffered 8 M guanidine hydrochloride (containing 2mM phenylmethylsulfonyl fluoride; 36raM Tris-HCl, pH 8.4; I% mercaptoethanol), dialyzed against water, lyophilized, and resuspended in 10M urea sample buffer for isoelectrofocusing. Ultrapure urea (Schwartz Mann) was lyophilizedand stored with silica gel. The urea sample buffer was decyanated using Rexyn 1-300 (Sigma Chemical Co.), aliquoted, and frozen until use. lsoelectrofocusing Isoelectrofocusing was performed as described by Cox and Willis (1985) in slab gels containing 2% LKB ampholines and 6.3 M urea. The urea concentration was varied in experimental gels to learn its influence on protein separation. 2D gel analysis The second dimension separation was performed in SDSpolyacrylamide gels using modifications of the O'Farrell (1975) method. Modifications were the inclusion of urea in the stacking and resolving gels, change in the pH of the resolving gel buffer, and fixation of the IEF strip before equilibration with SDS sample buffer. The resolving gels contained either 11 or 12% acrylamide (30:0.8, acrylamide:N,N'-methylene bisacrylamide). Properties of the cuticular proteins from larval dorsal abdomens were studied by varying the urea concentration in both the stacking and resolving gel from 0 to 7.5 M urea.
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DIANA L. Cox and JUDITH H. WILLIS
The effect of pH was determined by varying the pH of the resolving gel buffer from pH 8.45 to 9.0. The influence of increased ionic strength and buffering capacity in the resolving gel was determined by using a modification of the Goldsmith et al. (1979) technique; the resolving gel contained 0.3% SDS and 0.675 M Tris-HC1, pH 8.9, along with 6.4 M urea. This formulation was adopted for most comparisons of different anatomical regions. After isoelectrofocusing, IEF sample strips (1 cm wide) were cut from the IEF gel using long razor blades (BioRad), fixed in 0.04% Serva Blue W in 20% TCA for 10rain, followed by a 5 min rinse in 0.4% TCA and then a 5 rain rinse in 0.025% TEMED, after a 10min equilibration in SDS sample buffer (0.0625 M Tris HCI, pH 6.8; 12% SDS; 8.5% glycerol; 5% MSH) the strips were frozen in parafilm. (Strips equilibrated without prior fixation yielded gels with diffuse and missing spots; longer fixation in TCA generated multiple spots.) Immediately before use, two further 10 min equilibrations were carried out, and the sample strips loaded onto the 3% acrylamide stacking gels. Filter paper wicks (4 × 4 mm) were loaded with 5 #1 (2.3 #g/iLl) of molecularweight protein standards (Sigma, MW-SDS-70L), and one wick placed at each end of the stacking gel. (See legend of Fig. 1 for standards used.) The strip and wicks were sealed in place with 1% agarose (Sigma, type V) in SDS sample buffer, and a small amount of hot agarose containing bromophenol blue pipetted along the top. Electrophoresis was performed at 20 mA until the Serva Blue W ran off and the bromophenol blue was about 1 cm from the bottom of the gel (5-9 hr). Gels made using the modified Goldsmith et al. (1979) technique were clamped to a plastic cooling plate with water circulating at 7'C and run al 40 mA for about 4 hr. Following electrophoresis, gels were stained overnight in 0.1% Coomassie Brilliant Blue in 50% methanol and 16% acetic acid, destained in 10% methanol and 7.5% acetic acid, and photographed using Polaroid Type 55, positive negative film.
RESULTS
Effect of urea Cuticular proteins from the larval dorsal abdomen ( L D A ) focused into distinct bands only when the isoelectrofocusing gels contained urea, with 6.3 M urea giving optimal resolution. Except for improved resolution, urea concentrations from 0.75 to 8 M did not affect the banding pattern, and caused only a minor increase in the apparent isoelectric points (gels not shown). Unexpectedly, the behavior of proteins was profoundly affected by the urea concentration in the SDS gel used for the second dimension. This was in sharp contrast to the behavior of the molecular weight standards which maintained a linear relationship between mobility and log of their molecular weights. Relative to molecular weight markers, the mobilities of many individual cuticular proteins of the larval dorsal abdomen were reduced by increasing concentration of urea in the second dimension (Fig. 1). This change in mobility was not due to overloading the sample, for under at least one set of conditions, the proteins measured migrated as distinct spots, and all 2D gels were prepared with comparable strips from a single I E F gel. Different proteins responded differently to the changing conditions, four distinct patterns were apparent and are illustrated in Fig. 2. Although the measurements in Fig. 2 were made from
the " t o p " of each protein, similar shifts were obtained when bottoms of spots were compared. For example, using the bottom of the spot, the relative molecular weight of protein No. 11, changes from 3 9 k D in 2 M urea to 64 kD in 7 M urea gels. The mobility of one of the elongated spots (No. 10) likewise changes from approx. 15 kD at 2 M urea to 26 kD at 7 M urea. Also conspicuous was the streaking of a group of acidic proteins (proteins 8-10: pls 4.8-5) which began as the urea concentration approached 6 M. With increased urea concentration, the apparent molecular weights of thc cuticular proteins from the hard larval tubercles and pupal wing were affected comparably to proteins from the larval dorsal abdomen under these gel conditions (gels not shown).
E[:[ects of pH and ionic strength The mobility of all proteins, including the molecular weight standards, was decreased as pH of the resolving gel was increased, with the mobility of the cuticular proteins once again decreasing disproportionately to the molecular weight standards (Fig. 3). Streaking in the molecular weight dimension was reduced as the pH was increased. Increasing the ionic strength of the Tris in the resolving gel buffer from 0.375 to 0.675 M also decreased the amount ot' streaking, improved the resolution of many proteins, and split each component of the charge train set into 2 3 spots (gels not shown).
Compar&on of the cuticular proteins .fi'om the larval dorsal abdomen and,lore- and hindguts We previously showed that the larval fore- and hindgut cuticles are flexible and composed of proteins with acidic isoelectric points, many of which are identical in isoelectric point to the proteins of the flexible larval dorsal abdomen (Cox and Willis, 1985). In 2D gels, 22 spots with identical isoelectric points and molecular weights were shared between the larval foregut ( L F G ) and the larval dorsal abdomen (LDA: Fig. 4). The correspondence in mobility of the most acidic proteins (pls 4.4 4.6) from larval foregut and abdominal cuticle was verified in 2D gels with first dimensions of pH 4 ~ . 5 (not shown). Even though many bands from the larval hindgut cuticle (LHG) share identical isoelectric points with bands from both the larwd dorsal abdomen and foregut (Cox and Willis, 1985), only one spot had identical molecular weight with its counterpart from the other two larval regions (Fig. 4, arrows). This unanticipated difference m molecular weights when the general pattern of spots remained the same was observed in fours sets of standard gels and in 2D gels without urea. The larvcl hindgut and foregut also shared high molecular weight proteins that were poorly resolved under these gel conditions.
Comparison of cuticular regions/i'om di~,rent metamorphic stages Rigid cuticular regions. In our previous analysis (Cox and Willis, 1985) larval tubercles, head capsules, and pupal sclerites shared at least seven spots with identical molecular weights and isoelectric points. In the present study with different 2D gel conditions and greater resolution even more similarities were ob-
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Fig. 1. Effects of urea concentration on the separation of cuticular proteins taken from the larval abdomen. First dimension: pH 4-45; second dimension: 12% acrylamide, 0.375 M Tris-HCl, pH 8.6. Urea concentrations are indicated on each panel. Numbers indicate comparable spots and show the point at which the measurements reported in Fig. 2 were made. Molecular weight standards: (A) bovine serum albumin (66,000); (B) ovalbumin (45,000); (C) glyceraldehyde-3-phosphate dehydrogenase (36,000); (D) carbonic anhydrase (29,000); (E) trypsinogen (24,000); (F) trypsin inhibitor (20,100); (G) ~-lactalbumin (14,100).
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Fig. 3. Effect of increasing pH in the second dimension SDS gel. First dimension: pH 4-6; second dimension: 12% acrylamide, 6 M urea, 0.375 M Tris HCI. (A) pH 8.45, (B) pH 8.6, (C) pH 8.8, (D) pH 9.0. pH values are approximate. Cuticular proteins and molecular weight markers designated as in Fig. 1.
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Fig. 4. 2D gels displaying cuticular proteins of the larval dorsal abdomen (LDA), larval foregut (LFG), larval hindgut (LHG). First dimension: pH b6; second dimension: 12% acrylamide, 7.5 M urea, 0.375 M Tris-HCI, pH 8.6.
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14Fig. 5. 2D gels displaying cuticular proteins from rigid cuticular regions of larvae, pupae and adults. Larval tubercles (LTU), larval head capsule (LHC), pupal sclerite (PSC), adult medial sclerite (AMS), pharate adult thorax (ATH). First dimension: pH 5 8; second dimension: 11% acrylamide, 6.4 M urea, 0.675 M Tris-HCl, pH 8.9.
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Fig. 6. 2D gels of cuticular proteins from flexible cuticular regions of larvae, pupae and adults. Larval dorsal abdomen (LDA), pupal intersegmental membrane (PIN), inner cuticle of pupal wing (PWI), adult intersegmental membrane (AIN). First dimension: pH 4-6; second dimension: 11% acrylamide, 6.4 M urea, 0.675 M Tris-HCl, pH 8.9.
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UREA CONCENTRATION ( M )
Fig. 2. Changes in apparent molecular weights of cuticular proteins from the larval dorsal abdomen separated in 2D gels with increasing urea concentration (see Fig. 1). (A) Major proteins with pIs 4.4-4.6 (Nos 4--7); (B) major proteins with pIs 4.8-5.0 (Nos 8-10); (C) charge train protein No. 11 and protein No. 12; (D) minor proteins Nos 13, 14. served (Fig. 5). The 2D gels of extracts from larval head capsules (LHC) were identical to those of the larval tubercles (LTU), except for four minor spots; head capsules had three additional spots (white lines) and lacked one spot which is common to larval tubercles and pupal sclerites (double black lines). Comparing larval head capsules, larval tubercles, and pupal sclerites (PSC), 15 spots had identical isoelectric points and molecular weights (see black lines). Extracts of pupal sclerites had five low molecular weight spots (white lines) not found in any other regions that were examined, except for extracts of dorsal pupal forewings which were identical to those of pupal sclerites (gel not shown). In the adult, two fairly rigid cuticular regions were examined, the medial sclerite (AMS) and notum or dorsal thorax (ATH; Fig. 5). In 2D gel comparisons, the adult medial sclerite had 11 spots (black lines on AMS) of the 15 common spots found in larval tubercles, larval head capsules, and pupal sclerites and also had three unique spots (white lines). The adult medial sclerite is unique among rigid cuticles in having a few minor acidic (pls 4.4-4.6) proteins (Cox and Willis, 1985); although evidence of these proteins was not found in the 2D gel illustrated, they were identified as minor elongated streaks in 2D gels from both pH 4-6 and 3.5-10 IEF gels. No proteins from the notum could be extracted after post-ecdysial sclerotization had taken place; therefore, proteins extracted from pharate adults (day 19) were analyzed. Only spots with more basic isoelectric points (pH 6.4-7.4) were detected. All spots from the notum, except for one (white line on ATH) were identical to spots found in extracts of adult medial sclerite. Six of these spots (black lines) were shared with extracts from the rigid regions of the other stages.
Flexible cuticles. Many proteins from flexible cuticular regions from larvae, pupae, and adults have identical isoelectric points (Cox and Willis, 1985); and 2D gels revealed that most of these proteins also shared identical molecular weights. Figure 6 shows extracts of cuticular proteins from four regions of flexible cuticle, larval dorsal abdomen (LDA), intersegmental membranes from pupae (PIN) and adults (AIN) and the flexible, colorless membrane which lies internally, separating the fore- and hindwings (PWI). Comparison of banding patterns is facilitated by recognizing that there are distinct classes of spots. Prominent acidic proteins (pls 4-4.6) were found in all four regions. Extracts from larval dorsal abdomen and pupal intersegmental membranes shared six spots in this region, four of which were found in the adult intersegmental membrane, and one (black line) in the pupal wing membrane. There were charge trains consisting of spots with nearly identical molecular weights but with step-wise differences in isoelectric points found in the larval dorsal abdomen and pupal and adult intersegmental membranes. The larval and adult charge trains (double-shafted arrows) were similar in charge distribution and molecular weight, the pupal intersegmental membrane charge train was more basic, had a lower molecular weight, and was composed of fewer and more prominent spots. Spots in another group can be recognized because of their tendency to form elongated streaks. Such proteins (arrows) were found in all but the pupal intersegmental membrane, but with different properties. Those from larval cuticle form a distinct cluster slightly more basic than the most acidic group of spots. In extracts from adult intersegmental mem-
466
DIANA L. COX and JUDITH H. WILLIS
branes, proteins forming elongated streaks were interspersed with well defined spots in the most acidic group. In the pupal wing membrane, while such streaking spots had pIs equivalent to the larval group, most had apparent molecular weights double those of the other regions. There were minor spots shared between regions; larval dorsal abdomen and pupal intersegmental membrane shared six such spots. Lastly, high molecular weight material which remained close to the sample application site was observed in extracts from pupal intersegmental membranes. Flexible versus rigid cuticles. The preceding sections compared flexible or rigid cuticular regions using, for the first dimension, IEF gradients which gave best resolution for each type of cuticle (pH 4-6 for flexible cuticles and pH 5-8 for rigid cuticles). To help comparisons between flexible and rigid cuticles, extracts of nine regions from the three metamorphic stages were also compared in 2D gels in which the first dimension was from pH 3.5-10 IEF gels (gels not shown). With but two exceptions, none of the major spots were shared between the flexible and rigid cuticular regions. Proteins which formed elongated streaks occurred as major proteins in the flexible adult cuticles and as minor proteins in the adult medial sclerite. A set of minor spots was shared between the cuticles of the pupal sclerite and the intersegmental membranes.
DISCUSSION
Effects o f urea in protein electrophoresis Urea is routinely used for isoelectrofocusing of proteins when denaturation is not a problem. Shawky and Vincent (1978) found, as we have, that increased urea concentration in IEF gels resulted in greater resolution of cuticular proteins and they attributed this to changes in conformation and/or the aggregation of the proteins in the presence of urea. Proteins which shift in apparent isoelectric point in the presence of urea, as did our cuticular proteins, are generally assumed to have charged groups buried within the native structure which are exposed only under denaturing conditions (Ui, 1971a, b). In contrast to the relatively minor influence of urea (4-8 M) on the isoelectrofocusing separations, substantial changes in the mobilities of cuticular proteins occurred in SDS gels as the urea concentration was increased. The migration of different proteins was affected in different ways by the increased urea concentration, summarized in Fig. 2. Only the molecular weight standards and a few minor cuticular proteins showed no shift in mobility in relation to each other, all other cuticular proteins were profoundly affected by urea concentration. One explanation for the odd behavior of most cuticular proteins is that they had been incompletely saturated with SDS (Pau and Crambach, personal communication). We have eliminated this possibility by subjecting both the molecular weight standards and the cuticular protein extracts from the larval dorsal abdomen to varying times of boiling in SDS (0-60 min) and then running them on a standard SDS gel. There was no influence of treatment time on the
mobility of any of the proteins, although 60 min of boiling caused some minor degradation (unpublished observations). These changes in mobility of the cuticular proteins with increased concentrations of urea can be explained by the complex mechanism of urea denaturation (Ghelis and Yon, 1982). Denaturation by urea alone causes proteins to unfold by interacting with hydrophobic regions of the protein and increasing the protein's solubility in aqueous solutions. The degree to which urea causes proteins to unfold is affected by temperature, pH, and ionic strength of the solution (Creighton, 1979; Ghelis and Yon, 1982; Goldenberg and Creighton, 1984). The effect of each of these variables differs for each protein (Creighton, 1979). The apparent unfolding of the cuticular proteins was reversible since proteins focused in the unfolded state in 6.3 M urea IEF gels assumed lower molecular weights in SDS gels without urea than in gels with urea. Like urea, detergents such as SDS are also considered to be strong denaturants. As opposed to urea which disrupts the secondary structure of the protein, SDS may not completely unfold the protein but may instead form a highly ordered complex with the protein (Reynolds and Tanford, 1970). The use of both urea and SDS in gels has been found to improve the resolution of several proteins, including insect chorion proteins (Goldsmith ct al., 1979). The changes in mobility of cuticular proteins with increasing urea concentrations possibly can he explained by the unfolding of hydrophobic regions. Previous evidence for globular conformations of cuticular proteins comes from X-ray diffraction studies (Rudall and Kenchington, 1973). Properties o f individual cuticular proteins The preceding information and our results provide insights into the properties of the different groups of cuticular proteins from the larval dorsal abdomen. Some proteins (e.g. 13 and 14, Fig. 2) showed little change with increasing urea concentrations. Members of the charge train group of cuticular proteins changed their relative mobilities with increased urea concentration, and, therefore, may have different degrees of hydrophobicity, or alternatively, these differences may have been due to differences in glycosylation (Cox and Willis, 1987). The most acidic proteins (pls 4.4 -4.6) appeared to be completely unfolded near 6 M urea concentrations; whereas, the proteins with pls 4.8--5.0 were only unfolded at higher urea concentrations or with increased pH in the resolving gel. The streaking behavior of the proteins with pls 4.8 5.0 may have been due to the trapping of intermediates in the unfolding process (Goldenberg and Creighton, 1984). The shift in the mobilities of all the proteins, including the molecular weight markers, may, in part, be owing to the increased viscosity of the gel solvent with increased urea concentration which adds to the molecular sieving properties of the gel (Goldenberg and Creighton, 1984). The amino acid composition of individual cuticular proteins (Willis et al., 1981) raises questions about the role hydrophobicity plays in the urea-dependent shifts in molecular weight, for when net hydro-
Cuticular proteins
467
phobicity is calculated according to the method used by Andersen (1979), proteins showing major shifts are relatively hydrophilic.
separated rigid and flexible cuticles from the different stages, different conclusions might have been reached.
Cuticular proteins and functions In previous studies using IEF gels, we found different banding patterns from extracts of different cuticular regions of a single individual and suggested that the proteins could be related to the function of the cuticle (Cox and Willis, 1985). Chihara et al. (1982) and Roter et al. (1985) showed regional differences in cuticular protein composition in Drosophila melanogaster; Shawky and Vincent (1978) and Bordereau and Andersen (1978) found differences in proteins from locust and termite sclerites and intersegmental membranes. The restriction of particular proteins to specific layers within the cuticle has been reported by Andersen and Weis-Fogh (1964) and Wolfgang and Riddiford (1986). In H. cecropia larvae, the dorsal abdominal cuticle forms a barrier between the external environment and the insect's internal anatomy. This cuticle is thick, has limited permeability, and also provides for muscle attachment. The thin foregut cuticle is also a barrier on a more limited scale, protecting the animal against microbes. In this area, desiccation is less of a problem since contact with air is limited. In comparison to these two areas, the hindgut cuticle must also be permeable to water since the epidermal cells in this area take up water from the gut contents. These differences in function were found to correspond to differences in cuticular protein compositions. Most cuticular proteins from the foregut were a subset of the proteins from the dorsal abdomen and only contained a few unique minor spots. The hindgut, on the other hand, had only one spot in common with either the foregut or dorsal abdomen.
Significance of clusters of spots Clustering of chorion protein spots in 2D gels has been correlated with their membership in a multigene .family, more formally recognized by nucleotide and amino acid sequence data (see Goldsmith and Kafatos, 1984). In Drosophila, three of four larval cuticular protein genes that have been sequenced belong to a multigene family (Snyder et al., 1982). Sequences and amino acid compositions of Sarcophaga bullata cuticular proteins also indicate relatedness (Henzel et al., 1985). In the present study, 2D gels showed clustering of cuticular proteins similar to that seen for chorion proteins. Within each group, the proteins also behaved similarly under different gel conditions lending additional support for the existence of multigene families in H. cecropia cuticular proteins. It is probable that each protein within most of these clusters corresponds to a product of a single gene, since the only clusters with evidence of post-translational modifications are the charge trains and elongated streak proteins (Cox and Willis, 1987). In the flexible cuticular regions, there are three clusters or putative multigene families: the acidic proteins (pls 4.4~4.6, 20,000-24,000 daltons), proteins which formed elongated streaks, and those that formed charge trains. Additional evidence that the most acidic cuticular proteins belong to a multigene family has come from amino acid compositions (Willis et al., 1981). The flexible cuticular regions from pupae and adults had only subsets of this most acidic group (Fig. 6), and the larval hindgut had none of these spots. The observation that not all of these acidic spots were present in every flexible cuticle suggests that the synthesis of each protein is under independent regulation and/or that each protein of this group may have a unique function within the cuticular matrix. Formation of elongated streaks occurred in clusters of spots from every flexible cuticle examined except the pupal intersegmental membrane; however, the isoelectric points and molecular weights of these spots were variable among the regions. In addition to this unique behavior, these proteins also shared the characteristics of being released from gels by 7% acetic acid, 1 N nitric acid, and 50-95% ethanol (unpublished observations). Such streaking behavior was also found in some chorion proteins (Goldsmith et al., 1979); with both types of proteins, streaking could be reduced by increasing the ionic strength of the resolving gel buffer. The conformations of these proteins may be similar, and they may function in their respective matrices in similar ways. The extent of sequence similarity deserves further analysis. In the rigid cuticles, it is more difficult to distinguish discrete groups of proteins. The clusters are not tightly organized and do not have special characteristics, although a cluster of six major proteins was found in every region. Many of the minor, basic proteins with similar molecular weights also appeared to be shared among all the regions. Interestingly, they did not exhibit the independent regulation that was seen with the acidic proteins. A group of proteins (pIs
Lack of stage specificity in cuticular proteins Based upon isoelectrofocusing data, we have previously discounted claims that metamorphic stage is a major determinant of which cuticular proteins will be present (Cox and Willis, 1985). In the present study, data from 2D gels further strengthens the evidence that the synthesis of cuticular proteins is less related to metamorphic stage than to the function and properties of the cuticle. Flexible cuticles had many more spots with identical molecular weights and isoelecteric points than they shared with rigid cuticles. The similarity among the protein patterns of the rigid cuticles was even greater than the similarity among the flexible cuticles. Most of the major cuticular proteins were common to all rigid cuticles, irrespective of metamorphic stage. None of these shared spots was found in the extracts of flexible cuticles. The few unique spots found in each region may be related to other properties besides rigidity. Recent studies have reported stage-specific differences in the synthesis of lepidopteran cuticular proteins. Kiely and Riddiford (1985a,b) examined the synthesis of cuticular proteins using dorsal abdominal epidermis of Manduca larvae and pupae. In another study, Sridhara (1985) found about 40% of the mRNAs taken during cuticle synthesis from wings of second pupae or adults of A. polyphemus were unique. If these analyses had been based on
468
DIANA L. Cox and JUDITH H. WILLIS
5.0--6.0, 14,000-18,000 daltons) was f o u n d in every rigid cuticle examined, except for the p h a r a t e n o t u m . CONCLUSIONS F o r the analysis of cuticular proteins, I E F is a sensitive a n d reproducible technique a n d less affected by m i n o r fluctuations in gel c o m p o n e n t s t h a n SDS electrophoresis. Hence, this is an a p p r o p r i a t e technique if one wants to c o m p a r e a large n u m b e r of cuticular protein samples from different regions, meta m o r p h i c stages or species (Cox a n d Willis, 1985). The c o m b i n a t i o n of the two electrophoretic separations in 2D gels, however, offers significant inform a t i o n o n the c o m p o s i t i o n a n d properties of these proteins; but it is absolutely necessary to control all possible variables, especially p H and urea concentration, to o b t a i n consistent results. W h e n this is done, d a t a from 2D gels indicated t h a t greater similarity exists between the cuticular proteins of cuticles with similar mechanical properties and functions t h a n a m o n g dissimilar cuticles from the same stage. U n i q u e proteins f o u n d in various regions m a y confer u n i q u e properties to the cuticle and, hence, are not necessarily synthesized u n d e r stage-specific regulation. Acknowledgements--We thank Stanley Friedman, Ellis G. MacLeod and Richard N. Pau for their advice and appreciate the able technical assistance of Laura K. Moehling. This paper is based in part on a thesis submitted by D. L. C. for the doctoral degree at the University of Illinois at Urbana-Champaign. The research was supported by grant PCM 8201934 from the National Science Foundation. REFERENCES
Andersen S. O. (1971) Resilin. In Comprehensive Biochemistry (Edited by Florkin M. and Stoz E. H.), Vol. 26C, pp. 633~57. Elsevier, New York. Andersen S. O. (1979) Biochemistry of insect cuticle. A. Rev. Ent. 24, 29-61. Andersen S. O. and Weis-Fogh T. (1964) Resilin. A rubberlike protein in arthropod cuticle. Adv. Insect Physiol. 2, 1~5. Bordereau C. and Andersen S. O. (1978) Structural cuticular proteins in termite queens. Comp. Biochem. Physiol. 60B, 251-256. Chihara C. J., Silvert D. J. and Fristrom J. W. (1982) The cuticle proteins of Drasophila melanogaster : stage specificity. Devl Biol. 89, 379-388. Cox D. L. and Willis J. H. (1985) The cuticular proteins of Hyalophora cecropia from different anatomical regions and metamorphic stages. Insect Biochem. 15, 349-362. Cox D. L. and Willis J. H. (1987) Post-translational modifications of the cuticular proteins of Hyalophora cecropia from different anatomical regions and metamorphic stages. Insect Biochem. 17, 469484. Creighton T. E. (1979) Electrophoretic analysis of the unfolding of proteins by urea. J. molec. Biol. 129, 235-264. Ghelis C. and Yon J. (1982) Protein Folding, pp. 223 296, 395418. Academic Press, New York.
Gotdenberg D. P. and Creighton T. E. (1984) Gel electrophoresis in studies of protein conformation and folding. Analyt. Bioehem. 138, 1-18. Goldsmith M. R. and Kafatos F. C. (1984) Developmentally regulated genes in silkmoths..4. Rer. Genel. 18, 443487. Goldsmith M. R., Rattner E. C., Koehler M. M. D., Balikov S. R. and Bock S. C. (1979) Two-dimensional electrophoresis of small-molecular-weight proteins. Analyt. Bioehem. 99, 3340. Henzel W. J., Mole J. E., Mulligan K. and Lipke H. (1985) Sarcophagid larval proteins: partial sequence homologies among three cuticle proteins and related structures of Drosophilids. J. molec. Evol. 22, 39 45. Hillerton J. E. and Vincent J. F. V. (1983) Consideration of the importance of hydrophobic interactions in stabilizing insect cuticle. Int. J. Biol. Macromol. 5, 163 166. Kiely M. L. and Riddiford L. M. (1985a) Temporal patterns of protein synthesis in Manduca epidermis during the change to pupal commitment in vitro: their modulation by 20-hydroxyecdysone and juvenile hormone. Wilhelm Roux's Arch. 194, 325.335. Kiely M. L. and Riddiford L. M. (1985b) Temporal programming of epidermal cell protein synthesis during the larval pupal transformation of Manduca sexta. Wilhelm Roux's Arch. 194, 336 343. O'Farrell P. H. (1975) High resolution two-dimensional electrophoresis of proteins. J. biol. Chem. 250, 4007 -1021. Reynolds J. A. and Tanford C. (1970) The gross conformation of protein-sodium dodecyl sulfate complexes. J. biol. Chem. 245, 5161 5165. Roter A. H., Spofford J. B. and Swift H. (1985) Synthesis of the major adult cuticle proteins of Drosophila melanogaster during hypoderm differentiation. DeN Biol. 107, 420-43 l. Rudall K. M. and Kenchington W. (1973) The chitin system. Biol. Rev. 49, 597 636. Shawky N. A. F. and Vincent J. F. V. (1978) The proteins of the urea-soluble fraction of locust intersegmental membrane. Insect Biochem. 8, 255-261. Snyder M., Hunkapiller M., Yuen D., Silvert D., Fristrom J. and Davidson N. (1982) Cuticle protein genes of Drosophila: structure, organization and evolution of four clustered genes. Cell 29, 102% 1040. Sridhara S. (1985) Evidence that pupal and adult cuticular proteins are coded by different genes in the silkmoth Antheraea polyphemus. Insect Biochem. 15, 333 339. Ui N. (1971a) Isoetectric points and conformation of proteins. I. Effect of urea on the behavior of some proteins in isoelectric focusing. Biochim. hiophys. Acta 229, 567 581. Ui N. (197lb) Isoelectric points and conformation of proteins. II. Isoelectric focusing of ct-chymotrypsin and its inactive derivative. Bioehim. biophys. Acta 229, 582 589. Vincent J. F. V. (1980) Insect cuticle: a paradigm for natural composites. Symp. Soc. exp. Biol. 34, 183 210. Willis J. H., Regier J. C. and Debrunner B. A. (1981) The metamorphosis of arthropodin. In Current Topics in Insect Endocrinology and Nutrition (Edited by Bhaskaran G., Friedman S, and Rodriquez J. G.), pp. 27 -46. Plenum Press, New York. Wolfgang W. J. and Riddiford L. M. (1986) Larval cuticular morphogenesis in the tobacco hornworm, Manduca sexta. and its hormonal regulation. Devl Biol. 113, 305 316.
0020-1790/87 $3.00+0.00 Copyright © 1987 PergamonJournals Ltd
ANALYSIS OF THE CUTICULAR PROTEINS OF H Y A L O P H O R A CECROPIA WITH TWO DIMENSIONAL
ELECTROPHORESIS DIANA L. COX* and JUDITH H. WILLIS Department of Entomology, University of Illinois, Urbana, IL 61801, U.S.A.
(Received 2 January 1986; revised and accepted 25 June 1986)
Abstract--The soluble cuticular proteins of defined anatomical regions from different metamorphic stages of the giant silkmoth, Hyalophora cecropia, were characterized by two dimensional electrophoresis. As urea concentrations in 2D gels were increased, some of the cuticular proteins from the larval dorsal abdomen decreased in mobility relative to the molecular weight standards. This decrease was also influenced by the pH and ionic strength of the resolving gel. Clustering of proteins into groups, whose members showed similar behavior under different electrophoretic conditions, was indicative of membership in multigene families. By such criteria, common families were found in cuticles with similar mechanical properties from different metamorphic stages, yet there was evidence that different members of a single family were independently regulated.
Key Word Index: Cuticle, cuticular proteins, metamorphosis, 2D gels, protein hydrophobicity
INTRODUCTION Proteins extracted from different cuticular regions within a single metamorphic stage yield quite different electrophoretic patterns. We have been able to correlate the isoelectric points of these proteins with the different mechanical and functional properties of the regions (Cox and Willis, 1985). Indeed, there is an increasing body of evidence that the particular proteins found in cuticles are more related to the functions and properties of the cuticle than they are to the metamorphic stage of the insects (Andersen and Weis-Fogh, 1964; Andersen, 1971; Shawky and Vincent, 1978; Bordereau and Andersen, 1978; Willis et al., 1981; Chihara et al., 1982; Roter et al., 1985; Cox and Willis, 1985; Wolfgang and Riddiford, 1986). The hydrophobicities of the amino acids from hydrolyzed cuticle also have been correlated with different cuticular properties (Andersen, 1979; Vincent, 1980; Hillerton and Vincent, 1983). Yet, to date, such analyses have failed to consider the role of individual proteins or the possibility of different hydrophobicities among the cuticular proteins. In this paper, the conditions for best resolution of cuticular proteins in isoelectrofocusing and SDS gels are described. Using 2D gels with various concentrations of urea and made with buffers of different pHs and ionic strengths, we present evidence consistent with differences in hydrophobicities among individual proteins extracted from the larval dorsal abdomen of the giant silkmoth, Hyalophora cecropia. Using the optimal conditions established, we have *Present address: Department of Molecular Biology, Vanderbilt University, P.O. Box 1820, Station B, Nashville, TN 37235, U.S.A. 457
examined the cuticular proteins from several anatomical regions in each metamorphic stage. We found that there is even greater similarity between the cuticular proteins of regions having similar properties than was apparent from isoelectrofocusing. MATERIALS AND METHODS
Sample preparation Cuticular proteins from H. cecropia were prepared as described in Cox and Willis (1985). In brief, proteins were extracted from scrupulously cleaned cuticles using buffered 8 M guanidine hydrochloride (containing 2mM phenylmethylsulfonyl fluoride; 36raM Tris-HCl, pH 8.4; I% mercaptoethanol), dialyzed against water, lyophilized, and resuspended in 10M urea sample buffer for isoelectrofocusing. Ultrapure urea (Schwartz Mann) was lyophilizedand stored with silica gel. The urea sample buffer was decyanated using Rexyn 1-300 (Sigma Chemical Co.), aliquoted, and frozen until use. lsoelectrofocusing Isoelectrofocusing was performed as described by Cox and Willis (1985) in slab gels containing 2% LKB ampholines and 6.3 M urea. The urea concentration was varied in experimental gels to learn its influence on protein separation. 2D gel analysis The second dimension separation was performed in SDSpolyacrylamide gels using modifications of the O'Farrell (1975) method. Modifications were the inclusion of urea in the stacking and resolving gels, change in the pH of the resolving gel buffer, and fixation of the IEF strip before equilibration with SDS sample buffer. The resolving gels contained either 11 or 12% acrylamide (30:0.8, acrylamide:N,N'-methylene bisacrylamide). Properties of the cuticular proteins from larval dorsal abdomens were studied by varying the urea concentration in both the stacking and resolving gel from 0 to 7.5 M urea.
458
DIANA L. Cox and JUDITH H. WILLIS
The effect of pH was determined by varying the pH of the resolving gel buffer from pH 8.45 to 9.0. The influence of increased ionic strength and buffering capacity in the resolving gel was determined by using a modification of the Goldsmith et al. (1979) technique; the resolving gel contained 0.3% SDS and 0.675 M Tris-HC1, pH 8.9, along with 6.4 M urea. This formulation was adopted for most comparisons of different anatomical regions. After isoelectrofocusing, IEF sample strips (1 cm wide) were cut from the IEF gel using long razor blades (BioRad), fixed in 0.04% Serva Blue W in 20% TCA for 10rain, followed by a 5 min rinse in 0.4% TCA and then a 5 rain rinse in 0.025% TEMED, after a 10min equilibration in SDS sample buffer (0.0625 M Tris HCI, pH 6.8; 12% SDS; 8.5% glycerol; 5% MSH) the strips were frozen in parafilm. (Strips equilibrated without prior fixation yielded gels with diffuse and missing spots; longer fixation in TCA generated multiple spots.) Immediately before use, two further 10 min equilibrations were carried out, and the sample strips loaded onto the 3% acrylamide stacking gels. Filter paper wicks (4 × 4 mm) were loaded with 5 #1 (2.3 #g/iLl) of molecularweight protein standards (Sigma, MW-SDS-70L), and one wick placed at each end of the stacking gel. (See legend of Fig. 1 for standards used.) The strip and wicks were sealed in place with 1% agarose (Sigma, type V) in SDS sample buffer, and a small amount of hot agarose containing bromophenol blue pipetted along the top. Electrophoresis was performed at 20 mA until the Serva Blue W ran off and the bromophenol blue was about 1 cm from the bottom of the gel (5-9 hr). Gels made using the modified Goldsmith et al. (1979) technique were clamped to a plastic cooling plate with water circulating at 7'C and run al 40 mA for about 4 hr. Following electrophoresis, gels were stained overnight in 0.1% Coomassie Brilliant Blue in 50% methanol and 16% acetic acid, destained in 10% methanol and 7.5% acetic acid, and photographed using Polaroid Type 55, positive negative film.
RESULTS
Effect of urea Cuticular proteins from the larval dorsal abdomen ( L D A ) focused into distinct bands only when the isoelectrofocusing gels contained urea, with 6.3 M urea giving optimal resolution. Except for improved resolution, urea concentrations from 0.75 to 8 M did not affect the banding pattern, and caused only a minor increase in the apparent isoelectric points (gels not shown). Unexpectedly, the behavior of proteins was profoundly affected by the urea concentration in the SDS gel used for the second dimension. This was in sharp contrast to the behavior of the molecular weight standards which maintained a linear relationship between mobility and log of their molecular weights. Relative to molecular weight markers, the mobilities of many individual cuticular proteins of the larval dorsal abdomen were reduced by increasing concentration of urea in the second dimension (Fig. 1). This change in mobility was not due to overloading the sample, for under at least one set of conditions, the proteins measured migrated as distinct spots, and all 2D gels were prepared with comparable strips from a single I E F gel. Different proteins responded differently to the changing conditions, four distinct patterns were apparent and are illustrated in Fig. 2. Although the measurements in Fig. 2 were made from
the " t o p " of each protein, similar shifts were obtained when bottoms of spots were compared. For example, using the bottom of the spot, the relative molecular weight of protein No. 11, changes from 3 9 k D in 2 M urea to 64 kD in 7 M urea gels. The mobility of one of the elongated spots (No. 10) likewise changes from approx. 15 kD at 2 M urea to 26 kD at 7 M urea. Also conspicuous was the streaking of a group of acidic proteins (proteins 8-10: pls 4.8-5) which began as the urea concentration approached 6 M. With increased urea concentration, the apparent molecular weights of thc cuticular proteins from the hard larval tubercles and pupal wing were affected comparably to proteins from the larval dorsal abdomen under these gel conditions (gels not shown).
E[:[ects of pH and ionic strength The mobility of all proteins, including the molecular weight standards, was decreased as pH of the resolving gel was increased, with the mobility of the cuticular proteins once again decreasing disproportionately to the molecular weight standards (Fig. 3). Streaking in the molecular weight dimension was reduced as the pH was increased. Increasing the ionic strength of the Tris in the resolving gel buffer from 0.375 to 0.675 M also decreased the amount ot' streaking, improved the resolution of many proteins, and split each component of the charge train set into 2 3 spots (gels not shown).
Compar&on of the cuticular proteins .fi'om the larval dorsal abdomen and,lore- and hindguts We previously showed that the larval fore- and hindgut cuticles are flexible and composed of proteins with acidic isoelectric points, many of which are identical in isoelectric point to the proteins of the flexible larval dorsal abdomen (Cox and Willis, 1985). In 2D gels, 22 spots with identical isoelectric points and molecular weights were shared between the larval foregut ( L F G ) and the larval dorsal abdomen (LDA: Fig. 4). The correspondence in mobility of the most acidic proteins (pls 4.4 4.6) from larval foregut and abdominal cuticle was verified in 2D gels with first dimensions of pH 4 ~ . 5 (not shown). Even though many bands from the larval hindgut cuticle (LHG) share identical isoelectric points with bands from both the larwd dorsal abdomen and foregut (Cox and Willis, 1985), only one spot had identical molecular weight with its counterpart from the other two larval regions (Fig. 4, arrows). This unanticipated difference m molecular weights when the general pattern of spots remained the same was observed in fours sets of standard gels and in 2D gels without urea. The larvcl hindgut and foregut also shared high molecular weight proteins that were poorly resolved under these gel conditions.
Comparison of cuticular regions/i'om di~,rent metamorphic stages Rigid cuticular regions. In our previous analysis (Cox and Willis, 1985) larval tubercles, head capsules, and pupal sclerites shared at least seven spots with identical molecular weights and isoelectric points. In the present study with different 2D gel conditions and greater resolution even more similarities were ob-
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Fig. 2. Changes in apparent molecular weights of cuticular proteins from the larval dorsal abdomen separated in 2D gels with increasing urea concentration (see Fig. 1). (A) Major proteins with pIs 4.4-4.6 (Nos 4--7); (B) major proteins with pIs 4.8-5.0 (Nos 8-10); (C) charge train protein No. 11 and protein No. 12; (D) minor proteins Nos 13, 14. served (Fig. 5). The 2D gels of extracts from larval head capsules (LHC) were identical to those of the larval tubercles (LTU), except for four minor spots; head capsules had three additional spots (white lines) and lacked one spot which is common to larval tubercles and pupal sclerites (double black lines). Comparing larval head capsules, larval tubercles, and pupal sclerites (PSC), 15 spots had identical isoelectric points and molecular weights (see black lines). Extracts of pupal sclerites had five low molecular weight spots (white lines) not found in any other regions that were examined, except for extracts of dorsal pupal forewings which were identical to those of pupal sclerites (gel not shown). In the adult, two fairly rigid cuticular regions were examined, the medial sclerite (AMS) and notum or dorsal thorax (ATH; Fig. 5). In 2D gel comparisons, the adult medial sclerite had 11 spots (black lines on AMS) of the 15 common spots found in larval tubercles, larval head capsules, and pupal sclerites and also had three unique spots (white lines). The adult medial sclerite is unique among rigid cuticles in having a few minor acidic (pls 4.4-4.6) proteins (Cox and Willis, 1985); although evidence of these proteins was not found in the 2D gel illustrated, they were identified as minor elongated streaks in 2D gels from both pH 4-6 and 3.5-10 IEF gels. No proteins from the notum could be extracted after post-ecdysial sclerotization had taken place; therefore, proteins extracted from pharate adults (day 19) were analyzed. Only spots with more basic isoelectric points (pH 6.4-7.4) were detected. All spots from the notum, except for one (white line on ATH) were identical to spots found in extracts of adult medial sclerite. Six of these spots (black lines) were shared with extracts from the rigid regions of the other stages.
Flexible cuticles. Many proteins from flexible cuticular regions from larvae, pupae, and adults have identical isoelectric points (Cox and Willis, 1985); and 2D gels revealed that most of these proteins also shared identical molecular weights. Figure 6 shows extracts of cuticular proteins from four regions of flexible cuticle, larval dorsal abdomen (LDA), intersegmental membranes from pupae (PIN) and adults (AIN) and the flexible, colorless membrane which lies internally, separating the fore- and hindwings (PWI). Comparison of banding patterns is facilitated by recognizing that there are distinct classes of spots. Prominent acidic proteins (pls 4-4.6) were found in all four regions. Extracts from larval dorsal abdomen and pupal intersegmental membranes shared six spots in this region, four of which were found in the adult intersegmental membrane, and one (black line) in the pupal wing membrane. There were charge trains consisting of spots with nearly identical molecular weights but with step-wise differences in isoelectric points found in the larval dorsal abdomen and pupal and adult intersegmental membranes. The larval and adult charge trains (double-shafted arrows) were similar in charge distribution and molecular weight, the pupal intersegmental membrane charge train was more basic, had a lower molecular weight, and was composed of fewer and more prominent spots. Spots in another group can be recognized because of their tendency to form elongated streaks. Such proteins (arrows) were found in all but the pupal intersegmental membrane, but with different properties. Those from larval cuticle form a distinct cluster slightly more basic than the most acidic group of spots. In extracts from adult intersegmental mem-
466
DIANA L. COX and JUDITH H. WILLIS
branes, proteins forming elongated streaks were interspersed with well defined spots in the most acidic group. In the pupal wing membrane, while such streaking spots had pIs equivalent to the larval group, most had apparent molecular weights double those of the other regions. There were minor spots shared between regions; larval dorsal abdomen and pupal intersegmental membrane shared six such spots. Lastly, high molecular weight material which remained close to the sample application site was observed in extracts from pupal intersegmental membranes. Flexible versus rigid cuticles. The preceding sections compared flexible or rigid cuticular regions using, for the first dimension, IEF gradients which gave best resolution for each type of cuticle (pH 4-6 for flexible cuticles and pH 5-8 for rigid cuticles). To help comparisons between flexible and rigid cuticles, extracts of nine regions from the three metamorphic stages were also compared in 2D gels in which the first dimension was from pH 3.5-10 IEF gels (gels not shown). With but two exceptions, none of the major spots were shared between the flexible and rigid cuticular regions. Proteins which formed elongated streaks occurred as major proteins in the flexible adult cuticles and as minor proteins in the adult medial sclerite. A set of minor spots was shared between the cuticles of the pupal sclerite and the intersegmental membranes.
DISCUSSION
Effects o f urea in protein electrophoresis Urea is routinely used for isoelectrofocusing of proteins when denaturation is not a problem. Shawky and Vincent (1978) found, as we have, that increased urea concentration in IEF gels resulted in greater resolution of cuticular proteins and they attributed this to changes in conformation and/or the aggregation of the proteins in the presence of urea. Proteins which shift in apparent isoelectric point in the presence of urea, as did our cuticular proteins, are generally assumed to have charged groups buried within the native structure which are exposed only under denaturing conditions (Ui, 1971a, b). In contrast to the relatively minor influence of urea (4-8 M) on the isoelectrofocusing separations, substantial changes in the mobilities of cuticular proteins occurred in SDS gels as the urea concentration was increased. The migration of different proteins was affected in different ways by the increased urea concentration, summarized in Fig. 2. Only the molecular weight standards and a few minor cuticular proteins showed no shift in mobility in relation to each other, all other cuticular proteins were profoundly affected by urea concentration. One explanation for the odd behavior of most cuticular proteins is that they had been incompletely saturated with SDS (Pau and Crambach, personal communication). We have eliminated this possibility by subjecting both the molecular weight standards and the cuticular protein extracts from the larval dorsal abdomen to varying times of boiling in SDS (0-60 min) and then running them on a standard SDS gel. There was no influence of treatment time on the
mobility of any of the proteins, although 60 min of boiling caused some minor degradation (unpublished observations). These changes in mobility of the cuticular proteins with increased concentrations of urea can be explained by the complex mechanism of urea denaturation (Ghelis and Yon, 1982). Denaturation by urea alone causes proteins to unfold by interacting with hydrophobic regions of the protein and increasing the protein's solubility in aqueous solutions. The degree to which urea causes proteins to unfold is affected by temperature, pH, and ionic strength of the solution (Creighton, 1979; Ghelis and Yon, 1982; Goldenberg and Creighton, 1984). The effect of each of these variables differs for each protein (Creighton, 1979). The apparent unfolding of the cuticular proteins was reversible since proteins focused in the unfolded state in 6.3 M urea IEF gels assumed lower molecular weights in SDS gels without urea than in gels with urea. Like urea, detergents such as SDS are also considered to be strong denaturants. As opposed to urea which disrupts the secondary structure of the protein, SDS may not completely unfold the protein but may instead form a highly ordered complex with the protein (Reynolds and Tanford, 1970). The use of both urea and SDS in gels has been found to improve the resolution of several proteins, including insect chorion proteins (Goldsmith ct al., 1979). The changes in mobility of cuticular proteins with increasing urea concentrations possibly can he explained by the unfolding of hydrophobic regions. Previous evidence for globular conformations of cuticular proteins comes from X-ray diffraction studies (Rudall and Kenchington, 1973). Properties o f individual cuticular proteins The preceding information and our results provide insights into the properties of the different groups of cuticular proteins from the larval dorsal abdomen. Some proteins (e.g. 13 and 14, Fig. 2) showed little change with increasing urea concentrations. Members of the charge train group of cuticular proteins changed their relative mobilities with increased urea concentration, and, therefore, may have different degrees of hydrophobicity, or alternatively, these differences may have been due to differences in glycosylation (Cox and Willis, 1987). The most acidic proteins (pls 4.4 -4.6) appeared to be completely unfolded near 6 M urea concentrations; whereas, the proteins with pls 4.8--5.0 were only unfolded at higher urea concentrations or with increased pH in the resolving gel. The streaking behavior of the proteins with pls 4.8 5.0 may have been due to the trapping of intermediates in the unfolding process (Goldenberg and Creighton, 1984). The shift in the mobilities of all the proteins, including the molecular weight markers, may, in part, be owing to the increased viscosity of the gel solvent with increased urea concentration which adds to the molecular sieving properties of the gel (Goldenberg and Creighton, 1984). The amino acid composition of individual cuticular proteins (Willis et al., 1981) raises questions about the role hydrophobicity plays in the urea-dependent shifts in molecular weight, for when net hydro-
Cuticular proteins
467
phobicity is calculated according to the method used by Andersen (1979), proteins showing major shifts are relatively hydrophilic.
separated rigid and flexible cuticles from the different stages, different conclusions might have been reached.
Cuticular proteins and functions In previous studies using IEF gels, we found different banding patterns from extracts of different cuticular regions of a single individual and suggested that the proteins could be related to the function of the cuticle (Cox and Willis, 1985). Chihara et al. (1982) and Roter et al. (1985) showed regional differences in cuticular protein composition in Drosophila melanogaster; Shawky and Vincent (1978) and Bordereau and Andersen (1978) found differences in proteins from locust and termite sclerites and intersegmental membranes. The restriction of particular proteins to specific layers within the cuticle has been reported by Andersen and Weis-Fogh (1964) and Wolfgang and Riddiford (1986). In H. cecropia larvae, the dorsal abdominal cuticle forms a barrier between the external environment and the insect's internal anatomy. This cuticle is thick, has limited permeability, and also provides for muscle attachment. The thin foregut cuticle is also a barrier on a more limited scale, protecting the animal against microbes. In this area, desiccation is less of a problem since contact with air is limited. In comparison to these two areas, the hindgut cuticle must also be permeable to water since the epidermal cells in this area take up water from the gut contents. These differences in function were found to correspond to differences in cuticular protein compositions. Most cuticular proteins from the foregut were a subset of the proteins from the dorsal abdomen and only contained a few unique minor spots. The hindgut, on the other hand, had only one spot in common with either the foregut or dorsal abdomen.
Significance of clusters of spots Clustering of chorion protein spots in 2D gels has been correlated with their membership in a multigene .family, more formally recognized by nucleotide and amino acid sequence data (see Goldsmith and Kafatos, 1984). In Drosophila, three of four larval cuticular protein genes that have been sequenced belong to a multigene family (Snyder et al., 1982). Sequences and amino acid compositions of Sarcophaga bullata cuticular proteins also indicate relatedness (Henzel et al., 1985). In the present study, 2D gels showed clustering of cuticular proteins similar to that seen for chorion proteins. Within each group, the proteins also behaved similarly under different gel conditions lending additional support for the existence of multigene families in H. cecropia cuticular proteins. It is probable that each protein within most of these clusters corresponds to a product of a single gene, since the only clusters with evidence of post-translational modifications are the charge trains and elongated streak proteins (Cox and Willis, 1987). In the flexible cuticular regions, there are three clusters or putative multigene families: the acidic proteins (pls 4.4~4.6, 20,000-24,000 daltons), proteins which formed elongated streaks, and those that formed charge trains. Additional evidence that the most acidic cuticular proteins belong to a multigene family has come from amino acid compositions (Willis et al., 1981). The flexible cuticular regions from pupae and adults had only subsets of this most acidic group (Fig. 6), and the larval hindgut had none of these spots. The observation that not all of these acidic spots were present in every flexible cuticle suggests that the synthesis of each protein is under independent regulation and/or that each protein of this group may have a unique function within the cuticular matrix. Formation of elongated streaks occurred in clusters of spots from every flexible cuticle examined except the pupal intersegmental membrane; however, the isoelectric points and molecular weights of these spots were variable among the regions. In addition to this unique behavior, these proteins also shared the characteristics of being released from gels by 7% acetic acid, 1 N nitric acid, and 50-95% ethanol (unpublished observations). Such streaking behavior was also found in some chorion proteins (Goldsmith et al., 1979); with both types of proteins, streaking could be reduced by increasing the ionic strength of the resolving gel buffer. The conformations of these proteins may be similar, and they may function in their respective matrices in similar ways. The extent of sequence similarity deserves further analysis. In the rigid cuticles, it is more difficult to distinguish discrete groups of proteins. The clusters are not tightly organized and do not have special characteristics, although a cluster of six major proteins was found in every region. Many of the minor, basic proteins with similar molecular weights also appeared to be shared among all the regions. Interestingly, they did not exhibit the independent regulation that was seen with the acidic proteins. A group of proteins (pIs
Lack of stage specificity in cuticular proteins Based upon isoelectrofocusing data, we have previously discounted claims that metamorphic stage is a major determinant of which cuticular proteins will be present (Cox and Willis, 1985). In the present study, data from 2D gels further strengthens the evidence that the synthesis of cuticular proteins is less related to metamorphic stage than to the function and properties of the cuticle. Flexible cuticles had many more spots with identical molecular weights and isoelecteric points than they shared with rigid cuticles. The similarity among the protein patterns of the rigid cuticles was even greater than the similarity among the flexible cuticles. Most of the major cuticular proteins were common to all rigid cuticles, irrespective of metamorphic stage. None of these shared spots was found in the extracts of flexible cuticles. The few unique spots found in each region may be related to other properties besides rigidity. Recent studies have reported stage-specific differences in the synthesis of lepidopteran cuticular proteins. Kiely and Riddiford (1985a,b) examined the synthesis of cuticular proteins using dorsal abdominal epidermis of Manduca larvae and pupae. In another study, Sridhara (1985) found about 40% of the mRNAs taken during cuticle synthesis from wings of second pupae or adults of A. polyphemus were unique. If these analyses had been based on
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5.0--6.0, 14,000-18,000 daltons) was f o u n d in every rigid cuticle examined, except for the p h a r a t e n o t u m . CONCLUSIONS F o r the analysis of cuticular proteins, I E F is a sensitive a n d reproducible technique a n d less affected by m i n o r fluctuations in gel c o m p o n e n t s t h a n SDS electrophoresis. Hence, this is an a p p r o p r i a t e technique if one wants to c o m p a r e a large n u m b e r of cuticular protein samples from different regions, meta m o r p h i c stages or species (Cox a n d Willis, 1985). The c o m b i n a t i o n of the two electrophoretic separations in 2D gels, however, offers significant inform a t i o n o n the c o m p o s i t i o n a n d properties of these proteins; but it is absolutely necessary to control all possible variables, especially p H and urea concentration, to o b t a i n consistent results. W h e n this is done, d a t a from 2D gels indicated t h a t greater similarity exists between the cuticular proteins of cuticles with similar mechanical properties and functions t h a n a m o n g dissimilar cuticles from the same stage. U n i q u e proteins f o u n d in various regions m a y confer u n i q u e properties to the cuticle and, hence, are not necessarily synthesized u n d e r stage-specific regulation. Acknowledgements--We thank Stanley Friedman, Ellis G. MacLeod and Richard N. Pau for their advice and appreciate the able technical assistance of Laura K. Moehling. This paper is based in part on a thesis submitted by D. L. C. for the doctoral degree at the University of Illinois at Urbana-Champaign. The research was supported by grant PCM 8201934 from the National Science Foundation. REFERENCES
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