Paramyosin isoforms of Schistosoma mansoni are phosphorylated and localized in a large variety of muscle types

Jürgen SCHMIDT 2, Otto BODOR 1, Lutz GOHR 1 and Werner KUNZ 1

1 Institute of Genetics and Biological-Medical Research Center, Heinrich-Heine-University, Universitäts-Straße 1, D-40225 Düsseldorf, Germany 2 Institute of Zoology, Heinrich-Heine-University, Universitäts-Straße 1, D-40225 Düsseldorf, Germany

Key words: Schistosoma mansoni, paramyosin, tegument, muscle, phosphorylation.

SUMMARY

Paramyosin, although a widely distributed muscle component among invertebrates, has hitherto not clearly been shown to occur in the muscles of schistosomes. Instead, it has been reported to occur in the tegument. In the present study, a specific antibody reacting with each of ten isoforms of paramyosin was used for light microscopical immunolocalization in sections of Schistosoma mansoni. Specimens were fixed by a new method to immobilize antigens with uranyl acetate-trehalose-methanol. In cercariae, schistosomula, and adults, the circular and longitudinal muscles of the body wall, the dorsoventral muscles and those surrounding the gut and the pharynx as well as the fast moving cross striated muscles of the tail of cercariae intensely reacted with the antibody. However, neither immunohistologically nor on Western blots of isolated tegument, were indications found for the presence of paramyosin in the tegument. In vivo phosphorylation and binding of anti-phospho-tyrosine and anti-phospho-serine antibodies show phosphorylation of paramyosin which probably is responsible for the generation of the isoforms.

INTRODUCTION

Paramyosin of schistosomes is an immunogenic and highly protective antigen in most of the infected hosts (Pearce et al. 1988). Protectivity is surprising since paramyosin in invertebrates is a structural component of the muscles. In S.mansoni, however, paramyosin has been localized in the tegument and suggested to be a secretory product (Matsumoto et al. 1988; Nara et al. 1994). Surprisingly, in previous publications containing immunohistological localization of paramyosin in schistosomes, the muscles, if at all, are always only weakly labelled.

In this study, immunolocalization of paramyosin was performed with worms that were prepared for light microscopical examination by fixation techniques that avoid crosslinking of proteins with aldehydes and that also yield low background. High resolution was achieved with semithin sections of specimens embedded in LR Gold and labelled with immunogold. We found a distinct staining of each muscle type in cercariae, schistosomula and adult schistosomes. On the other hand, there was no evidence for an extramuscular occurrence of paramyosin.

MATERIALS AND METHODS

Parasites

Adult S.mansoni (Liberian strain) were perfused with RPMI medium containing 10% fetal calf serum from hamsters about 7 weeks after infection. Cercariae were shed from Biomphalaria glabrata in water and collected by sedimentation for about 20 min on ice. Schistosomula were produced by transformation in vitro, essentially as described by Samuelson & Stein (1989) and Ogbunude et al. (1989). Cercariae were treated in 1 ml RPMI medium for 5 min at 42°C, vortexed for 30 s to shear off the tails, and then incubated for 3 h at 37°C.

Antibodies and Western blots

Preparation of protein extracts from schistosomes, gel electrophoresis and Western blotting were carried out as described by Kusel (1972) and Köster et al. (1988). Paramyosin was purified essentially as described by Levine et al. (1982). Anti-schistosome antiserum was produced by immunization of rabbits with homogenates of male schistosomes (Köster et al. 1988). This antiserum was used for Western blots in a dilution of 1 : 5000. Furthermore, this antiserum was utilized for affinity purification of specific anti-paramyosin antibodies. For this purpose, the paramyosin cDNA sequence (accession number M35499) cloned in the plasmid expression vector pUR 290 (recombinant plasmid TB3-1, obtained from J.P.Laclette, Mexico City) was expressed in E.coli XL1Blue (Stratagene). Antibodies were purified to monospecificity with filter replicas of plate lysates containing the paramyosin fusion proteins (Köster et al. 1988). For Western blots, anti-paramyosin antibodies were diluted 1 : 50. Anti-phospho-tyrosine and anti-phospho-serine antibodies (Sigma) were diluted following the instructions of the manufacturer. Bound antibodies were detected using alkaline phosphataseconjugated goat antirabbit or goat anti-mouse antibody, and colour was developed by the azo-dye reaction with naphtholASMX phosphate and Fast Brown (West, Schröder & Kunz, 1990).

Labelling of S.mansoni proteins with 32P

Worms were incubated for 6 h in phosphate-reduced in vitro culture medium at 37°C: RPMI 1640 (Mercer & Chappell, 1985), diluted with an equal volume of 0.9% NaCl, containing 200 mCi of H332PO4. After ten washes in 0.9% NaCl, proteins were extracted, and unincorporated radioactive phosphate was removed by centrifugation through an NMWL 10,000 membrane (Millipore).

Immunoprecipitation

200 mg protein A-Sepharose beads and 2 mg anti-paramyosin antibodies were incubated in 500 ml 40 mM HEPES, pH 8.0, at 4°C overnight. After two washes in HEPES, the beads were resuspended in 400 ml adsorption buffer (0.1 M H3BO3, 25 mM Na2B4O7, 75 mM NaCl, 0.5% NP40, and 0.05% ovalbumin, pH 8.3), and 20 mg of schistosome extracts were added. Following an incubation at 4°C for 4 h, the beads were washed two times in adsorption buffer and two times in 40 mM HEPES.

2-D electrophoresis

2-D electrophoresis was carried out in the Multiphor II (Pharmacia, Uppsala, Sweden) as described by Westermeier (1990). Fifteen mg of purified paramyosin was separated by isoelectric focusing in the first dimension in a pH gradient 4-6, generated with Ampholine in the presence of urea. Separation in the second dimension was by SDS electrophoresis. The gels were stained with silver or blotted and probed with antibodies against paramyosin.

Immunocytochemistry

Fixation

Worms were transferred to the fixatives (1), (2), or (3) immediately after collection.

(1) Fixation with aldehydes. Worms were treated with formaldehyde and glutaraldehyde according to Matsumoto et al. (1988) or were incubated with 1% or 4% of depolymerized paraformaldehyde in a solution of 150 mM NaCl, 1.5 mM CaCl2, and 25 mM 1,4-piperazinediethanesulphonic acid (PIPES), pH 7.35, for 3 h at 4°C. Residual aldehydes were quenched with 20 mM ammonium chloride in buffer for 1 h before dehydration of the probes or, alternatively, by treating the semithin sections with 50 mM sodium borohydride for 15 min at 4°C. Using a CS-auto-apparatus (Reichert-Jung, Wien, Austria), the probes were dehydrated in a graded series of ethanol with progressively lowering temperature, and were then infiltrated with LR Gold (London Resin Co., Basingstoke, England) with an intermediate step of equal parts of ethanol and resin at -20°C. The resin was polymerized with UV light for 18 h.

(2) Cryofixation. Worms sticking at the tip of a needle with little medium fluid were rapidly immersed in liquid propane cooled with nitrogene. They were transferred to the CS-auto-apparatus and dehydrated by cryosubstitution with methanol at -89°C for 8 h and at -62°C for 4.5 h, and were then infiltrated with resin.

(3) Immobilization with uranyl acetate-trehalose-methanol. Specimens were incubated for 10 min in an ice-cold solution of 1% uranyl acetate, 240 mM trehalose and 25 mM PIPES in distilled water, which had been adjusted to pH 5.8 by adding NaOH while continuously stirring. Dehydration steps were in 50% methanol with 1% uranyl acetate, 50 mM trehalose and 10 mM PIPES, pH 5.8, for 7 min at 0°C, subsequently in 75% methanol with 0.5% uranyl acetate, 25 mM trehalose and 5 mM PIPES for 30 min at -20°C, and in 100% methanol with 0.25% uranyl acetate for 90 min at -20°C. The probes were incubated in 100% methanol without additives for 15 min prior to infiltration of resin. Results from current studies on the immobilization procedure indicate that the given schedule may have to be modified to accomodate properly to the tonicity of the examined tissues.

Immunogold techniques

Immunogold was produced using conventional techniques by complexing affinity pure goat anti-rabbit IgG (Dianova, Hamburg, FRG) to colloidal gold particles with a diameter of about 9 nm (Horisberger & Rosset, 1977; De Mey, 1983; Slot & Geuze, 1985). Semithin sections, about 0.5 mm thick, were cut with an OmU3 microtome (Reichert-Jung) transferred to chrome alum-gelatine-coated slides and dried at 45°C. Immunolabelling was performed in a humid chamber at room temperature. The sections were wetted with incubation buffer, which consisted of 25 mM Tris, 40 mM glycine, 150 mM NaCl and 0.01% Tween 20, pH 7.5. They were then pretreated with 3% ovalbumin hydrolysate (Sigma) or 3% nonfat dry milk in incubation buffer for 30 min to block nonspecific binding sites. The sections were covered with 60 ml of anti-paramyosin antibodies in buffer containing blocking agents and were incubated for 90 min. Incubations for control of binding specificity were run in parallel, but primary antibodies were omitted. Subsequently, the slides were rinsed with buffer, kept in a cuvette with buffer for 7 min, and rinsed again. Gold-conjugated secondary antibodies were diluted from stock solution with buffer containing 1% bovine serum albumin to an optical density of about 0.2 at 520 nm. Sixty ml of this immunogold solution were pipetted onto the sections on each slide. After incubation for 60 min with occasionally moving the slides by hand, the gold solution was removed, and after immersion in buffer for 4 min, the slides were rinsed with distilled water and air-dried. The bound immunogold was visualized by silver enhancement using the IntenSE BL kit (Amersham, Braunschweig, FRG).

RESULTS

Paramyosin is not detected in isolated tegument

In previous investigations on muscle-specific gene expression in S.mansoni, we characterized myosin (Weston et al. 1993) and paramyosin clones. Since our paramyosin clones were partial, we continued our studies with the full-length clone TB3-1 of S.mansoni, kindly provided by J. P. Laclette (Laclette et al. 1991)). Fusion proteins produced in E.coli XL1-Blue, were used to affinity purify a rabbit antiserum raised against homogenates of adult schistosomes. The antibodies obtained were tested on Western blots of total extracts of schistosomes and strongly reacted with a band of about 100 kDa which is in the size range of paramyosin. In addition, a minor reaction with a band of 200 kDa was observed which probably resulted from crossreaction with myosin. To obtain monospecificity, the antibodies were postadsorbed on preparative Western blots from which the paramyosin band had been excised. The antibodies remaining in solution were again tested on Western blots of S.mansoni proteins, and now reacted only with the 100 kDa band (Fig. 1A, lane S) which demonstrates their selectivity. In addition, the antibodies clearly reacted with purified paramyosin of S.mansoni (Fig. 1A, lane P), documenting their specificity.

Being a highly protective antigen, paramyosin has to be exposed to the immune system and is suggested to be excreted or located at the worm surface. We separated teguments from adult male schistosomes, using the freeze-thaw method (Kusel, 1972). About 10 mg each of SDS-solubilized proteins from isolated teguments and from carcasses were fractionated on polyacrylamide gels, blotted and probed with anti-paramyosin antibodies (Fig. 1B). Parallel blots of teguments and carcasses were probed with antiserum raised against homogenates of total schistosomes to document that sufficient quantities of proteins were loaded to visualize immune reactions (Fig. 1C). Unexpectedly, no paramyosin band was detected in the tegument fraction (Fig. 1B, lane T), whereas a distinct paramyosin band was visible in the lanes of the carcasses (Fig. 1B, lane Car).

Isoforms of paramyosin

To search for isoforms, purified paramyosin was separated by two-dimensional electrophoresis and stained for protein and antigen profiles. At 105 kDa, about ten spots were resolved in the pH range of 4.7 to 5.2 (Fig. 2A). From the amino acid composition of paramyosin, a pI value of 5.2 has been calculated (Laclette et al. 1991). On parallel immunoblots, a positive reaction was observed with our antibodies in the entire corresponding pH range, indicating that the antibodies recognize approximately the complete spectrum of different paramyosin isoforms (Fig. 2B).

To determine whether different isoforms are generated by phosphorylation, adult male worms were incubated in vitro with inorganic 32P. Extracts of these worms, separated on polyacrylamide gels, show that many proteins are phosphorylated (Fig. 3A, lane 1), particularly paramyosin that has been immunoprecipitated from these extracts (Fig. 3A, lane 2). Furthermore, monoclonal antibodies against phospho-tyrosine and phospho-serine reacted with purified paramyosin of schistosomes (Fig. 3B). These experiments show that paramyosin is phosphorylated which is predictable from its amino acid sequence. Differential phosphorylation appears to be responsible for the generation of paramyosin isoforms. Phosphorylation would explain why the pI values of the isoforms lie below the calculated value of 5.2.

The location of paramyosin has been investigated immunocytologically in adult worms, in in vitro transformed schistosomula, and in cercariae. Paramyosin has been detected in semithin sections by indirect immunogold labelling, followed by silver enhancement. In adult worms, the outer circular muscles and the broader underlying longitudinal muscle layer were intensely labelled (Fig. 4A-C). The numerous dorsoventral muscle strands reacted with similar intensity. Also the thin muscle layer surrounding the intestine was clearly visible (Fig. 4C). In females, the number of outer longitudinal and dorsoventral muscle cells was distinctly lower (Fig. 4C). In addition, the muscles of the suckers and the genital tract were labelled (not shown).

In cercariae, the antibodies recognized paramyosin in the outer circular and longitudinal muscle cells (Fig. 5A, B). The complex of oral sucker and pharynx containing particularly well developed muscles, was also labelled (Fig. 5A, B). Furthermore, muscles surrounding the secretory ducts of the penetration glands (Fig. 5B) and the gut were positively stained. In the tail of cercariae, paramyosin was distinctly visible in the sarcomers of the cross striated longitudinal muscles (Fig. 5C). In schistosomula, anti-paramyosin antibodies recognized essentially the same tissues as in cercariae (Fig. 4F, G).

These results clearly show that the large variety of muscle types of different developmental stages of S.mansoni contain paramyosin. However, muscle cells are the only type of tissue that do contain paramyosin. We found no indication for the existence of paramyosin in the teguments of cercariae (Fig. 5), schistosomula (Fig. 4F, G), and adults (Fig. 4D).

DISCUSSION

Paramyosin exclusively occurs in invertebrate organisms where it is widely distributed. As a structural component of the muscles, it is found in varying quantities in different muscle types ranging from smooth to cross-striated muscles (Winkelman, 1976). It interacts with the core proteins within the thick myofilaments as well as with the surrounding myosin molecules, thus stabilizing the thick myofilaments (Deitiker & Epstein, 1993). In contrast to actin and myosin, paramyosin has hitherto not been found as a component of the cytoskeleton.

In the platyhelminths Taenia, Echinococcus and Schistosoma, however, an extramuscular localization of paramyosin has repeatedly been reported, and, hence, suggesting additional, yet unknown functions in this animal phylum. In Taenia solium larvae, paramyosin (formerly named antigen B) has been found to be widely distributed (Laclette, Merchant & Willms, 1987). With immunofluorescence, it mainly has been localized to the external surface of the bladder wall of the cysticercus and in the tegumentary cytons. Notably, the tegument of the scolex did not show any immunofluorescence. No positive reaction with the muscles was mentioned. In Echinococcus granulosus, Mühlschlegel et al. (1993) concluded from their immunofluorescence studies the presence of paramyosin in the tegument of the protoscolex larva. However, their given figure shows a strong reaction of the muscles of the four oral suckers, and also at the body wall the fluorescence seems to be subtegumental. The tegument covering the oral suckers and the body wall appears to be negative.

In larvae of Schistosoma, paramyosin has been localized in various tissues. A monoclonal antibody against paramyosin from adult worms has been reported to bind to the surface of living schistosomula in a radioimmunoassay, but the signal was not intense (Wiest et al. 1991). However, the examined worms have been incubated in PBS which might cause partial tegument damages (Simpson et al. 1981) and, therefore, might expose subtegumental muscle filaments. By immunoelectron microscopy using a monoclonal antibody, Nara et al. (1994) described the presence of paramyosin in schistosomula in the postacetabular glands as well as in the tegument and muscle layers. However, the reaction with the tegument and muscles was relatively weak, and the postacetabular glands were labelled in only 5% of the larvae.

In adult S.mansoni, paramyosin has been localized by indirect immunofluorescence using monoclonal antibodies against the antigen Sm97 (Pearce et al. 1986). These authors observed most intense fluorescence just beneath the surface of adult worms. However, in the 6 µm frozen sections, it was not possible to unequivocally discriminate between the tegument syncytium and the underlying muscle layer.

A more detailed localization has been performed by Matsumoto et al. (1988) at the ultrastructural level. By immunogold labelling, they found binding of anti-paramyosin antibodies in the membrane-bounded elongate vesicles of the tegument in cercariae, schistosomula, and adults of S.mansoni, whereas antibodies only rarely bound to muscles.

These findings conflict with our observations, which may be explained by several differences in technical details. Matsumoto et al. (1988) used heterologous antibodies against paramyosin of Limulus and the polychaete Owenia, whereas we used antibodies against S.mansoni paramyosin. We focussed on the preservation of native epitope structure and maintenance of membrane integrity. Therefore, we applied two alternative methods: the cryofixation-freeze-substitution method avoids covalent crosslinking and, therefore, maintains epitope structure (Quintana, Lopez-Iglesias & Laine-Delaunay, 1991); the uranyl acetate-trehalose technique stabilizes membranes by reversible bonding. Both methods had the advantage of high resolution labelling and low background and yielded identical results with respect to reproducible localization of paramyosin antigens. This could not be achieved with aldehyde-fixed material (Bodor, Schmidt & Kunz, 1995). Fixation of specimens with formaldehyde and glutaraldehyde following the protocol of Matsumoto et al. (1988) and Nara et al. (1994), in our hands resulted in high background and low specific binding, since aldehydes may mask epitopes containing basic amino acids. Furthermore, in the procedure to localize paramyosin in the tegument and the postacetabular glands (Matsumoto et al. 1988; Nara et al. 1994), the sections have been treated with metaperiodate prior to immunolabelling. Metaperiodate is employed occasionally to restore immunoreactivity in osmium-fixed tissues embedded in epoxy resins (Bendayan & Zollinger, 1983). This treatment is critical, since metaperiodate oxidizes many carbohydrates into aldehydes, which, if not properly quenched, may non-specifically bind antibodies. In fact, the elongate vesicles in the tegument of adult worms contain glycans with terminal oxidizable galactose residues (Schmidt, 1995). Glycans are also a major constituent of the tegument surface and the contents of the postacetabular glands in cercariae of S.mansoni and S.japonicum (Stirewalt & Walters, 1973; Schmidt, unpublished). Thus, application of periodates best is avoided in immunocytochemistry of schistosomes. Even then, we observed an enhanced background over the penetration glands of cercariae, which could be due to proteases interacting with the antibodies and, more important, silver ions may be reduced by calcium salts in the glands.

In contrast to Drosophila and Caenorhabditis, it is remarkable that in most of the above mentioned papers on platyhelminths, paramyosin has not been localized predominantly in the muscles. This is particularly surprising since wide distribution and sequence conservation suggest a ubiquitous function of paramyosin in the thick muscle filaments throughout invertebrates.

With our approach, a distinct staining of each muscle type in cercariae, schistosomula and adult schistosomes has been obtained. On the other hand, we got no evidence for an extramuscular occurrence of paramyosin. We can, of course, not exclude minor amounts. It might be argued that the tegument contains an isoform of paramyosin that is not recognized by the antibodies used. In our experiments with 2-D electrophoresis, about ten different isoforms of paramyosin were detected. All of them reacted with our antibodies documenting that the muscles of schistosomes contain several different isoforms of paramyosin. The generation of the isoforms may be explained by differential phosphorylation. Phosphorylation of paramyosin is also found in Caenorhabditis (Schriefer & Waterson, 1989) and Drosophila (Vinós et al. 1991). In Caenorhabditis, different binding affinities of paramyosin in the thick muscle filaments were shown (Deitiker & Epstein, 1993) which probably are due to a different degree of phosphorylation.

The postulated occurrence of paramyosin in the tegument has also been investigated with tegument extracts isolated by careful application of the freeze-thaw technique. Subsequent analysis of immunoblots revealed no paramyosin in isolated teguments of adult schistosomes. In similar experiments with schistosomula, however, paramyosin was found in the tegument (Flanigan et al. 1989). The schistosomula were extensively washed in PBS and tegument material was extracted with Triton X-100. Therefore, it cannot be excluded that molecules from subtegumental tissues were extracted. In addition, it has been shown in S.mansoni that PBS induces cellular disruptions resulting in the release of subtegumental material into the tegumental fraction (Simpson et al. 1981).

There is little evidence to suppose that paramyosin might be a secretory protein. The absence of a signal peptide in the paramyosin sequence for entry into the endoplasmic reticulum does not support this assumption (Laclette et al. 1991). Genomic sequence data predict a specific hybridization pattern on genomic Southern blots which could be verified (7). The pattern indicates that paramyosin in S.mansoni is a single copy gene (Gohr, Maniak & Kunz, 1995). This excludes the existence of other paramyosin genes coding for isoforms with a signal sequence, although alternative splicing may still be possible.

If paramyosin does not occur in the tegument or at the surface of S.mansoni, it is difficult to explain protective immunity. A possible explanation is based on the homology between paramyosin and the protein backbone of the immunogenic membrane glycoprotein GP38. GP38 shed from the schistosomulum surface could induce an immuno response of paramyosin-specific antibodies or T cells (Sher et al. 1989). Furthermore, a 200 kDa antigen (IrV5) at the surface of schistosomula having partial homology to myosin heavy chain sequences (Amory Soisson et al. 1992) could exhibit cross-reactivity to paramyosin antibodies.

In general, some schistosomula die during invasion into the vertebrate host. Hence, immunogens that are expressed solely in internal parasite tissues become exposed to the immune system. Stimulation of cell mediated defence mechanisms then could attack intact schistosomula, that need not necessarily bear the immunogen on their surface.

We are indebted to Prof. Dr. W. Peters for enabling us to perform cytological experiments in his laboratory. This investigation received financial supports from the Deutsche Forschungsgemeinschaft (grants Pe 254/3-1 and Ku 282/13-2) and from the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases.

REFERENCES

AMORY SOISSON, L.M., MASTERSON, C.P., TOM, T.D., MCNALLY, M.T., LOWELL, G.H. & STRAND, M. (1992). Induction of protective immunity in mice using a 62kDa recombinant fragment of a Schistosoma mansoni surface antigen. Journal of Immunology 149, 3612-20.

BENDAYAN, M. & ZOLLINGER, M. (1983). Ultrastructural localization of antigenic sites on osmiumfixed tissues applying the protein Agold technique. Journal of Histochemistry and Cytochemistry 31, 10-18.

BODOR, O., SCHMIDT, J. & KUNZ, W. (1995). Localization of paramyosin in S.mansoni. Zentralblatt für Bakteriologie 182, 130-1.

DE MEY, J. (1983). Colloidal gold probes. In Immunocytochemistry: practical applications in pathology and biology, (ed. Polak, J.M. & Van Norden, S.) pp. 82112. Bristol: Wright PSG.

DEITIKER, P.R. & EPSTEIN, H.F. (1993). Thick filament substructures in Caenorhabditis elegans: evidence for two populations of paramyosin. The Journal of Cell Biology 123, 303-11.

FLANIGAN, T.P., KING, C.H., LETT, R.R., NANDURI, J. & MAHMOUD, A.A.F. (1989). Induction of resistance to Schistosoma mansoni infection in mice by purified parasite paramyosin. Journal of Clinical Investigation 83, 101-4.

GOHR, L. (1995). Studies on the multifunctionality of paramyosin, on transient gene transfer and on the regulation of the eggshell genes in Schistosoma mansoni (text in German). Thesis, University of Düsseldorf.

GOHR, L., MANIAK, M. & KUNZ, W. (1995). Paramyosin of Schistosoma mansoni: multifunctionality and host-parasite interaction. Zentralblatt für Bakteriologie 282, 148.

HORISBERGER, M. & ROSSET, J. (1977). Colloidal gold, a useful marker for transmission and scanning electron microscopy. Journal of Bistochemistry and Cytochemistry 25, 295-305.

KÖSTER, B., DARGATZ, H., SCHRÖDER, J., HIRZMANN, J., HAARMANN, C., SYMMONS, P. & KUNZ, W. (1988). Identification and localisation of the products of a putative eggshell precursor gene in the vitellarium of Schistosoma mansoni. Molecular and Biochemical Parasitology 31, 183-98.

KUSEL, J.R. (1972). Protein composition and protein synthesis in the surface membranes of Schistosoma mansoni. Parasitology 65, 55-69.

LACLETTE, J.P., LANDA, A., ARCOS, L., WILLMS, K., DAVIS, A.E. & SHOEMAKER, C.B. (1991). Paramyosin is the Schistosoma mansoni (Trematoda) homologue of antigen B from Taenia solium (Cestoda). Molecular and Biochemical Parasitology 44, 287-96.

LACLETTE, J.P., MERCHANT, M. & WILLMS, K. (1987). Histological and ultrastructural localization of antigen B in the metacestode of Taenia solium. The Journal of Parasitology 73, 12-19.

LEVINE, R.J.C., ELFVIN, M.J. & SAWYNA, V. (1982). Preparation and Assay of Paramyosin. Methods in Enzymology 85, 14964.

MATSUMOTO, Y., PERRY, G., LEVINE, R.J.C., BLANTON, R., MAHMOUD, A.A.F. & AIKAWA, M. (1988). Paramyosin and actin in schistosomal teguments. Nature 333, 76-8.

MERCER, J.G. & CHAPPELL, L.H. (1985). Schistosoma mansoni: effect of maintenance in vitro on the physiology and biochemistry of adult worms. Parasitology 90, 339-49.

MÜHLSCHLEGEL, F., SYGULLA, L., FROSCH, P., MASSETTI, P. & FROSCH, M. (1993). Paramyosin of Echinococcus granulosus: cDNA sequence and characterization of a tegumental antigen. Parasitology Research 8, 6606.

NARA, T., MATSUMOTO, N., JANECHARUT, T., MATSUDA, H., YAMAMOTO, K., IRIMURA, T., NAKAMURA, K., AIKAWA, M., OSWALD, I., SHER, A., KITA, K. & KOJIMA, S. (1994). Demonstration of the target molecule of a protective IgE antibody in secretory glands of Schistosoma japonicum larvae. International Immunology 6, 963-71.

OGBUNUDE, P.O.J., SELIM, A., HAQ, A. & BAER, H.P. (1989). A simple procedure for the isolation of schistosomules for biochemical studies or culture. Tropical Medicine and Parasitology 40, 337-8.

PEARCE, E.J., JAMES, S.L., DALTON, J., BARRALL, A., RAMOS, C., STRAND, M. & SHER, A. (1986). Immunochemical characterization and purification of Sm97, a Schistosoma mansoni antigen monospecifically recognized by antibodies from mice protectively immunized with a nonliving vaccine. Journal of Immunology 86, 3593-600.

PEARCE, E.J., JAMES, S.L., HIENY, S., LANAR, D.E. & SHER, A. (1988). Induction of protective immunity against Schistosoma mansoni by vaccination with schistosome paramyosin (Sm97), a nonsurface parasite antigen. Proceedings of the National Academy of Sciences, USA 85, 5678-82.

QUINTANA, C., LOPEZIGLESIAS, C.L. & LAINEDELAUNAY, M.C. (1991). Cryomethods (cryofixation, cryosubstitution and cryoembedding) for processing of tissues for ultrastructural and immunocytochemical studies. Application to oviduct cells of laying quail. Biologie Cellulaire 72, 167-80.

SAMUELSON, J.C. & STEIN, L.D. (1989). Schistosoma mansoni: increasing saline concentration signals cercariae to transform to schistosomula. Experimental Parasitology 69, 23-9.

SCHMIDT, J. (1995). Glycans with Nacetyllactosamine type 2 like residues covering adult Schistosoma mansoni, and glycomimesis as a putative mechanism of immune evasion. Parasitology 111, 325-36.

SCHRIEFER, L.A. & WATERSON, R.H. (1989). Phosphorylation of the Nterminal region of Caenorhabditis elegans paramyosin. Journal of Molecular Biology 207, 451-4.

SHER, A., JAMES, S.L., CORREAOLIVEIRA, R., HIENY, S. & PEARCE, E.J. (1989). Schistosome vaccines: current progress and future prospects. Parasitology 98, 61-8.

SIMPSON, A.J.G., SCHRYER, M.D., CESARI, I.M., EVANS, W.H. & SMITHERS, S.R. (1981). Isolation and partial characterization of the tegumental outer membrane of adult Schistosoma mansoni. Parasitology 83, 163-77.

SLOT, J.W. & GEUZE, H.J. (1985). A new method of preparing gold probes for multiplelabeling cytochemistry. European Journal of Cell Biology 37, 87-93.

STIREWALT, M.A. & WALTERS, M. (1973). Schistosoma mansoni: Histochemical analysis of the postacetabular gland secretion of cercariae. Experimental Parasitology 33, 56-72.

VINóS, J., DOMINGO, A., MARCO, R. & CERVERA, M. (1991). Identification and characterization of Drosophila melanogaster paramyosin. Journal of Molecular Biology 220, 687-700.

WEST, S., SCHRÖDER, J. & KUNZ, W. (1990). A multiplestaining procedure for the detection of different DNA fragments on a single blot. Analytical Biochemistry 190, 254-8.

WESTERMEIER, R. (1990). ElektrophoresePraktikum. Weinheim: VCH.

WESTON, D., SCHMITZ, J., KEMP, W.M. & KUNZ, W. (1993). Cloning and sequence of a complete myosin heavy chain cDNA from Schistosoma mansoni. Molecular and Biochemical Parasitology 58, 161-164.

WIEST, P.M., WISNEWSKI, A.V., JOHNSON, J.H., RAMIREZ, B., KRESINA, T.F. & OLDS, G.R. (1991). Screening of murine monoclonal antibodies against living schistosomula of Schistosoma mansoni by radioimmunoassay. International Journal for Parasitology 21, 449-454.

WINKELMAN, L. (1976). Comparative studies of paramyosins. Comparative Biochemistry and Physiology [B] 55, 391-397.

EXPLANATIONS OF FIGURES

Fig. 1. Western blots of homogenates of whole Schistosoma mansoni (S), of purified paramyosin (P), of isolated teguments (T), and of carcasses (Car), incubated with anti-paramyosin antibodies (A, B) or antisera against total worm homogenates (C). The anti-paramyosin antibody reacts only with the carcasses, whereas no paramyosin is detected in the tegument fraction. M, high molecular mass marker (Sigma).

Fig. 2. Two-dimensional electrophoresis of purified paramyosin of Schistosoma mansoni. (A) Silver-stained gel showing about ten spots; all of these spots react with the anti-paramyosin antibodies in the corresponding immunoblot (B).

Fig. 3. (A) Autoradiographs of extracts from total adult male Schistosoma mansoni (1) and immunoprecipitated paramyosin (2) that have been incubated with inorganic 32P. (B) Western blot of purified paramyosin, probed with anti-phospho-tyrosin (1) and anti-phospho-serine (2) antibodies. M, marker proteins.

Fig. 4. Localization of paramyosin in Schistosoma mansoni. Binding of anti-paramyosin antibodies to semithin sections of worms is detected with immunogold/silver by light microscopy. (A) Cross section of a male worm shows intense reactions with circular (CM), longitudinal (LM), and dorsoventral muscles (DVM). (B) Tangential section along the dorsal part of a male worm demonstrates staining of the longitudinal muscle cells. (C) Section through a worm pair shows muscle staining as in (A) and also a thin muscle layer surrounding the intestine (IM). (D) High magnification documents that the tegument (T) of the adult worm does not react with the paramyosin specific antibody, whereas the muscles are prominently stained. Phase contrast. (E) Negative control without primary antibody. (F) Longitudinal section through an in vitro transformed schistosomulum with staining of muscles in the periphery of the body, at the oral sucker (OS) and the pharynx (P). (G) High magnification of a schistosomulum shows absence of staining in the tegument (T). Muscle fibers of the body (M) and the pharynx are positive. Lightly counterstained with azocarmine to visualize the tegument. (A) Cryofixation, (B-G) uranyl acetate-trehalose-methanol fixation.

Fig. 5. Paramyosin in cercariae of Schistosoma mansoni. Sections labelled with anti-paramyosin antibodies and stained with immunogold/silver for light microscopy. (A) Longitudinal section shows staining of muscles of the oral sucker (OS), the pharynx (P), and the peripheral muscles (M). (B) Cross section through the anterior part of a cercaria shows muscle staining as in (A). Also muscle cells around the secretory ducts (SD) of the penetration glands are labelled. (C) Longitudinal section through the tail reveals intense reaction of paramyosin within the sarcomers of cross striated muscles (CS).