Biochemical properties of a 24 kilodalton membrane glycoprotein antigen complex from Schistosoma mansoni

Molecular and Biochemical Parasitology, 31 (1988) 163-172 Elsevier

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Biochemical properties of a 24 kilodalton membrane glycoprotein antigen complex from Schistosoma mansoni S t e v e n R . K a r c z , B a r b a r a J. B a r n a r d a n d R o n B. P o d e s t a Cell Sciences Laboratory, Department of Zoology. University of Western Ontario, London, Ontario, Canada (Received 16 March 1988; accepted 15 June 1988)

Antibodies were affinity purified from crude antiserum by elution from the 24 kDa region of preparative one-dimensional Western blots containing immobilized adult Schistosoma mansoni inner bilayer membrane proteins. They were shown to be specific for a single acidic polypeptide complex, Smgp24, following immunoblotting from two-dimensional polyacrylamide gels. These antibodies were then used to detect the presence of the Smgp24 complex in fractions prepared from lectin affinity chromatography, phase separation in Triton X-114 and chemical and enzymatic carbohydrate modification treatments. The 24 kDa antigen was bound and specifically eluted from both concanavalin A and lentil lectin affinity matrices. In addition, the electrophoretic mobility of the antigen was shifted to approximately 20 kDa after treatment with endoglycosidase F and N-glycanase, but was not appreciably altered following treatment with endoglycosidase H, neuraminidase, or sodium meta-periodate. The 20 kDa species produced by endoglycosidase F or N-glycanase treatment no longer bound to the lectin affinity resins. The Smgp24 complex also partitioned almost quantitatively into the detergent-enriched phase after phase separation in Triton X-114 solutions. These results indicate that the Smgp24 complex is an antigenic integral membrane glycoprotein and may consist of a single polypeptide backbone which is extensively post- or co-translationally modified. Key words: Schistosoma mansoni; Membrane glycoprotein; Antigen characterization; Immunoblotting; Affinity-purified antibody

Introduction Interest in the characterization of surface glycoproteins of Schistosoma mansoni derives from the suggestion that these molecules may be targets of a protective immune response [1,2]. Current approaches using experimental models of schistosome immunity have shown that antibodies are formed against epitopes residing on surCorrespondence address: R.B. Podesta, Department of Zoology, University of Western Ontario, London. Ontario, Canada N6A 5B7. Abbreviations: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; IEF, isoelectric focussing; Con A. concanavalin A; Endo H, endoglycosidase H; Endo F, endoglycosidase F; BSA, bovine serum albumin; BC1P, bromochloroindolyl phosphate; NBT, nitro blue tetrazolium; RalB, rabbit anti-bilayer serum; MalB, mouse anti-inner bilayer serum; IMS, infected mouse serum; NMS, normal mouse serum; NRS, normal rabbit serum.

face proteins or glycoconjugates found on different developmental stages of the parasite [3,4]. In addition, other apparently non-membrane proteins have been implicated in the mediation of protective immunity [5-7]. It seems possible, therefore, that an effective molecular vaccine incorporating defined epitopes can be developed

I2,81. In addition to being the potential targets of protective immunity, membrane proteins are also involved in signal transduction, ion and molecular transport and immune evasion [9,10]. Despite this fact, very little is known about the biochemical properties of individual schistosome membrane proteins. In this light, we have used a technique [11,12] for the preparation of small quantities of specific antibodies in conjunction with immunoblotting methods to investigate some qualitative biochemical properties of an antigenic membrane glycoprotein complex which we have called Smgp24. We have studied this polypeptide

11166-6851/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

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complex with respect to its lectin binding characteristics, its sensitivity to carbohydrate modifying agents and its behaviour in solutions of Triton X-114 using microgram quantities of a defined apical membrane fraction [13,14] as starting material. As such, similar methods may be useful in the study of other schistosome membrane proteins for which antibody reagents are available. Materials and Methods

Chemicals and biochemicals. All chemicals were from standard sources and were reagent or analytical grade. Electrophoresis chemicals, blotting materials, nitrocellulose (0.2 Ixm pore size), bromochloroindolyl phosphate (BCIP), and nitro-blue tetrazolium (NBT) were obtained from BioRad. Alkaline phosphatase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were from Jackson Immunoresearch. Concanavalin A (Con A) agarose, lentil lectin agarose, Triton X114, sodium meta-periodate, neuraminidase and protease inhibitors were supplied by Sigma. Endoglycosidase H (Endo H) and endoglycosidase F (Endo F) were products of Boehringer Mannheim. N-Glycanase was from Genzyme. Prestained molecular weight markers from Bethesda Research Laboratories were used throughout the study and were visualized directly in gels or on blots. Parasites and membrane removal. Syrian hamsters were infected with approximately 1400 S. mansoni cercariae (Puerto Rican strain) obtained from light-stressed infected Biomphalaria glabrata which were supplied by the Center for Tropical Diseases, University of Lowell, MA. Adult parasites were eluted from the cut portal and mesenteric veins of infected hamsters and maintained on ice in Krebs-Ringer phosphate buffer p H 7.4, for one hour or less. Fractions enriched in outer and inner bilayer membranes from the apical m e m b r a n e complex of adult S. mansoni were obtained as previously described [13,14], using a buffered digitonin solution. Only the inner bilayer fraction was used in this study. Protein was estimated by the method of Bradford [15], using bovine serum albumin (BSA) as standard. Alkaline phosphatase activity was de-

termined colorimetrically using p-nitrophenyl phosphate as substrate.

Preparation of antisera. Crude, anti-inner bilayer antisera were raised in both rabbits and mice. New Zealand white rabbits were immunized with inner bilayer protein which was denatured by boiling with 2% sodium dodecyl sulfate (SDS) (w/v), 25 mM dithiothreitol in 100 mM Tris pH 6.8, and precipitated with five volumes of acetone. An initial injection of 1 mg of protein in Freund's complete adjuvant was administered followed by three subsequent injections of 500 ~xg protein in Freund's incomplete adjuvant after 3, 5, and 7 weeks. The animals were bled 7 days after the final boost and the sera stored at -70°C. These sera were called rabbit anti-inner bilayer sera (RalB). BALB/c mice were immunized following the same schedule. In this case, Freund's adjuvants were omitted, the proteins were not denatured or precipitated and the initial injection was 50 ~g while subsequent injections contained 25 txg protein. Sera were collected and stored at -70°C and were called mouse anti-inner bilayer sera (MedB). Infected mouse serum (IMS) was collected from BALB/c mice which were infected 11 weeks previously with 40 cercariae. Normal rabbit and normal mouse serum (NRS and NMS, respectively) were collected from animals which were injected with buffer alone, buffer in Freund's adjuvants or an irrelevant protein (rat transferrin) in Freund's adjuvants according to the same immunization schedule. Electrophoresis and immunoblotting. Inner bilayer membrane protein samples were analyzed by SDS polyacrylamide gel electrophoresis (SDSP A G E ) , using the buffer system of Laemmli [16], and gels of 12% acrylamide. Electroblotting of S D S - P A G E fractionated material to nitrocellulose was carried out using the transfer buffer previously described [17] for 1 h at 100 V. Two-dimensional electrophoresis was performed essentially as described by O'Farrell [18]. Nitrocellulose blots containing immobilized schistosome membrane proteins were processed using standard immunoblotting methods. Blots were reacted with either a 1/200 dilution of RcdB,

165 a 1/500 dilution of M~IB, a 1/100 dilution of IMS or affinity purified antibodies prepared as described below. Bound antibody was detected with a 1/5000 dilution of alkaline phosphatase-conjugated goat anti-rabbit or goat anti-mouse IgG and the B C I P / N B T substrate system. In some cases blots were stained in 0.1% amido black in 50% methanol, 10% acetic acid for 5 min and destained in 50% methanol, 10% acetic acid.

Affinity purification of antibodies specific for Smgp24. Antibodies specific for Smgp24 were prepared using a modification of published methods [11,12] involving low pH elution of antibodies from preparative Western blots. Blots were blocked, washed, and incubated in a 1/200 dilution of R~IB or a 1/500 dilution of M~IB. Antibodies binding to Smgp24 were then eluted by vigorous agitation for 1 rain in a solution of 50 mM glycine pH 2.8. The eluates were neutralized, and adjusted to 500 mM NaCI, 0.05% Tween 20, 1% gelatin, and 0.05% sodium azide. These antibodies are referred to as rabbit or mouse affinity purified antibodies.

Lectin affinity chromatography. Absorption of solubilized membrane glycoproteins to immobilized lectin affinity matrices was carried out on a small scale in batch fashion at room temperature. Inner bilayer membrane proteins were solubilized and adsorbed to concanavalin A (Con A) or lentil lectin agarose beads in 20 mM Tris pH 7.4, 150 mM NaCI, 0.1% Triton X-100, 1 mM CaCI 2, 1 mM MnC12. The beads were washed and eluted with 0.5 M ~-methylmannoside. Aiiquots of the unbound fractions and ~-methylmannoside eluates were analyzed by S D S - P A G E and immunoblotting using affinity purified antibodies.

aprotinin (each at 5 jxg ml ~) and 1 mM phenylmethylsulfonyl fluoride. These protease inhibitors had no effect on the reaction and were omitted in subsequent experiments. In some experiments, membrane samples which had been treated with Endo F or N-glycanase were boiled to inactivate the enzymes, and adsorbed to lectin affinity matrices as described above. Neuraminidase treatment was carried out on intact membranes in 50 mM sodium acetate pH 5.1. Enzyme concentration and incubation time was titrated over a tenfold range.

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Treatment of membranes with carbohydrate modifying agents. Digestion of membrane glycoconjugates with Endo H, Endo F and N-glycanase was carried out in 0.1 M sodium phosphate pH 6.5, 10 mM E D T A , 0.5% Triton X-100, 0.05% SDS, 1% 2-mercaptoethanol at 37°C. Optimal enzyme concentrations and incubation times were determined empirically for each enzyme. In initial experiments, the reaction mixture was supplemented with pepstatin, leupeptin, antipain, and

18 m Fig. 1. Immunoblotdetection of inner bilayer polypeptides of

S. rnansonirecognized by different mouse antisera. Lane 1, infected mouse serum (IMS), 1/100 dilution: lane 2, mouse anti-inner bilayer serum, 1/500dilution; lane 3, normal mouse serum (NMS), 1/100dilution. Each lane of the gel was loaded with 2 p~gprotein.

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Treatment with sodium meta-periodate was performed in 50 mM sodium acetate buffer. Periodate concentrations were titrated up to 100 mM and oxidations were performed for 1 h at room temperature in the dark at pH 4.5 and 5.8. The membranes were collected by centrifugation and then treated with excess sodium borohydride in 50 mM sodium phosphate pH 7.4, 150 mM NaCI (PBS) for 30 min at room temperature in the dark. All membrane samples treated with carbohydrate modifying agents were analyzed by SDSP A G E and immunoblotting using affinity purified antibodies.

Phase separation of inner bilayer proteins in Triton X-114. Stock Triton X-114 was pre-condensed and used as described by Bordier [19]. Proteins partitioning into the detergent-depleted phase were then precipitated with trichloroacetic acid in the presence of 5 p~g BSA, while those in the detergent-enriched phase were precipitated with acetone also in the presence of carrier BSA. Protein samples were then solubilized and analyzed by S D S - P A G E and immunoblotting using

affinity purified antibodies against the Smgp24 complex. Results

We first examined the profiles of inner bilayer polypeptides which were recognized by infection serum (IMS) and anti-inner bilayer serum (ModB). As illustrated in Fig. 1, lanes 1 and 2, these sera clearly recognized a unique subset of membrane-associated proteins. Both these antisera, however, also recognized common polypeptides of 190 kDa, 85 kDa, 65 kDa and a 24 kDa component with a diffuse migration pattern. Within the 24 kDa region, however, discrete bands could be resolved. No background bands could be detected using NMS (lane 3). The results from immunoblotting using RodB (not shown) also demonstrated that the 24 kDa complex was a major antigen in this fraction. In order to study the components and properties of the antigenic 24 kDa complex, we prepared antibodies by low pH elution from preparative one-dimensional Western blots containing the 24 kDa antigen which were probed with ModB

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Fig. 2. Two-dimensional blot analysis of S. mansoni inner bilayer polypeptides. (A) A m i d o black stained blot of inner bilaycr associated polypeptides. (B) Two-dimensional i m m u n o b l o t probed with rabbit affinity purified antibodies prepared as described in Materials and Methods. T h e first dimensional gel in A was loaded with 40 ~g protein while that in B was loaded with 5 p~g protein.

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or RcxIB. We then tested the specificity of these antibodies by two-dimensional electrophoresis of inner bilayer proteins followed by immunoblotting (Fig. 2B). Both rabbit and mouse affinity purified antibodies bound exclusively to a single antigen complex with approximate isoelectric points between 4.2 and 4.6 which corresponded to a major stained polypeptide complex with the same mobility properties (Fig. 2A). This complex exhibited migrational heterogeneity in both the isoelectric focusing (IEF) dimension and the SDS dimension. Using antibodies prepared in this manner, we were able to generate a rapid immunological signal on background-free immunoblots from both one- and two-dimensional gels. Analysis of lectin binding followed by immunoblotting using affinity-purified antibodies (Fig. 3), demonstrated that the 24 kDa polypeptide complex bound and was specifically eluted from both lentil lectin and Con A agarose affinity matrices (Fig. 3, lanes 3 and 5). Further investigation I

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18 m Fig. 3. Detection of Smgp24 in fractions from lectin affinity chromatography by i m m u n o b l o n i n g using m o u s e affinity purified antibodies. Lane 1, total inner bilayer protein; lane 2, unbound fraction; and lane 3, a-methylmannoside eluate from affinity c h r o m a t o g r a p h y on C o n A agarose. Lane 4, u n b o u n d fraction and lane 5, ~ - m e t h y l m a n n o s i d e eluate from chromatography on lentil lectin agarose. Each lane of the gel was loaded with approximately 500 ng inner bilayer protein.

of the glycoprotein nature of the 24 kDa antigen was studied following treatment with carbohydrate modifying agents and assaying for an electrophoretic mobility shift. Experiments using membranes treated with sodium meta-periodate (Fig. 4A) followed by the detection of the 24 kDa complex with affinity purified antibodies indicated that the mobility pattern of the complex was condensed and shifted slightly to a more rapidly migrating position. Treatment with neuraminidase or Endo H (Fig. 4B, lanes 1-4), did not induce a mobility shift. Identical results were obtained with these enzymes even upon extended incubation for up to 18 h. However, incubation of membranes with Endo F or N-glycanase resulted in the rapid appearance of a discrete 20 kDa component (Fig. 4B, lanes 5 and 6; Fig. 4C). The appearance of the 20 kDa species correlated with the disappearance of the 24 kDa complex under the indicated conditions. The presence of protease inhibitors in the incubation mixtures did not inhibit the appearance of the 20 kDa polypeptide during digestion with Endo F or N-glycanase. Moreover, the 20 kDa polypeptide produced by Endo F or N-glycanase treatment no longer bound to Con A agarose and was found primarily in the unbound fraction following lectin affinity adsorption experiments (Fig. 5, lanes 4 and 6). The 20 kDa species was also a minor component of the Con A eluates (Fig. 5, lanes 5 and 7). Since the 24 kDa complex was glycosylated and was associated with the inner bilayer membrane fraction, we investigated the nature of the membrane association by examination of the properties of the complex in solutions of Triton X-114 (Fig. 6). These experiments demonstrated that the 24 kDa complex could not be completely solubilized from the membrane by treatment with 1% Triton X-114 for 10-30 min on ice (Fig. 6, lane 2). However, the rest of the 24 kDa complex could be almost quantitatively recovered in the detergent-enriched phase (Fig. 6, lane 3), suggesting that it bound significant quantities of the detergent. In addition, no 24 kDa antigen could be detected in the detergent-depleted phase (Fig. 6, lane 4).

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Fig. 4. I m m u n o b l o t analysis of the effect of carbohydrate modifying agents on the electrophoretic mobility of the Smgp24 complex using m o u s e affinity purified antibodies. (A) Effect of sodium meta-periodate; m e m b r a n e s were treated with 0, 10, 50, or 100 mM periodate in lanes 1-4, respectively and analyzed by immunoblotting. (B) Effect of neuraminidase, Endo H, and Endo F: lane 1, treatment of m e m b r a n e s with 1 unit ml L neuraminidase and lane 2, undigested control. Lane 3, digestion of m e m b r a n e s with 0.25 units ml 1 Endo H and lane 4, undigested control. Lane 5, treatment of m e m b r a n e s with 1 unit/ml ~ Endo F and lane 6, undigested control. (C) Effect of N-glycanase; lane 1, undigested control and lane 2. treatment of m e m b r a n e s with 1 unit ml N-glycanase. All enzyme digestions were carried out at 37°C for 1 h. Each lane of the gel was loaded with approximately 500 ng inner bilayer protein.

Discussion

The characterization of adult schistosome membrane glycoproteins is important for at least two reasons. Firstly, it is a prerequisite for studies aimed at examining the structural role of the kDa

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surface membrane in development and immune evasion. Secondly, it has been shown that immunization with adult membrane glycoproteins can confer protection in the mouse model of schistosomiasis [20]. Consequently, biochemical study of individual membrane polypeptides has

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Fig. 5. I m m u n o b l o t analysis of the effect of Endo F and Nglycanase on the Con A binding properties of the Smgp24 complex using mouse affinity purified antibodies. Lane 1, total inner bilayer protein. Lane 2, u n b o u n d fraction and lane 3, c~-methylmannoside eluate from Con A chromatography of inner bilayer proteins incubated 1 h at 37°C in the absence of glycohydrolase. Lane 4, u n b o u n d fraction and lane 5, ~methylmannoside eluate from Con A chromatography using inner bilayer proteins treated for 1 h at 37°C with 1 unit ml Endo F. Lane 6. u n b o u n d fraction and lane 7, e~-methylmannoside e[uate from Con A chromatography using inner bilayer treated with 1 unit ml ~ N-glycanase for 1 h at 37°C. Each lane of the gel was loaded with approximately 500 ng inner bilayer protein.

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TEMPERATURE-INDUCED PHASE SEPARATION OF SCHISTOSOMA MANSONI MEMBRANE PROTEINS IN TRITON-X114 SOLUTION Isolated, washed H rna=nsoni

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Fig. 6. Phase partitioning of the Smgp24 complex in solutions of Triton X-114. (A) Flow diagram illustrating the method as applied to S. mansoni membrane proteins. (B) Immunoblot detection of gp24 in Triton X-114 phases using rabbit affinity purified antibodies. Lane 1, total inner bilayer proteins. Lane 2, Triton X-114 insoluble material, lane 3, detergent-enriched phase from Triton X-114 phase separation, lane 4, detergent-depleted phase from Triton X-114 phase separation. Lane 5, total inner bilayer proteins probed with NRS. Each lane of the gel was loaded with the entire fraction derived from phase separation using 2 ~g inner bilayer protein as starting material. implications for both vaccine development and the elucidation of biological function of individual membrane components. Using antibodies affinity purified from antigen immobilized on one-dimensional Western blots, we have described some qualitative biochemical properties of a 24 kDa antigen (Smgp24) which is a natural immunogen in a murine schistosome infection. The method with which antibodies were affinity purified merits some discussion. In initial experiments using serially diluted inner bilayer protein concentrations (not shown), we found that the immunological signal detectable at the lowest protein concentration tested was restricted to the 24 kDa region using either Rc~IB or McdB. This suggested that Smgp24 might be the most immunogenic component in this fraction as judged by Western blot analysis since no other bands could be detected at these low levels. This information was then used to calculate the protein concentration for the preparative Western blots from which antibodies were affinity purified.

Consequently, it is possible that we were selecting from the entire repertoire of antibodies against Smgp24, those with the most favourable binding properties for the antigen immobilized on Western blots. This may explain the specificity of the antibodies for Smgp24, the absence of detectable antibodies against other polypeptides in the 24 kDa region and the high sensitivity of the assay system. Smgp24 exhibited heterogeneity in both the IEF dimension and the SDS dimension on one- and two-dimensional gels. The heterogeneity in the SDS dimension is particularly evident on one-dimensional blots and is manifested by the presence of apparently related multiple bands. These electrophoretic properties are at least partly due to carbohydrate moieties since glycohydro[ase treatment abolishes the heterogeneity in the SDS dimension and can effect an electrophoretic shift to a more rapid relative mobility (20 kDa). Based on these data, we suggest that the Smgp24 complex which we have described consists of a single

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polypeptide backbone which is differentially postor co-translationally modified and that the 20 kDa species is a largely deglycosylated form of the polypeptide. The specificity of N-glycanase and Endo F and the effect of these enzymes on the electrophoretic mobility of Smgp24 suggest that presence of N-linked carbohydrate moieties. These oligosaccharides may be primarily of the "complex' class rather than the 'high mannose" class since Endo H had no effect using the mobility shift assay [21,22]. The magnitude of the electrophoretic shift probably reflects removal of carbohydrate chains from asparagine residues and conversion to aspartic acid residues. Given the limitations of the mobility shift assay, we were unable to convincingly demonstrate the presence of periodate or neuraminidase sensitive carbohydrate structures, since these treatments did not induce an appreciable change in electrophoretic mobility. In addition, we cannot rule out the presence of other oligosaccharide structures residing on Smgp24 since a small fraction of the 20 kDa species produced by glycohydrolase treatment retained the ability to bind to Con A agarose. The behaviour of the Smgp24 complex in solutions of Triton X-114 is consistent with that of integral membrane proteins [19]. There are at least two known possibilities which could explain the phase partitioning properties of Smgp24 in Triton X-114. It is possible that the Smgp24 complex possesses a transmembrane segment(s) or alternatively is attached to the membrane via a phosphatidylinositol glycolipid anchor [23]. These possibilities are not mutually exclusive, since at least one example is known [24], where a glycoprotein is thought to have both a transmembrane domain and a phospholipid anchor. Well-characterized membrane proteins with transmembrane domains as well as those with phospholipid attachment moieties are known to exhibit similar

properties in Triton X-l14 as those described here for Smgp24 [19,25]. The elucidation of the exact mechanism by which Smgp24 is anchored to the membrane may have important implications for its biochemical function. Polypeptides with similar electrophoretic and antibody recognition characteristics as Smgp24 have been described from S. mansoni. A 25 kDa molecule which can be Iodogen labelled from live lung stage as well as isolated adult membranes can be immunoprecipitated using chronic mouse infection serum and an anti-tegument membrane antiserum [26]. In addition, a 25 kDa membrane glycoprotein antigen has also been described [2(I]. The Smgp24 complex also has similar mobility on two dimensional gels as Sj23, a molecule from S. japonicum, which is the target of an immunodiagnostic monoclonal antibody [27,28]. However, the relationship of Smgp24 to these molecules will have to await further studies and sequence analysis of the corresponding cloned genes. In conclusion, we have characterized an antigenic membrane glycoprotein from S. mansoni with an apparent molecular mass of 24 kDa. While the Smgp24 complex is recognized by antibodies from chronically infected mice and by anti-inner bilayer antisera, the role of the polypeptide in the irradiated vaccine model of resistance has not yet been investigated. This, along with further structural studies will be required to elucidate a possible biological role for Smgp24 in S. mansoni.

Acknowledgements This study was supported by the Natural Sciences and Engineering Research Council of Canada. The authors would like to thank Michael Ansell for his gift of McdB and Dr. Michael Clarke for critical reading of the manuscript.

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3 Dalton, J.P. and Strand, M. (1987) Schistosoma mansoni polypeptides immunogenic in mice vaccinated with radiation-attenuated cercariae. J. Immunol. 139, 2474-2481. 40mer-Ali, P., Magee, A.I., Kelly, C. and Simpson, A.J.G. (1986) A major role for carbohydrate epitopes preferentially recognized by chronically infected mice in the deter-

171 mination of Sch&tosoma mansoni schistosomula surface antigenicity. J. Immunol. 137, 3601-3607. 5 Pearce, E.J., James, S.L., Dalton, J., Barrall, A., Ramos, C., Strand, M. and Sher, A. (1986) Immunochemical characterization and purification of Sm-97, a Schistosoma mansoni antigen monospecifically recognized by mice protectively immunized with a non-living vaccine. J. Immunol. 137, 3593-36(10. 6 Lanar, D.E., Pearce, E.J., James, S.L. and Sher, A. (1986) Identification of paramyosin as the schistosome antigen recognized by intradermally vaccinated mice. Science 234, 593-596. 7 Balloul, J.M., Sondermeyer, P., Dreyer, D., Capron, M., Grzych, J.M., Pierce, R.J., Carvallo, D., Lecocq, J.P. and Capron, A. (1987) Molecular cloning of a protective antigen of schistosomes. Nature 326, 149-153. 8 Capron, A., Dessaint, J.P., Capron, M., Ouma, J.H. and Butterworth, A.E. (1987) Immunity to schistosomes: Progress toward vaccine. Science 238, 1065-1072. 9 Cornford, E.M. (1987) Glucose metabolism in parasitic trematodes: Unique specializations of schistosomes. U C L A Symp. Mol. Cell. Biol. 60,457-476. 10 Podesta, R., Karcz, S., Ansell, M. and Silva, E. (1987) Schistosoma mansoni: Apical membrane/envelope synthesis, signal transduction and protein phosphorylation. UCLA Symp. Mol. Cell. Biol. 611,241-255. 11 Olmsted, J.B. (1981) Affinity purification of antibodies from diazotized paper blots of heterogeneous protein sampies. J. Biol. Chem. 256, 11955-11957. 12 Beall, J.A. and Mitchell, G.F. (1986) Identification of a particular antigen from a parasite cDNA library using antibodies affinity purified from selected portions of Western blots. J. Immunol. Methods 86, 217-223. 13 McDiarmid, S.S., Dean, L.L. and Podesta, R.B. (1983) Sequential removal of outer bilayer and apical plasma membrane from the surface epithelial syncytium of Schistosoma mansoni. Mol. Biochem. Parasitol. 7, 141-157. 14 Dean, L.L. and Podesta, R.B. (1984) Electrophoretic patterns of protein synthesis and turnover in apical plasma membrane and outer bilayer of Schistosoma mansoni. Biochim. Biophys. Acta 799, 106-114. 15 Bradford, M.M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72. 248-254.

16 Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 68/)-685. 17 Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350-4354. 18 O'Farrell, P.H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, ~07-4021. 19 Bordier, C. (1981) Phase separation of integral membrane proteins in Triton X-114 solution. J. Biol. Chem. 256, 1604-1607. 20 Kelly, C., Knight, M., Simpson, A., Hackett, F., Geisow, M. and Smithers, S.R. (1987) Purification and amino acid sequencing of Schistosoma mansoni surface antigens. UCLA Symp. Mol. Cell. Biol. 60, 257-266. 21 Trimble, R.B. and Maley, F. (1984) Optimizing hydrolysis of N-linked high mannose oligosaccharides by endo-~-Nacetyl-glucosaminidase H. Anal. Biochem. 141,515-522. 22 Chu. F.K. (1986) Requirements of cleavage of high mannose oligosaccharides in glycoproteins by peptide N-glycosidase F. J. Biol. Chem. 261, 172-177. 23 Cross, G.A.M. (1987) Eukaryotic protein modification and membrane attachmcnt via phosphatidybnositol. Cell 48, 179-181. 24 Dustin, M.L., Selvaraj, P., Mattaliano, R.J. and Springer, T.A. (1987) Anchoring mechanisms for LFA-3 cell adhesion glycoprotein at membrane surface. Nature 329, 846-848. 25 Conzelman, A., Spiazzi, A. and Bron, C. (1987) Glycolipid anchors are attached to Thy-1 glycoprotein rapidly after translation. Biochem. J. 246, 605-610. 26 Payares, G., McLaren, D.J., Evans, W.H. and Smithers, S.R. (1985) Antigenicity and immunogenicity of the tegumental outer membrane of adult Schistosoma mansoni. Parasite lmrnunol. 7, 45-61. 27 Mitchell, G.F. and Cruise, K.M. (1986) Schistosomiasis: antigens and host-parasite interactions. In: Parasite Antigens: Towards New Strategies for Vaccines (Pearson, T.W., ed.), pp. 275-316, Marcel Dekker, New York. 28 Cruisc, K.M., Mitchell, G.F., Garcia, E.G., Tiu, W.U., Hocking, R.E. and Anders, R.F. (1983) Sj23, the target antigen in Schistosoma japonicum adult worms of an immunodiagnostic hybridoma antibody. Parasite Immunol. 5, 37-46.