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A surface-labeled 18 kilodalton antigen in Schistosoma mansoni
Molecular and Biochemical Parasitology, 18 (1986) 55-67
55
Elsevier MBP 00614
A S U R F A C E - L A B E L E D 18 K I L O D A L T O N A N T I G E N IN S C H I S T O S O M A MANSONI
PIETRO LIBERTI, ALFREDO FESTUCCI, ANDREAS RUPPEL*, SALVATORE GIGANTE and DONATO CIOLI Institute of Cell Biology, CNR 18A Via RomagnosL 00196 Rome, Italy
(Received 18 April 1985; accepted 23 August 1985)
A mild surface-labeling procedure was applied to various developmental stages of Schistosoma mansoni. An 18 kDa protein was preferentially labeled in freshly transformed schistosomula.The labeled protein was equally present on skin-penetrated and mechanically prepared schistosomula and it disappeared upon digestion of intact parasites with proteolytic enzymes.The 18 kDa protein could be specificallyprecipitated with an antiserum raised against 3-h schistosomula. Six-daylung forms also presented a singlemajor labeled protein component, but the apparent molecular weight of this protein in acrylamide gels was higher than 18000. Fourteen-day-old and adult schistosomes showed only weak labeling distributed in several bands. The radioactivity pattern of adult worms (but not of schistosomula) could also be obtained by incubating fresh parasites in a medium which had previously been used to label schistosomes and to which a 100000-fold excess of 127Iover 125Ihad been added. Post-labeling incubation of parasites was found to be essential for the detection of stable surface proteins. Key words: Schistosoma mansoni; Surface antigens; Lactoperoxidase iodination
INTRODUCTION The schistosome surface is a site for i m p o r t a n t physiological a n d metabolic functions which are crucial for the survival of the parasite, e.g. reception of signals for m i g r a t i o n a n d pairing, a b s o r p t i o n , secretion, etc. In a d d i t i o n , surface structures represent the m a j o r interface between this i n t r a v a s c u l a r h e l m i n t h a n d the i m m u n e system of its m a m m a l i a n host. Thus, the study of surface m e m b r a n e s is aimed not only at a better description of a parasite area of m a j o r f u n c t i o n a l relevance, but also at a possible discovery of those m o l e c u l a r m e c h a n i s m s of adaptive e v o l u t i o n which have e n a b l e d the schistosomes to survive with e x t r a o r d i n a r y success in an i m m u n o l o g i c a l l y hostile e n v i r o n m e n t . C o n n e c t e d with the latter aspect, a more practical goal in the study of the schistosome surface is the possibility of identifying a n d isolating parasite * Present address: Institute of Immunology, University, Heidelberg, F.R.G. Abbreviations: EBSS, Earle's balanced salt solution; PBS, phosphate-buffered saline; SDS, sodium dodecyl
sulfate; NP-40, Nonidet P-40; PAGE, polyacrylamide gel electrophoresis.
56 antigens likely to act as effective immunogens for the induction of resistance to the infection. In recent years, several lines of evidence have contributed to the concept that juvenile forms of schistosomes are susceptible to immune attack, while older stages in the life cycle are more or less insensitive to immune mechanisms and thus result in survival of several years in the mesenteric veins of the host. Evidence from in vitro systems tends to restrict the period of larval vulnerability to the first few hours after skin penetration, while evidence from in vivo experiments tends, With some exception, to prolong this period to several days (reviewed in ref. 1). In any event, in the search for target antigens and potential immunogens, it seems reasonable to focus on the early stages of the parasite. Among the various chemical components of the schistosome surface, proteins are the most obvious object in the search for the critical immunogen(s), not only because in many systems they have been shown to fulfill such a role, but also because of the more practical reason that adequate technology is available for their identification, isolation and, possibly, large scale production. Surface labeling experiments of adult worms and young larvae have been reported by several authors in recent years [2-11]. The results are difficult to compare because of the different labeling protocols, isolation procedures and techniques of analysis used. In general, several different protein species are labeled both on the surface of adult worms and young schistosomula. On the basis of electrophoretic mobility, some of these proteins seem to be c o m m o n to different developmental stages. We report on some experiments of surface labeling performed with various stages of Schistosoma mansoni. Preliminary accounts of this work have appeared elsewhere [ 12]. MATERIALS AND METHODS The origin and the maintenance of the S. mansoni strain used in this study have been previously described [13]. Schistosomula were prepared either by skin penetration, or by syringe transformation. In vitro skin penetration was performed according to the technique of Clegg and Smithers [ 14]. Freshly isolated rat skin was employed. The medium in which schistosomula were collected consisted of Earle's balanced salt solution (EBSS) plus 0.5% lactalbumin hydrolyzate (E/lac) buffered with bicarbonate and containing no serum. Penetrated schistosomula were washed once in E/lac, resuspended in phosphate-buffered saline (PBS) and immediately used in the iodination reaction. The yield of schistosomula varied, in different experiments, between 10 and 20%. Syringe transformation was performed following the protocol of Ramalho-Pinto et al. [15]. The cercarial suspension was pelleted in the cold, EBSS was added to the sediment and the mixture passed 12 times through a 22G 11/4needle fitted to a 10-ml syringe. Tails were removed by repeated sedimentation at 1 X g and cercarial bodies were incubated for 3 h at 37°C in a 5% CO2 atmosphere in EBSS without serum. At the Parasite.
57 end of the incubation, schistosomula were washed once in PBS, suspended in the same medium and iodinated. Unless otherwise stated, mechanically prepared schistosomula were used throughout this study. The final yield of syringe schistosomula was usually around 50%. Six-day-old lung schistosomula were obtained from mice infected with 5 000 cercariae. The lungs of 20-25 mice were perfused, removed, minced and incubated at 37°C for 5 h in 200 ml of RPMI-1640 buffered with 25 mM Hepes. Lung pieces were eliminated by filtration through a 75 jam screen and schistosomula were further purified on a Percoll gradient [16]. A preliminary experiment showed that purification on Percoll gradients did not modify the labeling pattern of 3-h schistosomula. Fourteen-day-old and adult parasites were obtained by portal perfusion from mice infected with 2 500 and 150 cercariae, respectively.
Iodination. Unless otherwise stated, all iodinations were performed following the lactoperoxidase technique [17-19]. To 2.5 )< 104 schistosomula suspended in 280 jal PBS the following reagents were added: 5 jal of 5 )< 10-5 M KI; 10 jal lactoperoxidase (0.2 mg ml-1; Calbiochem) pre-dissolved in PBS and stored frozen in small aliquots; 50 jaCi Na125I (Amersham) in 5 jal; 2 jal 5 mM glucose; 2 jal glucose-oxidase (Sigma) corresponding to 2.8 U. The reaction was allowed to proceed for 10-15 min at room temperature in a conical glass tube. The organisms were then rapidly sedimented at 100 )< g for 1 min at room temperature. The pellet was washed once in Eagle's medium, resuspended in 10 ml of the same medium containing 10% newborn calf serum (Gibco) and incubated 1-3 h at 37°C in a 5% CO2 atmosphere in order to allow unreacted iodine and nonspecifically bound radioactivity to wash off from schistosomula. When the Bolton-Hunter reagent (New England Nuclear) was used, 2.5 × 104 larvae resuspended in PBS adjusted to pH 8.0 were added to 100 jaCi of dried reagent. After 10 min at room temperature, schistosomula were treated as in the case of the lactoperoxidase labeling. Viability of larvae, as judged by microscopic appearance and motility, was around 95% with either labeling technique.
Polyacrylamide gel electrophoresis (PAGE). At the end of the post-labeling incubation, parasites were collected by centrifugation and the pellet was resuspended in 100-jal sample buffer (62 mM Tris-HCl pH 6.8; 10% glycerol; 0.1 M dithiothreitol; 2% sodium dodecyl sulfate (SDS)), kept in a boiling water bath for 2 min and loaded on the gel in aliquots corresponding to 5 × 103 newly transformed schistosomula, 2.5 )< 10~ lung schistosomula, 5 X 102 14-day-old schistosomula, 2 male and 4 female adult worms. Gels were prepared and run according to published procedures [20,21]. Acrylamide concentrations varied between 12.5 and 14% in different gels. Continuous acrylamide gradients (10-25%) were also used. Gel staining was performed with Coomassie brilliant blue R-250 (Bio-Rad). Gels were dried and exposed to a Trimax type XD film (3M) in cassettes with T16 intensifying screens (3M). Exposure time varied between 2 h and 9 days at -70°C, according to the intensity of radioactive bands.
58 Two or more of the following molecular weight markers, either unlabeled or 125I-labeled, were employed in different experiments (Mr X 103 in parenthesis): lactoperoxidase (78); bovine serum albumin (67); ovalbumin (45); carbonic anhydrase (30); trypsin inhibitor (20.1); betalactoglobulin ( 18.4); horse myoglobin ( 17.2); lactalbumin (14.4); cytochrome c (11.7).
Treatment of surface-labeled schistosomula with proteolytic enzymes. Live labeled schistosomula were washed 3X with PBS at the end of the postlabeling incubation. 5 X 103 schistosomula were suspended in 100 lal PBS and incubated for 30 min at 37°C in the presence or absence of DNAase (75 tag ml-l; Calbiochem), chymotrypsin (500 lag ml-1; Worthington) or Staphylococcus aureus V 8 protease (500 ~tg ml-1; Miles). At the end of the incubation, control and enzyme-treated schistosomula were washed with PBS and analyzed by PAGE. Immunoprecipitation. 8 X 104 labeled schistosomula were extracted for 30 rain at 0°C with 300 lal of 1% Nonidet P-40 (NP-40) in 10 mM Tris-HCl pH 6.8 in the presence of 100 U m1-1 Trasylol (Bayer). After centrifugation at 200 X g for I min, the supernatant was adjusted to 1% SDS, kept for 2 min at 100°C and diluted with 5 vol of a buffer containing 5 mM Tris-HC1 pH 7.4; 0.15 M NaC1; 2 mM ethylenediaminotetraacetic acid; 100 U m1-1 Trasylol (buffer A). Aliquots corresponding to 104 schistosomula were incubated overnight at 4°C with 30 ~tl of normal, immune or infected serum. 30 mg (dry weight) of preswollen protein A-Sepharose beads (Pharmacia) were added and incubation was carried out for 24 h at 4°C. Sepharose beads were washed 3X with 0.1% SDS in buffer A and finally resuspended in 30 lal of sample buffer. The suspension was immersed for 3 min in a boiling water bath and the eluted material was analyzed by PAGE. Immune rabbit serum was obtained 9 days after the following course of four injections, each consisting of 2 X 105 schistosomula: the 1st injection was given subcutaneously in complete Freund's adjuvant; the second, 1 month later, in incomplete adjuvant; the 3rd, 10 days later, consisted of freeze-thawed schistosomula given intravenously; the 4th, 20 days later, consisted of live schistosomula injected intravenously. Infected rat serum was obtained 5 weeks after a triple infection (at monthly intervals) with 1 000 cercariae, and infected mouse serum from mice infected 10weeks previously with 100 cercariae. RESULTS Schistosomula, prepared by either the skin penetration or the syringe method, were labeled with 12sI using the lactoperoxidase technique. After the post-labeling incubation, schistosomula were analyzed by PAGE (Fig. la). Two major radioactive components were evident: one band running slightly ahead of the dye front, and a second band with an apparent M r of about 18 000. Minor components both of higher and lower M r could be seen, particularly after long autoradiographic exposures. The
59
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Fig. 1. Autoradiogramof SDS-PAGEof 3-h schistosomula. (a) Comparisonbetween skin-prepared (1) and syringe-prepared (2) schistosomula labeled with the lactoperoxidase technique. (b) Comparison between syringe-prepared schistosomula labeled with the lactoperoxidase technique (3) or with Bolton-Hunter reagent (4). The molecular weights (in kDa) of known standards run in gel (a) are indicated on the left.
radioactivity pattems of skin- and mechanically prepared schistosomula were very similar, except for a low M r band which was only present in mechanical schistosomula. Also, the addition of protease inhibitors (100 U m1-1Trasylol and 2 mM phenylmethylsulfonyl fluoride) did not modify the radioactivity pattern (results not shown). When the Bolton-Hunter reagent was used (Fig. lb), a much more extensive labeling was obtained. The two most heavily labeled bands appeared to possess the same mobility as the two major bands identified by the lactoperoxidase technique, but, in addition, at least 20 other distinct bands were detected with the Bolton-Hunter reagent. Further comparisons (e.g. by 2-dimensional gels or by peptide mapping) between the two components of about 18 kDa obtained by the two techniques were not attempted, considering the differing labeling sites, the different number of labeled residues and the different chemical structure of the radioactive moieties attached to the proteins. The major component running ahead of the gel front was identified with lipids, since it was extractable with organic solvents, as shown in Fig. 2. Lactoperoxidase-labeled schistosomula were vacuum dried and extracted twice with 90% ethanol for 10 min at 80°C. When the extracted and unextracted material was examined by PAGE, only the fast running spot turned out to be quantitatively and selectively removed by ethanol. To confirm the surface position of the 18 kDa protein, live labeled schistosomula
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10 Fig. 2. Autoradiogram of SDS-PAGE of 3-h schistosomula labeled with the lactoperoxidasetechnique. (I) Intact schistosomula; (2) residue after ethanol extraction; (3) ethanol fraction. Fig. 3. Autoradiogram of SDS-PAGE of 3-h schistosomula exposed to various enzymesafter labeling with the lactoperoxidase technique. (1) Undigested control; (2) DNAse; (3) chymotrypsin; (4) S. aureus V8 protease. were exposed to various enzymes for 30 min at 37°C. Fig. 3 shows that DNAse did not affect the 18 k D a protein (slot 2), while both chymotrypsin (slot 3) and S. a u r e u s V 8 protease (slot 4) caused the disappearance of the labeled band. None of the enzymes affected the overall protein pattern, as judged by Coomassie blue staining (not shown), and the lipid component running ahead of the dye front was also unmodified. To investigate the possible stage specificity of the 18 kDa protein present on the surface of 3-h schistosomula, 4 adult males, 8 adult females, 2.5 )< 104 3-h schistosomula, 1.5 × 104 6-day-old and 2.5 × 103 14-day-old worms were labeled in separate tubes and incubated for 2 h in serum-containing medium. Aliquots corresponding to similar protein contents were then examined on a 10-25% acrylamide gradient. As shown in Fig. 4, 3-h schistosomula (slot 3) exhibited the usual pattern with the 18 kDa protein as the dominant labeled component. On the contrary, very little radioactivity was bound to adult males (slot 1) and almost no radioactivity to adult females (slot 2). In particular, no bands were visible in the 18 k D a region. Six-day-old schistosomula (slot 4) showed a radioactivity pattern distinct from that exhibited by 3-h schistosomula. Radioactivity concentrated in the gel front and in a well-defined band running clearly behind the major component of3-h schistosomula. Two-dimensional gels (not shown)
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Fig. 4. Autoradiogram of a 10-25% acrylamide gradient of various S, mansoni stages labeled with the lactoperoxidase technique. (1) Adult males; (2) adult females; (3) 3-h schistosomula; (4) 6-day lung forms; (5) 14-day forms. Fig. 5. Autoradiogram of SDS-PAGE of different S, mansoni stages labeled with the lactoperoxidase technique, either separately or in the same tube. (1) Adult males labeled in the same tube as 3-h forms; (2) 3-h schistosomula labeled separately; (3) 3-h schistosomula labeled in the same tube as adult males. confirmed that the label of 6-day forms did not coincide with that of 3-h schistosomula. M i n o r c o m p o n e n t s of higher M r were also visible and seemed to be more prominent in 6-day forms. Since no attempts were made (e.g., by limited proteolysis) to confirm the surface location of the proteins labeled on lung schistosomula, their presumed surface nature rests, so far, only on the selectivity o f the technique used for labeling. Finally, 14-day w o r m s (slot 5) showed a radioactivity pattern similar to that o f adult worms in the sense that no prominent low M r protein, but only m i n o r components, especially in the high M r region, were observed. To rule out the possibility that efficient adult w o r m labeling might be inhibited by h e m o g l o b i n that may be present in the material regurgitated into the medium (A. Ruppel, Thesis, University of Freiburg, 1978), larvae were labeled either alone or mixed in the same tube with adult male worms. Fig. 5 shows that schistosomula gave essentially the same pattern in the two experimental conditions (slots 2 and 3). Adult worms iodinated in the same tube as schistosomula showed only faint bands throughout the gel and the usual intense b a n d in the position of lipids. Post-labeling incubation in serum-supplemented medium is an important step in
62 preventing artifactual sticking on the worm surface of proteins labeled in the reaction mixture. This is particularly true for adult worms. Fig. 6 shows the result of an experiment in which adult worms were surface-labeled with lactoperoxidase and examined by PAGE without further incubation (lane 1). The supernatant of this labeling reaction is analyzed in lane 2. Roughly 50 times more radioactivity was associated with the supernatant than with the worms themselves, so that - f o r the sake of illustration - only 1/50 of the sample was applied to lane 2. It can be appreciated that essentially all the radioactive bands which were present on adult worms were also present in the labeling supernatant. This observation suggested that the radioactivity associated with the worms might be the result of the adsorption.on the worm surface of proteins released by the worms and labeled in the reaction mixture. The possibility of absorption of labeled proteins onto schistosomes is illustrated by auto-iodinated lactoperoxidase. The enzyme represents by far the most heavily labeled component in the iodination supernatant and has the same mobility as the most heavily labeled schistosome protein. The other components labeled in the iodination supernatant are of parasite origin. Our interpretation that these proteins absorb back to the surface of schistosomes was confirmed by the results shown in lane 3. Unlabeled worms were incubated with an iodination supernatant to which nonradioactive K127I had been added at a final concentration of 10 mM. This represents a 100 000-fold excess over the radioactive Na125I and completely inhibits further iodination. It can be seen that the pattern of radioactivity sticking to the worms was virtually identical with the pattern obtained upon direct iodination of adult worms (compare lanes 1 and 3). A post-labeling incubation appreciably reduced the amount of radioactivity associated with either directly or indirectly labeled worms (compare lane 1 with 4 and lane 3 with 5). Labeled proteins were also present in the supernatant of the 3-h schistosomula iodination mixture (Fig. 7, slot 4). Two bands could be detected in the 18 kDa region. However, on the basis of bidimensional gels (not shown) these two bands appeared to be different from the 18 kDa protein labeled on 3-h schistosomula. More important, no radioactivity was associated with 3-h schistosomula after 15 min incubation of unlabeled larvae with a labeled supernatant, followed by a 2-h incubation in serumcontaining medium (Fig. 7, slot 3). Comparison of the radioactivity pattern ofschistosomula in which this final step was (slot 1) or was not (slot 2) performed, showed that post-labeling incubation not only abolished artifactual sticking of supernatant proteins, but also probably eliminated molecules with a fast turnover (as in the case of some components running ahead of the 18 k D a ) w i t h consequent selection of stable surface species. Labeled schistosomula were extracted with 1% NP-40 for 30 min at 0°C. Solubilized material was then precipitated with immune rabbit serum and with sera obtained from chronically infected mice and rats. As shown in Fig. 8, immune rabbit serum gave a significant precipitation of the 18 kDa component, whereas infected sera from mice and rats failed, under the conditions of the experiment, to specifically recognize the labeled band.
63
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Fig. 6. Autoradiogram of adult worms labeled with the lactoperoxidase technique. (1) Direct labeling of adult worms; (2) supernatant of the iodination reaction (the amount of supernatant analyzed on the gel corresponds to 1/50 of the material applied to the remaining lanes); (3) unlabeled adult worms incubated with the iodination supernatant shown in 2, after the addition of 10 mM K127I;(4) directly labeled adults, same as 1, after a 2-h post-labeling incubation; (5) indirectly-labeled adults, same as 3, after a 2-h post-labeling incubation. Fig. 7. Autoradiogram of SDS-PAGE of 3-h schistosomula labeled with the lactoperoxidase technique. (1) Standard condition with post-labeling incubation; (2) same as 1, but post-labeling incubation omitted; (3) unlabeled schistosomula incubated with the supernatant of the iodination reaction after the addition of 10 mM K127I; (4) supernatant of the iodination reaction. DISCUSSION T h e a p p l i c a t i o n o f the l a c t o p e r o x i d a s e i o d i n a t i o n t e c h n i q u e to n e w l y t r a n s f o r m e d s c h i s t o s o m u l a r e s u l t e d in a r e p r o d u c i b l e l a b e l i n g p a t t e r n u n d e r the e x p e r i m e n t a l c o n d i t i o n s e m p l o y e d in this s t u d y . R a d i o a c t i v i t y was p r e s e n t a l m o s t e x c l u s i v e l y in t w o r e g i o n s o f t h e gel: a h e a d o f t h e gel f r o n t a n d in a r e g i o n c o r r e s p o n d i n g to a M r o f a b o u t 18000. T h e m a t e r i a l at the gel f r o n t was i d e n t i f i e d w i t h lipids o n the basis o f its c o m p l e t e a n d selective e x t r a c t i o n w i t h e t h a n o l , thus l e a v i n g o n l y o n e m a j o r p r o t e i n b a n d l a b e l e d by s u r f a c e i o d i n a t i o n . M i n o r l a b e l e d c o m p o n e n t s c o u l d be d e t e c t e d u p o n longer autoradiographic
e x p o s u r e s , b u t t h e i r i n t e n s i t y was at least o n e o r d e r o f
m a g n i t u d e l o w e r t h a n the 18 k D a b a n d . S c h i s t o s o m u l a p r e p a r e d by skin p e n e t r a t i o n in v i t r o g a v e results w h i c h w e r e v e r y s i m i l a r to t h o s e o b t a i n e d w i t h the syringe t e c h n i q u e . T h a t the 18 k D a c o m p o n e n t was a s u r f a c e p r o t e i n was s t r o n g l y s u g g e s t e d
64
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Fig. 8. Autoradiogram of SDS-PAGE of immune precipitation with various sera of a NP-40 extract of surface-labeled 3-h schistosomula. Source of sera: (1) immune rabbit; (2) normal rabbit; (3) infected mouse; (4) normal mouse; (5) infected rat; (6) normal rat.
by its sensitivity to proteolysis in vivo, by the selectivity of the procedure which labeled only a very restricted number of components, and by the apparent viability of the organisms at the end of the procedure. The most conspicuous difference between this study and previous reports on the surface-labeling of schistosomes consists of the striking preferential (albeit not exclusive) labeling of a single protein species under our experimental conditions. This can be probably accounted for by a number of technical reasons. First of all, our use of 50 laCi iodide per reaction contrasts with the 10-20-fold higher concentrations reported by other investigators [2-11]. We chose to keep the molar ratio of iodide to proteins as low as possible in order to decrease the probability that the oxidation process might generate molecular iodine which could then permeate the tegumental membrane [22]. A low iodide concentration obviously gives a low amount of parasite-bound radioactivity, but the result may be qualitatively different from what could be obtained using, for instance, high iodide concentrations and short autoradiographic exposures. We tried to minimize tegumental damage by using very short exposures to plain PBS and by using enzymatically generated peroxide. The high parasite viability obtained at the end of the labeling procedures suggests that the amount oftegumental damage may have been rather limited under our conditions. The acrylamide concentration we used to analyze labeled proteins may also be relevant to the differences between ours and
65 previously reported results. A preferentially labeled 18 kDa protein can hardly be resolved by standard P A G E analysis if acrylamide concentrations lower than 12% are employed [2,5,7-9]. Since lipids migrate just ahead of the gel front and since they usually contain a large amount of radiolabel, their presence may easily obscure an adjacent low-M r component. This would encourage longer autoradiographic exposures and divert the attention from the preferentially labeled band. Finally, our protocol includes a post-labeling incubation in serum-containing medium, which contributes to reduce considerably the amount of 'unstable' radioactivity inevitably associated with schistosomula and, to a much greater extent, with adult schistosomes. This may represent a real problem, especially after drastic labeling conditions, as evidenced by the presence of a strong lactoperoxidase band in some of the previously published patterns [3,5]. In addition, we have demonstrated that, in the case of adult worms, most of the radioactivity associated with the schistosomes can be accounted for by molecules which may be labeled in the medium and subsequently absorbed to the parasite. The origin of these molecules within the schistosomes is unknown, in the sense that they may or may not be of surface origin. Their release from the worms has been shown to be triggered by the incubation in protein free medium (A. Ruppel, Thesis, University of Freiburg, 1978). The technical choices outlined above were made in an effort to use rational surfacelabeling criteria, but alternative choices may be equally legitimate in a field where the very concepts and definitions of surface molecules are still under debate. Since our results are clearly dependent on a defined set of experimental conditions, differences with previously published data are most likely due to different labeling protocols. The preferential labeling of one major protein species on the surface of 3-h schistosomula could be interpreted as an indication that the labeled protein possesses peculiar characteristics which are conducive to a high level of iodination, e.g. that the protein has a high content of exposed tyrosine residues. That this is probably not the case is shown by the fact that the B o l t o n - H u n t e r reagent, which binds to amino groups, also preferentially labeled a component of approximately 18 kDa, in addition to a large number of less prominent bands. This result suggests that the 18 k D a molecule is probably the most abundant protein exposed on the surface of 3-h schistosomula. It may be worth stressing that the 18 kDa component is not the only protein which is present on the surface of3-h schistosomula. As outlined in the Results, we routinely see on our radiograms several other bands, with mobilities compatible with those described by other investigators [5,6,9,11]. For some of these bands, experimental evidence for their surface location has been obtained with various criteria, and in one case a significant degree of protection has been achieved by passive administration of the corresponding monoclonal antibody [23]. Stage specificity seems to be an interesting feature of the 18 k D a component. No corresponding band was labeled on male or female adult worms and, in fact, when the post-labeling incubation was carried out, very little radioactivity was bound by adult schistosomes. Lipids, however, were substantially labeled. Since we have shown that
66 labeled proteins in the supernatant can stick to adult worms, the question may arise of whether surface labeling of adult schistosomes, as reported by several authors [2,5,8], is actually revealing surface molecules or secreted/excreted material present in the medium. Similarly, no 18 k D a c o m p o n e n t became labeled in 14-day-old schistosomula. Six-day-old lung forms presented an intriguing pattern, since a single protein was preferentially labeled, but its mobility in SDS was definitely (albeit slightly) lower than the 18 k D a of 3-h schistosomula. It is not possible to decide, with the data available so far, whether the 6-day b a n d represents a modification of the same protein labeled at 3 h, or whether it is a completely different molecule. In either case, stage-specific labeling obviously reflects the availability of the molecule to the reagents employed, and may be the result o f either the presence/absence of the protein or of its different exposure on the parasite surface. Interestingly, results published during the preparation o f this manuscript [11] listed the disappearance of a 17 k D a antigen and the appearance of a 20 k D a molecule a m o n g the changes observed in a complex pattern of surface-labeled schistosomula when 3-h forms were cultured for 48 h ih vitro. In the experiments reported here, sera from chronically infected animals failed to precipitate the 18 k D a protein of 3-h schistosomula. This result is o f dubious predictive value as to the immunological relevance of the 18 k D a protein, since such sera are never endowed with fully protective effects. The same protein, however, was specifically recognized by a serum from a rabbit immunized with 3-h larvae. These results could be the consequence of a short exposure of the 18 k D a protein on the surface of newly transformed schistosomula, in which case the immune system of infected hosts might be poorly stimulated. Additonal information is required on this point, as well as on the possible use of the stage-specific components described here as immunogens in host protection experiments. ACKNOWLEDGEMENTS This investigation received financial support from the U N D P / W o r l d B a n k / W H O Special P r o g r a m m e for Research and Training in Tropical Diseases. The authors are grateful to R o l a n d o M o r o n i for expert maintenance of the life cycle and to Adalberto Di Luzio for technical assistance. REFERENCES 1 2
3 4
Damian, R.T. (1984) Immunity in schistosomiasis: A holistic view. In: Contemporary Topics in lmmunobiology (Marchalonis, J.J., ed.), pp. 359-420, Plenum Publishing Corp., New York. Hayunga, E.G., Murrell, K.D., Taylor, D.W. and Vannier, W.E. (1979)Isolation and characterization of surface antigens from Schistosoma mansoni. I: Evaluation of techniques for radioisotope labeling of surface proteins from adult worms. J. Parasitol. 65, 488-496. Ramasamy, R. (1979) Surface proteins on schistosomula and cercariae of Schistosoma mansoni. Int. J. Parasitol. 9, 491-493. Brink,L.H., Krueger, K.L. and Harris, C. (1980) Stage-specific antigens of Schistosoma mansoni. In:
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The Host-Invader Interplay (Van den Bossche, H., ed.) pp. 393-404, Elsevier/North Holland Biomedical Press, Amsterdam. Snary, D., Smith, M.A. and C!egg, J.A. (1980) Surface proteins of Schistosoma mansoni and their expression during morphogenesis. Eur. J. Immunol. 10, 573-575. Dissous, C., Dissous, C. and Capron, A. (1981) Isolation and characterization of surface antigens from Schistosoma mansoni schistosomula. Mol. Biochem. Parasitol. 3, 215-225. Taylor, D.W., Hayunga, E.G. and Vannier, W.E. (1981) Surface antigens of Schistosoma mansoni. Mol. Biochem. Parasitol. 3, 157-168. Shah, J. and Ramasamy, R. (1982) Surface antigens on cercariae, schistosomula and adult worms of Schistosoma mansoni. Int. J. Parasitol. 12, 451-461. Simpson, A.J.G., James, S.L. and Sher, A. (1983) Identification of surface antigens of schistosomula of Schistosoma mansoni recognized by antibodies from mice immunized by chronic infection and by exposure to highly irradiated cercariae. Infect. Immun. 41, 591-597. Roberts, S.M., Aitken, R., Vojvodic, M., Wells, E. and Wilson, R.A. (1983) Identification of exposed components on the surface of adult Schistosoma mansoni by lactoperoxidase-catalysed iodination. Mol. Biochem. Parasitol. 9, 129-143. Simpson, A.J.G., Payares, G., Walker, T., Knight, M. and Smithers, S.R. (1984) The modulation of expression of polypeptide surface antigens on developing schistosomula of Schistosoma mansoni. 3. Immunol. 133, 2725-2730. Gigante, S., Ruppel, A. and Cioli, D. (1979) Marcatura di proteine superficiali di Schistosoma rnansoni mediante t251 e lattoperossidasi. Parassitologia 21, 111-114. Cioli, D. (1976) Transfer of Schistosoma mansoni into the mesenteric veins of hamsters. Int. J. Parasitol. 6, 349-354. Clegg, J.A. and Smithers, S.R. (1972) The effect of immune rhesus monkey serum on schistosomula of Schistosoma mansoni during cultivation in vitro. Int. J. Parasitol. 2, 79-98. Ramalho-Pinto, F.J., Gazzinelli, G., Howells, R.E., Mota-Santos, T.A. and Pellegrino, 3. (1974) Schistosoma mansoni: defined system for stepwise transformation of cercariae to schistosomula in vitro. Exp. Parasitol. 36, 360-372. Lazdins, J.K., Stein, M.J., David, J.R. and Sher, A. (1982) Schistosoma mansoni: Rapid isolation and purification of schistosomula of different development stages by centrifugation on discontinuous density gradients of Percoll. Exp. Parasitol. 53, 39-44. Marchalonis, J.J. (1969) An enzymatic method for the trace iodination of immunoglobulins and other proteins. Biochem. J. 113, 299-305. Hubbard, A.L. and Cohn, Z.A. (1972) The enzymatic iodination of the red cell membrane. J. Cell. Biol. 55, 390-405. Phillips, D.R. and Morrison, M. (1971) Exposed proteins on the intact human erythrocyte. Biochemistry 10, 1766-1771. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227, 680-685. Studier, F.W. (1973) Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J. Mol. Biol. 79, 237-248. Philipp, M. and Rumjaneck, F.D. (1984) Antigenic and dynamic properties of helminth surface structures. Mol. Biochem. Parasitol. 10, 245-268. Dissous, C., Grzych, J.M. and Capron, A. (1982) Schistosoma mansoni surface antigen defined by a rat monoclonal IgG2a. J. Immunol. 129, 2232-2234.
55
Elsevier MBP 00614
A S U R F A C E - L A B E L E D 18 K I L O D A L T O N A N T I G E N IN S C H I S T O S O M A MANSONI
PIETRO LIBERTI, ALFREDO FESTUCCI, ANDREAS RUPPEL*, SALVATORE GIGANTE and DONATO CIOLI Institute of Cell Biology, CNR 18A Via RomagnosL 00196 Rome, Italy
(Received 18 April 1985; accepted 23 August 1985)
A mild surface-labeling procedure was applied to various developmental stages of Schistosoma mansoni. An 18 kDa protein was preferentially labeled in freshly transformed schistosomula.The labeled protein was equally present on skin-penetrated and mechanically prepared schistosomula and it disappeared upon digestion of intact parasites with proteolytic enzymes.The 18 kDa protein could be specificallyprecipitated with an antiserum raised against 3-h schistosomula. Six-daylung forms also presented a singlemajor labeled protein component, but the apparent molecular weight of this protein in acrylamide gels was higher than 18000. Fourteen-day-old and adult schistosomes showed only weak labeling distributed in several bands. The radioactivity pattern of adult worms (but not of schistosomula) could also be obtained by incubating fresh parasites in a medium which had previously been used to label schistosomes and to which a 100000-fold excess of 127Iover 125Ihad been added. Post-labeling incubation of parasites was found to be essential for the detection of stable surface proteins. Key words: Schistosoma mansoni; Surface antigens; Lactoperoxidase iodination
INTRODUCTION The schistosome surface is a site for i m p o r t a n t physiological a n d metabolic functions which are crucial for the survival of the parasite, e.g. reception of signals for m i g r a t i o n a n d pairing, a b s o r p t i o n , secretion, etc. In a d d i t i o n , surface structures represent the m a j o r interface between this i n t r a v a s c u l a r h e l m i n t h a n d the i m m u n e system of its m a m m a l i a n host. Thus, the study of surface m e m b r a n e s is aimed not only at a better description of a parasite area of m a j o r f u n c t i o n a l relevance, but also at a possible discovery of those m o l e c u l a r m e c h a n i s m s of adaptive e v o l u t i o n which have e n a b l e d the schistosomes to survive with e x t r a o r d i n a r y success in an i m m u n o l o g i c a l l y hostile e n v i r o n m e n t . C o n n e c t e d with the latter aspect, a more practical goal in the study of the schistosome surface is the possibility of identifying a n d isolating parasite * Present address: Institute of Immunology, University, Heidelberg, F.R.G. Abbreviations: EBSS, Earle's balanced salt solution; PBS, phosphate-buffered saline; SDS, sodium dodecyl
sulfate; NP-40, Nonidet P-40; PAGE, polyacrylamide gel electrophoresis.
56 antigens likely to act as effective immunogens for the induction of resistance to the infection. In recent years, several lines of evidence have contributed to the concept that juvenile forms of schistosomes are susceptible to immune attack, while older stages in the life cycle are more or less insensitive to immune mechanisms and thus result in survival of several years in the mesenteric veins of the host. Evidence from in vitro systems tends to restrict the period of larval vulnerability to the first few hours after skin penetration, while evidence from in vivo experiments tends, With some exception, to prolong this period to several days (reviewed in ref. 1). In any event, in the search for target antigens and potential immunogens, it seems reasonable to focus on the early stages of the parasite. Among the various chemical components of the schistosome surface, proteins are the most obvious object in the search for the critical immunogen(s), not only because in many systems they have been shown to fulfill such a role, but also because of the more practical reason that adequate technology is available for their identification, isolation and, possibly, large scale production. Surface labeling experiments of adult worms and young larvae have been reported by several authors in recent years [2-11]. The results are difficult to compare because of the different labeling protocols, isolation procedures and techniques of analysis used. In general, several different protein species are labeled both on the surface of adult worms and young schistosomula. On the basis of electrophoretic mobility, some of these proteins seem to be c o m m o n to different developmental stages. We report on some experiments of surface labeling performed with various stages of Schistosoma mansoni. Preliminary accounts of this work have appeared elsewhere [ 12]. MATERIALS AND METHODS The origin and the maintenance of the S. mansoni strain used in this study have been previously described [13]. Schistosomula were prepared either by skin penetration, or by syringe transformation. In vitro skin penetration was performed according to the technique of Clegg and Smithers [ 14]. Freshly isolated rat skin was employed. The medium in which schistosomula were collected consisted of Earle's balanced salt solution (EBSS) plus 0.5% lactalbumin hydrolyzate (E/lac) buffered with bicarbonate and containing no serum. Penetrated schistosomula were washed once in E/lac, resuspended in phosphate-buffered saline (PBS) and immediately used in the iodination reaction. The yield of schistosomula varied, in different experiments, between 10 and 20%. Syringe transformation was performed following the protocol of Ramalho-Pinto et al. [15]. The cercarial suspension was pelleted in the cold, EBSS was added to the sediment and the mixture passed 12 times through a 22G 11/4needle fitted to a 10-ml syringe. Tails were removed by repeated sedimentation at 1 X g and cercarial bodies were incubated for 3 h at 37°C in a 5% CO2 atmosphere in EBSS without serum. At the Parasite.
57 end of the incubation, schistosomula were washed once in PBS, suspended in the same medium and iodinated. Unless otherwise stated, mechanically prepared schistosomula were used throughout this study. The final yield of syringe schistosomula was usually around 50%. Six-day-old lung schistosomula were obtained from mice infected with 5 000 cercariae. The lungs of 20-25 mice were perfused, removed, minced and incubated at 37°C for 5 h in 200 ml of RPMI-1640 buffered with 25 mM Hepes. Lung pieces were eliminated by filtration through a 75 jam screen and schistosomula were further purified on a Percoll gradient [16]. A preliminary experiment showed that purification on Percoll gradients did not modify the labeling pattern of 3-h schistosomula. Fourteen-day-old and adult parasites were obtained by portal perfusion from mice infected with 2 500 and 150 cercariae, respectively.
Iodination. Unless otherwise stated, all iodinations were performed following the lactoperoxidase technique [17-19]. To 2.5 )< 104 schistosomula suspended in 280 jal PBS the following reagents were added: 5 jal of 5 )< 10-5 M KI; 10 jal lactoperoxidase (0.2 mg ml-1; Calbiochem) pre-dissolved in PBS and stored frozen in small aliquots; 50 jaCi Na125I (Amersham) in 5 jal; 2 jal 5 mM glucose; 2 jal glucose-oxidase (Sigma) corresponding to 2.8 U. The reaction was allowed to proceed for 10-15 min at room temperature in a conical glass tube. The organisms were then rapidly sedimented at 100 )< g for 1 min at room temperature. The pellet was washed once in Eagle's medium, resuspended in 10 ml of the same medium containing 10% newborn calf serum (Gibco) and incubated 1-3 h at 37°C in a 5% CO2 atmosphere in order to allow unreacted iodine and nonspecifically bound radioactivity to wash off from schistosomula. When the Bolton-Hunter reagent (New England Nuclear) was used, 2.5 × 104 larvae resuspended in PBS adjusted to pH 8.0 were added to 100 jaCi of dried reagent. After 10 min at room temperature, schistosomula were treated as in the case of the lactoperoxidase labeling. Viability of larvae, as judged by microscopic appearance and motility, was around 95% with either labeling technique.
Polyacrylamide gel electrophoresis (PAGE). At the end of the post-labeling incubation, parasites were collected by centrifugation and the pellet was resuspended in 100-jal sample buffer (62 mM Tris-HCl pH 6.8; 10% glycerol; 0.1 M dithiothreitol; 2% sodium dodecyl sulfate (SDS)), kept in a boiling water bath for 2 min and loaded on the gel in aliquots corresponding to 5 × 103 newly transformed schistosomula, 2.5 )< 10~ lung schistosomula, 5 X 102 14-day-old schistosomula, 2 male and 4 female adult worms. Gels were prepared and run according to published procedures [20,21]. Acrylamide concentrations varied between 12.5 and 14% in different gels. Continuous acrylamide gradients (10-25%) were also used. Gel staining was performed with Coomassie brilliant blue R-250 (Bio-Rad). Gels were dried and exposed to a Trimax type XD film (3M) in cassettes with T16 intensifying screens (3M). Exposure time varied between 2 h and 9 days at -70°C, according to the intensity of radioactive bands.
58 Two or more of the following molecular weight markers, either unlabeled or 125I-labeled, were employed in different experiments (Mr X 103 in parenthesis): lactoperoxidase (78); bovine serum albumin (67); ovalbumin (45); carbonic anhydrase (30); trypsin inhibitor (20.1); betalactoglobulin ( 18.4); horse myoglobin ( 17.2); lactalbumin (14.4); cytochrome c (11.7).
Treatment of surface-labeled schistosomula with proteolytic enzymes. Live labeled schistosomula were washed 3X with PBS at the end of the postlabeling incubation. 5 X 103 schistosomula were suspended in 100 lal PBS and incubated for 30 min at 37°C in the presence or absence of DNAase (75 tag ml-l; Calbiochem), chymotrypsin (500 lag ml-1; Worthington) or Staphylococcus aureus V 8 protease (500 ~tg ml-1; Miles). At the end of the incubation, control and enzyme-treated schistosomula were washed with PBS and analyzed by PAGE. Immunoprecipitation. 8 X 104 labeled schistosomula were extracted for 30 rain at 0°C with 300 lal of 1% Nonidet P-40 (NP-40) in 10 mM Tris-HCl pH 6.8 in the presence of 100 U m1-1 Trasylol (Bayer). After centrifugation at 200 X g for I min, the supernatant was adjusted to 1% SDS, kept for 2 min at 100°C and diluted with 5 vol of a buffer containing 5 mM Tris-HC1 pH 7.4; 0.15 M NaC1; 2 mM ethylenediaminotetraacetic acid; 100 U m1-1 Trasylol (buffer A). Aliquots corresponding to 104 schistosomula were incubated overnight at 4°C with 30 ~tl of normal, immune or infected serum. 30 mg (dry weight) of preswollen protein A-Sepharose beads (Pharmacia) were added and incubation was carried out for 24 h at 4°C. Sepharose beads were washed 3X with 0.1% SDS in buffer A and finally resuspended in 30 lal of sample buffer. The suspension was immersed for 3 min in a boiling water bath and the eluted material was analyzed by PAGE. Immune rabbit serum was obtained 9 days after the following course of four injections, each consisting of 2 X 105 schistosomula: the 1st injection was given subcutaneously in complete Freund's adjuvant; the second, 1 month later, in incomplete adjuvant; the 3rd, 10 days later, consisted of freeze-thawed schistosomula given intravenously; the 4th, 20 days later, consisted of live schistosomula injected intravenously. Infected rat serum was obtained 5 weeks after a triple infection (at monthly intervals) with 1 000 cercariae, and infected mouse serum from mice infected 10weeks previously with 100 cercariae. RESULTS Schistosomula, prepared by either the skin penetration or the syringe method, were labeled with 12sI using the lactoperoxidase technique. After the post-labeling incubation, schistosomula were analyzed by PAGE (Fig. la). Two major radioactive components were evident: one band running slightly ahead of the dye front, and a second band with an apparent M r of about 18 000. Minor components both of higher and lower M r could be seen, particularly after long autoradiographic exposures. The
59
a
2
I b
3
4
67 45
30
20.1 14.4
Fig. 1. Autoradiogramof SDS-PAGEof 3-h schistosomula. (a) Comparisonbetween skin-prepared (1) and syringe-prepared (2) schistosomula labeled with the lactoperoxidase technique. (b) Comparison between syringe-prepared schistosomula labeled with the lactoperoxidase technique (3) or with Bolton-Hunter reagent (4). The molecular weights (in kDa) of known standards run in gel (a) are indicated on the left.
radioactivity pattems of skin- and mechanically prepared schistosomula were very similar, except for a low M r band which was only present in mechanical schistosomula. Also, the addition of protease inhibitors (100 U m1-1Trasylol and 2 mM phenylmethylsulfonyl fluoride) did not modify the radioactivity pattern (results not shown). When the Bolton-Hunter reagent was used (Fig. lb), a much more extensive labeling was obtained. The two most heavily labeled bands appeared to possess the same mobility as the two major bands identified by the lactoperoxidase technique, but, in addition, at least 20 other distinct bands were detected with the Bolton-Hunter reagent. Further comparisons (e.g. by 2-dimensional gels or by peptide mapping) between the two components of about 18 kDa obtained by the two techniques were not attempted, considering the differing labeling sites, the different number of labeled residues and the different chemical structure of the radioactive moieties attached to the proteins. The major component running ahead of the gel front was identified with lipids, since it was extractable with organic solvents, as shown in Fig. 2. Lactoperoxidase-labeled schistosomula were vacuum dried and extracted twice with 90% ethanol for 10 min at 80°C. When the extracted and unextracted material was examined by PAGE, only the fast running spot turned out to be quantitatively and selectively removed by ethanol. To confirm the surface position of the 18 kDa protein, live labeled schistosomula
60
1
2
3
1
2
3
4
m
10 Fig. 2. Autoradiogram of SDS-PAGE of 3-h schistosomula labeled with the lactoperoxidasetechnique. (I) Intact schistosomula; (2) residue after ethanol extraction; (3) ethanol fraction. Fig. 3. Autoradiogram of SDS-PAGE of 3-h schistosomula exposed to various enzymesafter labeling with the lactoperoxidase technique. (1) Undigested control; (2) DNAse; (3) chymotrypsin; (4) S. aureus V8 protease. were exposed to various enzymes for 30 min at 37°C. Fig. 3 shows that DNAse did not affect the 18 k D a protein (slot 2), while both chymotrypsin (slot 3) and S. a u r e u s V 8 protease (slot 4) caused the disappearance of the labeled band. None of the enzymes affected the overall protein pattern, as judged by Coomassie blue staining (not shown), and the lipid component running ahead of the dye front was also unmodified. To investigate the possible stage specificity of the 18 kDa protein present on the surface of 3-h schistosomula, 4 adult males, 8 adult females, 2.5 )< 104 3-h schistosomula, 1.5 × 104 6-day-old and 2.5 × 103 14-day-old worms were labeled in separate tubes and incubated for 2 h in serum-containing medium. Aliquots corresponding to similar protein contents were then examined on a 10-25% acrylamide gradient. As shown in Fig. 4, 3-h schistosomula (slot 3) exhibited the usual pattern with the 18 kDa protein as the dominant labeled component. On the contrary, very little radioactivity was bound to adult males (slot 1) and almost no radioactivity to adult females (slot 2). In particular, no bands were visible in the 18 k D a region. Six-day-old schistosomula (slot 4) showed a radioactivity pattern distinct from that exhibited by 3-h schistosomula. Radioactivity concentrated in the gel front and in a well-defined band running clearly behind the major component of3-h schistosomula. Two-dimensional gels (not shown)
61
1
2
3
4
ii~iiii
iiiiii!!
5
2
3
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Fig. 4. Autoradiogram of a 10-25% acrylamide gradient of various S, mansoni stages labeled with the lactoperoxidase technique. (1) Adult males; (2) adult females; (3) 3-h schistosomula; (4) 6-day lung forms; (5) 14-day forms. Fig. 5. Autoradiogram of SDS-PAGE of different S, mansoni stages labeled with the lactoperoxidase technique, either separately or in the same tube. (1) Adult males labeled in the same tube as 3-h forms; (2) 3-h schistosomula labeled separately; (3) 3-h schistosomula labeled in the same tube as adult males. confirmed that the label of 6-day forms did not coincide with that of 3-h schistosomula. M i n o r c o m p o n e n t s of higher M r were also visible and seemed to be more prominent in 6-day forms. Since no attempts were made (e.g., by limited proteolysis) to confirm the surface location of the proteins labeled on lung schistosomula, their presumed surface nature rests, so far, only on the selectivity o f the technique used for labeling. Finally, 14-day w o r m s (slot 5) showed a radioactivity pattern similar to that o f adult worms in the sense that no prominent low M r protein, but only m i n o r components, especially in the high M r region, were observed. To rule out the possibility that efficient adult w o r m labeling might be inhibited by h e m o g l o b i n that may be present in the material regurgitated into the medium (A. Ruppel, Thesis, University of Freiburg, 1978), larvae were labeled either alone or mixed in the same tube with adult male worms. Fig. 5 shows that schistosomula gave essentially the same pattern in the two experimental conditions (slots 2 and 3). Adult worms iodinated in the same tube as schistosomula showed only faint bands throughout the gel and the usual intense b a n d in the position of lipids. Post-labeling incubation in serum-supplemented medium is an important step in
62 preventing artifactual sticking on the worm surface of proteins labeled in the reaction mixture. This is particularly true for adult worms. Fig. 6 shows the result of an experiment in which adult worms were surface-labeled with lactoperoxidase and examined by PAGE without further incubation (lane 1). The supernatant of this labeling reaction is analyzed in lane 2. Roughly 50 times more radioactivity was associated with the supernatant than with the worms themselves, so that - f o r the sake of illustration - only 1/50 of the sample was applied to lane 2. It can be appreciated that essentially all the radioactive bands which were present on adult worms were also present in the labeling supernatant. This observation suggested that the radioactivity associated with the worms might be the result of the adsorption.on the worm surface of proteins released by the worms and labeled in the reaction mixture. The possibility of absorption of labeled proteins onto schistosomes is illustrated by auto-iodinated lactoperoxidase. The enzyme represents by far the most heavily labeled component in the iodination supernatant and has the same mobility as the most heavily labeled schistosome protein. The other components labeled in the iodination supernatant are of parasite origin. Our interpretation that these proteins absorb back to the surface of schistosomes was confirmed by the results shown in lane 3. Unlabeled worms were incubated with an iodination supernatant to which nonradioactive K127I had been added at a final concentration of 10 mM. This represents a 100 000-fold excess over the radioactive Na125I and completely inhibits further iodination. It can be seen that the pattern of radioactivity sticking to the worms was virtually identical with the pattern obtained upon direct iodination of adult worms (compare lanes 1 and 3). A post-labeling incubation appreciably reduced the amount of radioactivity associated with either directly or indirectly labeled worms (compare lane 1 with 4 and lane 3 with 5). Labeled proteins were also present in the supernatant of the 3-h schistosomula iodination mixture (Fig. 7, slot 4). Two bands could be detected in the 18 kDa region. However, on the basis of bidimensional gels (not shown) these two bands appeared to be different from the 18 kDa protein labeled on 3-h schistosomula. More important, no radioactivity was associated with 3-h schistosomula after 15 min incubation of unlabeled larvae with a labeled supernatant, followed by a 2-h incubation in serumcontaining medium (Fig. 7, slot 3). Comparison of the radioactivity pattern ofschistosomula in which this final step was (slot 1) or was not (slot 2) performed, showed that post-labeling incubation not only abolished artifactual sticking of supernatant proteins, but also probably eliminated molecules with a fast turnover (as in the case of some components running ahead of the 18 k D a ) w i t h consequent selection of stable surface species. Labeled schistosomula were extracted with 1% NP-40 for 30 min at 0°C. Solubilized material was then precipitated with immune rabbit serum and with sera obtained from chronically infected mice and rats. As shown in Fig. 8, immune rabbit serum gave a significant precipitation of the 18 kDa component, whereas infected sera from mice and rats failed, under the conditions of the experiment, to specifically recognize the labeled band.
63
1
iiii
2
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4
!
~i)~!i)i~i)i?
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Fig. 6. Autoradiogram of adult worms labeled with the lactoperoxidase technique. (1) Direct labeling of adult worms; (2) supernatant of the iodination reaction (the amount of supernatant analyzed on the gel corresponds to 1/50 of the material applied to the remaining lanes); (3) unlabeled adult worms incubated with the iodination supernatant shown in 2, after the addition of 10 mM K127I;(4) directly labeled adults, same as 1, after a 2-h post-labeling incubation; (5) indirectly-labeled adults, same as 3, after a 2-h post-labeling incubation. Fig. 7. Autoradiogram of SDS-PAGE of 3-h schistosomula labeled with the lactoperoxidase technique. (1) Standard condition with post-labeling incubation; (2) same as 1, but post-labeling incubation omitted; (3) unlabeled schistosomula incubated with the supernatant of the iodination reaction after the addition of 10 mM K127I; (4) supernatant of the iodination reaction. DISCUSSION T h e a p p l i c a t i o n o f the l a c t o p e r o x i d a s e i o d i n a t i o n t e c h n i q u e to n e w l y t r a n s f o r m e d s c h i s t o s o m u l a r e s u l t e d in a r e p r o d u c i b l e l a b e l i n g p a t t e r n u n d e r the e x p e r i m e n t a l c o n d i t i o n s e m p l o y e d in this s t u d y . R a d i o a c t i v i t y was p r e s e n t a l m o s t e x c l u s i v e l y in t w o r e g i o n s o f t h e gel: a h e a d o f t h e gel f r o n t a n d in a r e g i o n c o r r e s p o n d i n g to a M r o f a b o u t 18000. T h e m a t e r i a l at the gel f r o n t was i d e n t i f i e d w i t h lipids o n the basis o f its c o m p l e t e a n d selective e x t r a c t i o n w i t h e t h a n o l , thus l e a v i n g o n l y o n e m a j o r p r o t e i n b a n d l a b e l e d by s u r f a c e i o d i n a t i o n . M i n o r l a b e l e d c o m p o n e n t s c o u l d be d e t e c t e d u p o n longer autoradiographic
e x p o s u r e s , b u t t h e i r i n t e n s i t y was at least o n e o r d e r o f
m a g n i t u d e l o w e r t h a n the 18 k D a b a n d . S c h i s t o s o m u l a p r e p a r e d by skin p e n e t r a t i o n in v i t r o g a v e results w h i c h w e r e v e r y s i m i l a r to t h o s e o b t a i n e d w i t h the syringe t e c h n i q u e . T h a t the 18 k D a c o m p o n e n t was a s u r f a c e p r o t e i n was s t r o n g l y s u g g e s t e d
64
1
2
imm~
in
3
m
4
imb
5
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6
,~
~
Fig. 8. Autoradiogram of SDS-PAGE of immune precipitation with various sera of a NP-40 extract of surface-labeled 3-h schistosomula. Source of sera: (1) immune rabbit; (2) normal rabbit; (3) infected mouse; (4) normal mouse; (5) infected rat; (6) normal rat.
by its sensitivity to proteolysis in vivo, by the selectivity of the procedure which labeled only a very restricted number of components, and by the apparent viability of the organisms at the end of the procedure. The most conspicuous difference between this study and previous reports on the surface-labeling of schistosomes consists of the striking preferential (albeit not exclusive) labeling of a single protein species under our experimental conditions. This can be probably accounted for by a number of technical reasons. First of all, our use of 50 laCi iodide per reaction contrasts with the 10-20-fold higher concentrations reported by other investigators [2-11]. We chose to keep the molar ratio of iodide to proteins as low as possible in order to decrease the probability that the oxidation process might generate molecular iodine which could then permeate the tegumental membrane [22]. A low iodide concentration obviously gives a low amount of parasite-bound radioactivity, but the result may be qualitatively different from what could be obtained using, for instance, high iodide concentrations and short autoradiographic exposures. We tried to minimize tegumental damage by using very short exposures to plain PBS and by using enzymatically generated peroxide. The high parasite viability obtained at the end of the labeling procedures suggests that the amount oftegumental damage may have been rather limited under our conditions. The acrylamide concentration we used to analyze labeled proteins may also be relevant to the differences between ours and
65 previously reported results. A preferentially labeled 18 kDa protein can hardly be resolved by standard P A G E analysis if acrylamide concentrations lower than 12% are employed [2,5,7-9]. Since lipids migrate just ahead of the gel front and since they usually contain a large amount of radiolabel, their presence may easily obscure an adjacent low-M r component. This would encourage longer autoradiographic exposures and divert the attention from the preferentially labeled band. Finally, our protocol includes a post-labeling incubation in serum-containing medium, which contributes to reduce considerably the amount of 'unstable' radioactivity inevitably associated with schistosomula and, to a much greater extent, with adult schistosomes. This may represent a real problem, especially after drastic labeling conditions, as evidenced by the presence of a strong lactoperoxidase band in some of the previously published patterns [3,5]. In addition, we have demonstrated that, in the case of adult worms, most of the radioactivity associated with the schistosomes can be accounted for by molecules which may be labeled in the medium and subsequently absorbed to the parasite. The origin of these molecules within the schistosomes is unknown, in the sense that they may or may not be of surface origin. Their release from the worms has been shown to be triggered by the incubation in protein free medium (A. Ruppel, Thesis, University of Freiburg, 1978). The technical choices outlined above were made in an effort to use rational surfacelabeling criteria, but alternative choices may be equally legitimate in a field where the very concepts and definitions of surface molecules are still under debate. Since our results are clearly dependent on a defined set of experimental conditions, differences with previously published data are most likely due to different labeling protocols. The preferential labeling of one major protein species on the surface of 3-h schistosomula could be interpreted as an indication that the labeled protein possesses peculiar characteristics which are conducive to a high level of iodination, e.g. that the protein has a high content of exposed tyrosine residues. That this is probably not the case is shown by the fact that the B o l t o n - H u n t e r reagent, which binds to amino groups, also preferentially labeled a component of approximately 18 kDa, in addition to a large number of less prominent bands. This result suggests that the 18 k D a molecule is probably the most abundant protein exposed on the surface of 3-h schistosomula. It may be worth stressing that the 18 kDa component is not the only protein which is present on the surface of3-h schistosomula. As outlined in the Results, we routinely see on our radiograms several other bands, with mobilities compatible with those described by other investigators [5,6,9,11]. For some of these bands, experimental evidence for their surface location has been obtained with various criteria, and in one case a significant degree of protection has been achieved by passive administration of the corresponding monoclonal antibody [23]. Stage specificity seems to be an interesting feature of the 18 k D a component. No corresponding band was labeled on male or female adult worms and, in fact, when the post-labeling incubation was carried out, very little radioactivity was bound by adult schistosomes. Lipids, however, were substantially labeled. Since we have shown that
66 labeled proteins in the supernatant can stick to adult worms, the question may arise of whether surface labeling of adult schistosomes, as reported by several authors [2,5,8], is actually revealing surface molecules or secreted/excreted material present in the medium. Similarly, no 18 k D a c o m p o n e n t became labeled in 14-day-old schistosomula. Six-day-old lung forms presented an intriguing pattern, since a single protein was preferentially labeled, but its mobility in SDS was definitely (albeit slightly) lower than the 18 k D a of 3-h schistosomula. It is not possible to decide, with the data available so far, whether the 6-day b a n d represents a modification of the same protein labeled at 3 h, or whether it is a completely different molecule. In either case, stage-specific labeling obviously reflects the availability of the molecule to the reagents employed, and may be the result o f either the presence/absence of the protein or of its different exposure on the parasite surface. Interestingly, results published during the preparation o f this manuscript [11] listed the disappearance of a 17 k D a antigen and the appearance of a 20 k D a molecule a m o n g the changes observed in a complex pattern of surface-labeled schistosomula when 3-h forms were cultured for 48 h ih vitro. In the experiments reported here, sera from chronically infected animals failed to precipitate the 18 k D a protein of 3-h schistosomula. This result is o f dubious predictive value as to the immunological relevance of the 18 k D a protein, since such sera are never endowed with fully protective effects. The same protein, however, was specifically recognized by a serum from a rabbit immunized with 3-h larvae. These results could be the consequence of a short exposure of the 18 k D a protein on the surface of newly transformed schistosomula, in which case the immune system of infected hosts might be poorly stimulated. Additonal information is required on this point, as well as on the possible use of the stage-specific components described here as immunogens in host protection experiments. ACKNOWLEDGEMENTS This investigation received financial support from the U N D P / W o r l d B a n k / W H O Special P r o g r a m m e for Research and Training in Tropical Diseases. The authors are grateful to R o l a n d o M o r o n i for expert maintenance of the life cycle and to Adalberto Di Luzio for technical assistance. REFERENCES 1 2
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