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The carbohydrate moieties of glycosphingolipids from eggs of the human parasite, Schistosoma mansoni, were enzymatically released, labelled with 2-aminopyridine (PA), fractionated and analysed by linkage analysis, partial hydrolysis, enzymatic cleavage, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nano-electrospray ionization mass spectrometry. Apart from large, highly fucosylated structures with five to seven HexNAc residues, we found short, oligofucosylated species containing three to four HexNAc residues. Their structures have been determined as Fuc(alpha1-3)GalNAc(beta1-4)[ +/- Fuc (alpha1-3)]GlcNAc(beta1-3)GalNAc(beta1-4)Glc-PA, GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)GlcNAc(beta1-3)GalNAc(beta1-4) Glc-PA, Fuc(alpha1-3)GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-4) GlcNAc(beta1-3)GalNAc(beta1-4)Glc-PA, and Fuc(alpha1-3) GalNAc(beta1-4)[ +/- Fuc(alpha1-2) +/- Fuc(alpha1-2)Fuc(alpha1-3)]Glc NAc(beta1-3)GlcNAc(beta1-3)GalNAc(beta1-4)Glc-PA. The last structure exhibits a trifucosyl sidechain previously identified on the cercarial glycocalyx. These structures stress the importance of 3-fucosylated GalNAc as a terminal epitope in schistosome glycoconjugates. To what degree these glycans contribute to the pronounced antigenicity of S. mansoni egg glycolipids remains to be determined. In addition, we have identified the compounds GlcNAc(beta1-3)GalNAc(beta1-4)Glc-PA, Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3) GalNAc (beta1-4)Glc-PA, the latter of which is a Lewis X-pentasaccharide identical to that present on cercarial glycolipids, as well as Gal(beta1-3)GalNAc(1-4)Gal(1-4)Glc-PA, which corresponds to asialogangliotetraosylceramide and is most probably derived from the mammalian host.

The distribution of genetic diversity in a local population of the trematode Schistosoma mansoni was determined within and between individual wild rats at a microspatial geographic scale of a standing water transmission site. Using RAPD markers, molecular variance and canonical correspondence analysis were performed to test the significance of genetic differentiation between infrapopulations. Of total gene diversity, 8 and 11% was partitioned between hosts trapped at few metres distance from each other. Significant temporal differentiation (2%) was also detected among schistosomes sampled at 6 month intervals with more infrapopulation pairs differentiated during the dry season of parasite transmission than during the rainy season (45 and 12%, respectively). A combination of factors such as restricted displacement of rats, patchy spatial aggregation of infected snails and limited cercarial dispersion in standing water are likely to promote the genetic differentiation observed between infrapopulations at this microgeographic scale.

Voltage-gated calcium (Ca2+) channels contribute to impulse propagation in excitable cells and also regulate intracellular levels of Ca2+. High voltage-activated (HVA) Ca2+ channels are heteromultimeric membrane proteins. The pore-forming, voltage-sensing subunit is the alpha1 subunit. We have cloned 3 HVA Ca2+ channel alpha1 subunit cDNAs from Schistosoma mansoni. One of these sequences most closely resembles the L-type class of HVA alpha1 subunits. The other two sequences are most closely related to non L-type alpha1 subunits. These schistosome alpha1 subunits have many of the features common to HVA Ca2+ channels, but also have distinct structural motifs. Analysis of the structural and functional properties of schistosome Ca2+ channel subunits may provide information about these critical components of excitable cells.

A cDNA encoding a Na,K-ATPase alpha subunit homologue, designated SNaK1, was isolated from an adult cDNA library of Schistosoma mansoni. The 3.8 kb DNA contained a 3021 bp open reading frame potentially encoding a 1,007 amino acid protein that had an Mr of 111,817 and a pl of 5.48. Homology searches for SNaK1 revealed approximately 70% sequence identity with a variety of Na, K-ATPases from evolutionarily diverse organisms. SNaK1 is predicted to contain 10 transmembrane regions typical of this protein family as well as other conserved domains, such as the phosphorylation site and ATP binding domain. Antibodies raised against an amino terminal peptide detected the protein in membrane preparations of eggs, cercariae and adult males and females, suggesting a general role for SNaK1. The mobility of the protein differed in various life-stages suggestive of post-transcriptional or post-translational modification. Immunolocalization of SNaK1 in sections of adult worms using epifluorescence and electron microscopy, revealed antibody labelling in the subtegumental and peripheral layers. Strong staining was discernible in the peripheral muscle band indicating that SNaK1 plays a central role in muscle contraction in adult parasites and may be the primary target of ouabain action. Staining was also detected in the secretory bodies in sections of ducts in this region and over the RER of the presumed gastrodermis. Immunogold labelling was also localized over neuronal vesicles in axons associated with the peripheral muscle layer.

We have recently shown that soluble extracts from Schistosoma mansoni eggs (SmEA) triggered basophils from nonsensitized donors to rapidly release interleukin (IL)-4. Assuming that this mechanism might play a role in vivo in biasing the immune response towards a Th2 phenotype, we determined basic properties of the IL-4-inducing activity contained in SmEA. Sensitivity to pepsin digestion indicated protein nature. Binding to and specific elution from Concanavalin A-sepharose suggested that this protein contains mannose residues, thus being a glycoprotein. The IL-4-inducing activity was stable for 30 min at room temperature towards shifting the pH between 3 and 10. When incubated at 100 degrees C, it was stable at pH 3, but less stable at neutral and alkaline pH. Electroelution from an SDS-PAGE gel indicated an apparent molecular weight of the IL-4-inducing activity between 31 and 66 kDa. Although binding to purified human immunoglobulin E (IgE) and activating basophils IgE-dependently, SmEA appears to activate basophils in a non-antigen-specific way, since the cells were purified from noninfected donors. Because the IL-4-inducing activity was found to be released from eggs, it could be an important factor in the environment of the eggs skewing the immune response towards the Th2 phenotype.

The mouse major histocompatibility complex (MHC) class I sequence was detected in 8-week-old Schistosoma mansoni by in situ polymerase chain reaction (in situ PCR). The signals to the mouse class I MHC sequence were observed in the nuclei of the mesenchymal and reproductive cells of S. mansoni. Signals were also observed in the cytoplasm of the tegumental tubercles. This finding suggested the possibility of MHC gene transfer from the host to schistosomes. Furthermore, the class I MHC sequence was detected in the DNA extracted from the cercariae of S. mansoni by nested PCR. Neither the nucleotide sequence of class I MHC detected in adult worm DNA nor that of class I MHC detected in the host (mouse) DNA was identical with that of class I MHC detected in the cercarial DNA. From the data we assumed that S. mansoni may have retained their own mouse class I MHC sequence in their genome throughout their life-cycle.

The glycobiology of Schistosoma mansoni is dominated by developmentally regulated expression of various fucosylated structures, most notably the Lewis X epitope and a multifucosylated sequence, Fuc alpha1-->2Fuc alpha1-->, in its various forms. For the infective cercarial stage, Lewis X has been structurally identified on glycosphingolipids and N-glycans of total glycoprotein extracts, and a population of multifucosylated glycoproteins were found to carry a unique terminal sequence, +/-Fuc alpha1-->2Fuc alpha1-->[3GalNAc beta1-->4(Fuc alpha1-->2Fuc alpha1--> 2Fuc alpha1-->3) GlcNAc beta1-->3Gal alpha1-->](n), on their O-glycans. Using a mass spectrometry approach coupled with chromatographic separation, sequential exoglycosidase digestion, periodate oxidation, and other chemical derivatization, we demonstrate that Lewis X could also be carried on the cercarial O-glycans, but the two distinctive sets of fucosylated epitopes were conjugated to two different core structures. Lewis X, lacNAc, or single GlcNAc was found to attach directly to the -->3Gal beta1-->3GalNAc core and indirectly via another beta-Gal residue branching off from C6 of the reducing end GalNAc to give a biantennary-like structure. The -->3(+/-Gal beta1-->6)Gal beta1-->3(-->3Gal beta1-->6)GalNAc core thus characterized represents a novel core type for O-glycans. In contrast, the previously characterized multifucosylated terminal sequences were carried on conventional type 1 and 2 cores. The smallest structures of the reductively released O-glycans were defined as GalNAc beta1-->4GlcNAc beta1-->3Gal beta1-->3GalNAcitol with a total of two to four fucoses attached to the terminal lacdiNAc. alpha-Galactosylation of the nonreducing terminal beta-GalNAc instead of fucose capping leads to further elongation with another lacdiNAc unit that could also extend directly from C6 of the reducing end GalNAc and similarly elongated or terminated.

Smad proteins are essential intracellular signal transducers of the transforming growth factor-beta (TGF-beta) superfamily. The TGF-beta superfamily signals through phosphorylation and activation of R-Smad proteins, receptor-regulated Smads, by heteromeric complexes of ligand-specific type I and type II serine/threonine kinase receptors. R-Smads receive a signal from the activated receptor complex and transmit it to the nucleus. A cDNA was isolated that encodes a 649-amino acid protein found to be homologous to members of R-Smad subfamily with highest homology scored to clawed African frog and human Smad2. The Schistosoma mansoni homologue (SmSmad2) was overexpressed in bacteria as a Sj26-GST fusion protein and used to raise specific antibodies. The IgG fraction of the immunized rabbit serum identified 70- and 72-kDa protein bands in Western analysis of schistosome extracts. Treatment with alkaline phosphatase removed the 72-kDa band, which indicates that this band represents the phosphorylated form of schistosome Smad2. SmSmad2 was localized in the subtegument, parenchymal cells, and sex organs in both male and female worm cryosections. Similar results were also obtained from the analysis of the Smad2 mRNA distribution pattern revealed by in situ hybridization of adult worm pair paraffin sections. SmSmad2 mRNA levels were determined by reverse transcriptase-polymerase chain reaction in different mammalian host developmental stages and found to be constitutively expressed. SmSmad2 was also found to interact with a previously identified SmTbetaR-I, a serine/threonine type I kinase receptor. Furthermore, SmSmad2 was shown to undergo phosphorylation by constitutively active forms of SmTbetaR-I in vitro. In addition, SmSmad2 localized in the nuclei of mink lung epithelial cells upon treatment with TGF-beta(1). These data indicate that the SmSmad2 responds to the TGF-beta signals by interaction with receptor I, which phosphorylates it, whereupon it translocates into the nucleus presumably to regulate target gene transcription and consequently elicit a specific TGF-beta effect.

Schistosomal egg N-glycans are the only examples in nature that have been structurally shown to contain beta2-xylosylation, alpha6-fucosylation, and alpha3-fucosylation on the N,N'-diacetyl chitobiose core. We present evidence that core difucosylated and xylosylated N-glycans are characteristics of Schistosoma japonicum eggs but not of the cercariae and adults, for which neither core xylosylation nor alpha3-fucosylation could be readily detected. In contrast, a majority of the N-glycans from Schistosoma mansoni cercariae but not the adults are core xylosylated. Tandem mass spectrometry analysis coupled with chromatographic mapping, sequential exoglycosidase digestion, and methylation analysis were employed to unambiguously define the structures of core beta2-xylosylated, alpha6-fucosylated N-glycans from S. mansoni cercariae. Unexpectedly, a majority of these N-glycans were found to carry Lewis X determinant, Galbeta1-->4(Fucalpha1-->3)GlcNAcbeta1-->, on the nonreducing termini of mono- and biantennary structures. The Lewis X-containing glycoproteins were found to be distinct from those carrying the complex, multifucosylated glycocalyx O-glycans reported previously. The corresponding N-glycans from S. japonicum cercariae are likewise dominated by Lewis X termini but without the core xylosylation. We concluded that the invading cercariae present an important and abundant source of Lewis X antigens, which may contribute to the induced humoral response upon infection. Following transformation and development into the adults, the N-glycans synthesized comprise a significantly larger amount of high mannose and fucosylated pauci-mannose structures in comparison with the cercarial N-glycans. A portion of the mono- and biantennary complex types were identified to carry Lewis X and fucosylated LacdiNAc termini, which could also be detected by mass spectrometry analysis on larger, complex-type structures.

We have provided an account of the progress we and others have made over the last decade on the structural characterization of glycans from parasitic helminths. We hope to have illustrated a few principles and patterns governing helminth glycosylation, as well as the experimental approaches adopted and their associated strengths and limitations. Schistosomes remain the best studied systems but are still punctuated with gaps of knowledge. An important theme developed here is the regulated developmental stage-specific expression of various glycan epitopes and their interplay with immediate host environments for successful parasitism. It is anticipated that more novel or unusual structures will continuously be uncovered in the future and that despite many difficulties, current analytical techniques should be well up to meet the challenge in at least elucidating the major or key glycoconjugates from each of the diverse range of worms. The bottle neck will in fact reside in finding suitable experimental models to test their putative immunobiological functions from which the intricate host-parasite interactions can be delineated and rational vaccine design be achieved. The glycobiology of parasitic helminths is an area waiting to be more fully explored and the rewards should be sweet.

In previous studies it was shown that the recombinant molecule, r-Sm14, induces high levels of protection against Schistosoma mansoni infection in two outbred animal models and immune crossprotection against infection by Fasciola hepatica in Swiss outbred mice. r-Sm14 was derived from a living worm extract, called SE, and is being developed as the molecular basis of an anti-helminth bivalent vaccine against the two parasites, for medical and veterinary application. Present data refer to SDS-PAGE and Western Blotting analysis of four different preparations of S. mansoni adult worms focusing Sm14 identification. The extracts correspond to the initial fraction of the SE extraction process, containing products released by living worms (SEi); SE2, reextraction of adult worms in PBS; and SE of separated male and female adult worms. In all extracts it was possible to detect the component of 14 kDa, that was recognized by specific anti-rSm14 antibody raised in rabbits.

In parallel with massive research efforts in human schistosomiasis over the past 30 years, persistent efforts have been made to understand the basis for compatibility and incompatibility in molluscan schistosomiasis. Snail plasma contains molecules that are toxic to trematodes, but these seem to kill only species that never parasitize the mollusc used as the source of plasma. A sporocyst will be killed actively by haemocytes alone if they are from a snail that is resistant to the trematode. Oxygen-dependent killing mechanisms play a major role. Enzymes such as NADPH oxidase, superoxide dismutase, myeloperoxidase and nitric oxide synthase are critical components of the putative killing pathways. Metabolic intermediates such as hydrogen peroxide and nitric oxide appear to be more important against trematodes than the shorter-lived intermediates that are more important in anti-microbial defences. Products secreted by trematode larvae influence the physiology of snail haemocytes, implying active counter-defences mounted by the parasite, but these remain largely unexplored. A possible molecular basis for the susceptibility/resistance dichotomy in molluscan schistosomiasis is suggested to be deficient forms of enzymes in the respiratory burst pathway, and a selective disadvantage for schistosome resistance is an integral component of this model.

Sm13, a 13-kDa Schistosoma mansoni tegumental antigen, is one of the principal polypeptides recognized by antibodies from mice protectively vaccinated with adult-worm tegumental membranes. To obtain the complete gene encoding Sm13 we subcloned and sequenced a cDNA and a fragment of a genomic clone. The collated sequence contains 1,088 nucleotides and represents the full-length open reading frame of the gene, encoding a protein of 104 amino acids with a calculated molecular mass of 11,923 Da, compatible with the native protein identified in the tegumental membranes. The sequence derived from genomic DNA contains a 45-nucleotide intron. The analysis of the predicted protein suggests the presence of both N- and C-terminal hydrophobic membrane-spanning segments, and the coding region contains no homology in the currently available data bases. Additionally, the coding region is preceded by putative CCAAT and TATA boxes that may be involved in the control of expression. Western-blot analysis and indirect immunofluorescence resulted in the identification of a 13-kDa protein (Sm13) in the tegument of adult worms. The present study reveals that Sm13 behaves as an integral membrane protein upon partitioning in Triton X-1 14 and that it is present in worms of 3 weeks or older but not in schistosomula or miracidia. Moreover, it is also specifically recognized by sera from some schistosomiasis patients in enzyme-linked immunosorbent assay and Western-blot analysis, suggesting that it is immunogenic in human schistosomiasis.

Intermediate filaments (IFs) make up the cytoskeleton of most eukaryotic cells. In vertebrates, a number of IF proteins have been identified, showing distributions unique to tissue or cell type. Information on helminth IFs is limited to some nematode species. To observe immunofluorescent localization of IFs in helminth tissues, we selected a murine hybridoma clone producing IgM antibody to multiple types of mammalian IF proteins and examined cross-reactivity to helminth proteins. The selected monoclonal antibody (HUSM-9) cross-reacted well with IFs from nematode species such as Toxocara canis, Dirofilaria immitis, Anisakis simplex, and Trichinella britovi; strong immunofluorescence on cryostat sections was detected in the hypodermis, cords, body muscle, smooth muscle of the uterus, and other epithelial structures. In platyhelminths, i.e., adult Schistosoma mansoni, larval Taenia taeniaeformis, adult Taenia crassiceps, and Echinococcus multilocularis protoscolex, the reactivity was weaker than in nematodes, and localized in the body wall muscle and subtegumental tissue. Western blotting of 8 M urea extracts of parasites with the antibody detected a pair of clear bands in nematodes but not in S. mansoni or the cestodes. These results might be explained by sparse distribution of IFs in platyhelminths, or low affinity of the used antibody to platyhelminth IF proteins, or both.

Sm15 is a major Schistosoma mansoni 15 kDa tegumental antigen, resulting from the proteolytic processing of a larger precursor. The amino terminus of Sm15 was identified by direct amino acid sequencing, and the antigen was tentatively mapped to the segment spanning amino acids 362-497 of the precursor. This will allow subsequent studies to elucidate the possible immunological role of proteolytic processing in schistosomiasis.

The SmMAK16 gene from Schistosoma mansoni was cloned by chance when an adult worm cDNA library was probed with antiserum to affinity-purified S. mansoni GSH S-transferases. SmMAK16 encodes a hydrophilic protein of 259 amino acids with a molecular mass of 31 kDa. The protein shares 43% sequence identity and 66% similarity to the nuclear protein MAK16 of Saccharomyces cerevisiae that has been implicated both in cell cycle progression and biogenesis of 60S ribosomal subunits. Both proteins display a similar degree of sequence similar to the hypothetical protein CeMAK16 from Caenorhabditis elegans. These proteins share a number of apparent protein motifs, including two nuclear localization signals (NLS), multiple sites for phosphorylation by protein kinase CK2 and four conserved cysteine residues that resemble a zinc binding domain. SmMAK16 mRNA is more highly expressed in adult female worm than males. Recombinant SmMAK16 was phosphorylated by human protein kinase CK2. When chimeric constructs containing SmMAK16 fused the green fluorescent protein (GFP) were transiently transfected into COS-7s cells, the reporter was localized not in nuclei, but exclusively in nucleoli. The yeast and nematode homologues were likewise able to direct nucleolar accumulation of the fluorescent reporter. The high degree of sequence conservation together with the ability to direct nucleolar protein transport supports the hypothesis that MAK16 proteins play a key role in the biogenesis of 60S subunits.

Synthesis of parasite specific IgE plays a critical role in the defence against helminth infections. We report here that IgE from serum from Schistosoma mansoni infected mice and Haemonchus contortus infected sheep recognizes complex-type N-glycans from Arabidopsis thaliana, which contain R-GlcNAcbeta1-->4(Fucalpha1-->3)GlcNAcbeta1-Asn (core alpha1-->3-Fuc) and Xylbeta1-->2Manbeta1-->4GlcNAcbeta1-R (core beta1-->2-Xyl) modifications, and honeybee phospholipase A2, which carries N-glycans that contain the core alpha1-->3-Fuc epitope. Evidence is presented that core alpha1-->3-fucosylated N-glycans bind a substantial part of the parasite specific IgE in serum of H. contortus infected sheep. These results suggest that the core alpha1-->3-Fuc antigen may contribute to induction of a Th2 response leading to the production of IgE. In addition we show here that N-glycans carrying core alpha1-->3-Fuc and beta1-->2-Xyl antigens are synthesized by many parasitic helminths and also by the free living nematode Caenorhabditis elegans. Since N-glycans containing the core alpha1-->3-Fuc have also been implicated in honeybee and plant induced allergies, this conserved glycan might represent an important common IgE epitope.

Specialized regions of cellular membranes termed detergent-insoluble glycosphingolipid-enriched membrane domains (DIG) have been identified in mammalian cells and shown to contain signalling molecules, cholesterol, sphingolipids and caveolae. Here we report that the unusual double surface membrane of the tegument of the trematode parasite Schistosoma mansoni possesses biochemically distinct domains analogous to DIG. When subjected to sucrose density gradient centrifugation, a detergent-extracted tegument from adult parasites yielded a low-density fraction consisting of detergent-insoluble complexes (DIC). Several tegument proteins were concentrated in DIC and a subset of these were labelled when adult schistosomes were biotinylated using a membrane-impermeant reactive biotin prior to extraction. The GPI-linked proteins alkaline phosphatase (SmAP), Sm200, the membrane-bound protein Sm23, and a protein recognized by an antibody against human caveolin, co-purified with DIC whereas soluble proteins, such as paramyosin and aldolase, were found at the bottom of the gradient. Antibodies against DIC immunoprecipitated a subset of worm surface proteins and immunolabeled the dorsal tegument of adult worms. Transmission electron microscopy of DIC revealed caveolae-like structures in the double bilayer surface structure. These results suggest that the tegument of adult S. mansoni possesses specialized membrane domains that are resistant to detergent-extraction, contain a subset of total tegument membrane proteins, and bear caveola-like invaginations, and thus are analogous to DIG.

The ubiquitous vertebrate protein stathmin is expressed and phosphorylated in response to a variety of external and internal signals. Stathmin, in turn, controls cell growth and differentiation through its capacity to regulate microtubule assembly dynamics. This is the first report on the molecular cloning and characterization of a stathmin-like protein (SmSLP) in an invertebrate, the human blood fluke Schistosoma mansoni. SmSLP is first synthesized at high levels in the intermediate molluscan host and completely disappears 48 h after penetration into the mammalian host. The protein is preferentially iodinated in intact immature parasites using the Bolton-Hunter reagent, can be quantitatively extracted in high salt buffers, and remains soluble after boiling. Native SmSLP was partially sequenced, and its complete structure was derived from the cloning and sequencing of its cDNA. The sequence is up to 26% identical to vertebrate stathmin sequences and contains two potential phosphorylation sites. Native SmSLP is indeed phosphorylated because phosphatase digestion shifts its mobility in electrofocusing gels. SmSLP associates with tubulin, as suggested by immune co-precipitation results. In vitro experiments demonstrated that SmSLP inhibits tubulin assembly and causes the depolymerization of preassembled microtubules, thus probably fulfilling regulatory roles in critical steps of schistosome development.

Several amplicons with approximately 120 bp each, obtained from the upstream domain of Schistosoma mansoni female-specific gene F-10, were coupled to Dynabeads M-280 streptavidin. The beads were used as a matrix for affinity purification of nuclear proteins obtained from mixed populations of adult worms. A protein of approximately 12 kDa, bound to the DNA in a sequence-independent manner. In contrast, when the DNA matrix was narrowed down to smaller synthetic oligonucleotides, bearing sequences corresponding to the TATA box and the CAAT box, band-shift assays revealed that different nuclear proteins from either adult male or female worms formed complexes with the DNA adduct. In order to characterise the bound proteins, the same oligonucleotides were UV cross-linked to the male and female protein extracts. Whilst the band shift experiments showed that the proteins from each sex produced a distinct mobility pattern when the TATA box sequences were tested and a similar one when the CAAT box sequences were added to the proteins, UV cross-linking experiments revealed clear qualitative differences between both, male and female proteins and also between the proteins binding to the two motifs. These results are compatible with a model in which the differential expression of the F-10 gene might depend on individual sub-sets of proteins.

Localization of the type 2 Alu sequence (B2), a highly repetitive DNA sequence in the mouse genome, was examined by in situ polymerase chain reaction (in situ PCR) in schistosomes. The signals to the B2 sequence were detected in the cytoplasm of the tegumental membrane and in the nuclei of the mesenchymal, testicular, ovarian and vitelline cells of 8-week Schistosoma japonicum. In contrast, it was difficult to detect any signals of this sequence in 8-week S. mansoni, whereas in 24-week male S. mansoni the signals were observed in the cytoplasm of the tegumental tubercles and in the nuclei of the mesenchymal and testicular cells. On the other hand, in 24-week female S. mansoni the signals were found in the nuclei of the mesenchymal, ovarian and vitelline cells but not found in the tegument. On the contrary, no hybridization band of the B2 sequence was detected in the amplified DNA of 3-week schistosomula of either species. These observations proved that the host DNA sequences existed in restricted schistosome cells and were accumulated in the schistosome body during their development.

Sm15 and Sm25 are two of the principal tegumental antigens recognized by antibodies from mice protectively vaccinated with adult worm tegumental membranes and may therefore be potential vaccine candidate antigens. Using antibodies affinity purified from anti-tegumental membrane anti-sera, and antibodies raised against the recombinant antigens, Sm15 and Sm25 were shown to be located specifically in the tegument of adult worms being distributed throughout the syncitium but not associated with the outer membrane.

Two clones, totalling 8068 bp and spanning over half of the coding region of the mitochondrial genome of Schistosoma mansoni, have been sequenced. Complete sequences are presented of the large and small ribosomal RNA subunits, CO2, ND3, ND4, ND6 and ATPase 6 genes. Incomplete sequences were found for the CO1, ND2 and CytB genes. At least 10 tRNAs were also detected and alternative structures for some of these discussed. The gene order of S. mansoni is unique and differs from that of Fasciola hepatica, the only other trematode for which any information is available.

TFG was discovered as a fusion partner of NTRK1 in human papillary thyroid carcinoma. We assembled the mouse TFG cDNA from EST sequences and 5' end RACE product, identified full coding length TFG EST clones in pig (c17b07) and Schistosoma mansoni (SMNAS62), and analyzed the genomic structure of TFG in Caenorhabditis elegans (Y63D3A). The protein sequences of mouse, pig, and S. mansoni TFG are highly homologous to human TFG. The C. elegans sequence has diverged, but its predicted secondary structure is remarkably conserved. Human, mouse, and C. elegans TFG contain a putative trimeric N-terminal coiled-coil domain, glycosylation, myristylation, and phosphorylation sites, and SH2- and SH3-binding motifs. The SH2-binding motif is absent in C. elegans TFG. The expression of TFG does not vary among 7, 11, 15, and 19 day mouse embryonal stages. In situ hybridization with a TFG probe in 10, 5-day whole mouse embryos showed preferential staining of the limb buds, branchial arches, nasal processes, and brain, and weak staining of the primitive spinal cord and dorsal root ganglia., (Copyright 1999 Academic Press.)

Aim: To observe the distribution of Schistosoma mansoni glucose transport proteins, SGTP1 and SGTP4, in the tegument of Schistosoma japonicum., Methods: The rapidly frozen fixation technique and ultracryomicrotomy were adopted for preparing ultrathin cryosections of S. japonicum. Anti-SGTP1 and anti-SGTP4 antibodies were used to localize the corresponding antigens in the tegument of adult S. japonicum by immunocytochemical technique., Results: SGTP1 was localized on the basal membrane of the tegument and its infoldings, SGTP4 was localized on the apical membrane of the tegument and its invaginations of S. japonicum., Conclusion: The same localization for SGTP 1 and SGTP4 in the tegument of S. japonicum and S. mansoni exhibited apparent homology between SGTPs of the two schistosomes.

Schistosomes have lost the capability to synthesize fatty acids de novo, but they can modify fatty acids by chain elongation. This has a profound effect on the molecular species composition of the two main phospholipid fractions of schistosomes, phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Molecular species of phospholipids are increasingly recognized as important mediators, or precursors thereof, in signal transduction, immune response modulation, and events like membrane fusion. As these are all important aspects of schistosome membranes and of the tegumental membranes in particular, we analysed the PE and PC molecular species of the tegumental membranes, the worm body and the blood of the host. With the aid of on-line mass spectrometry, we unequivocally identified a large number of PC and PE species in schistosomes, among which considerable amounts of plasmalogen species. This was unexpected, as this lipid subclass has been assumed to be absent in the parasite. Species, like (20:1-16:0) diacyl PC and (16:0-20:1) plasmalogen PE, found to be main constituents in schistosomes, were absent from the blood of the host. Large differences were also found between the molecular species composition of the tegumental membranes and the membranes of the worm body. In the tegumental membranes, 1-hexadecyl 2-palmitoyl PC was detected, which could possibly function as a precursor for platelet activating factor (PAF).

The present study investigated the presence of ryanodine receptors in the trematode Schistosoma mansoni. [3H]Ryanodine specific binding sites were found in the four subcellular fractions of S. mansoni; however, more binding sites were recovered in the heterogeneous fraction P1 and the microsomal fraction P4, as was thapsigargin-sensitive (Ca2+-Mg2+)ATPase activity, marking the sarco/endoplasmic reticulum calcium ATPase (SERCA) pumps. This binding had an equilibrium dissociation constant (Kd) in the nanomolar range, an apparent maximal number of receptors (Bmax) of about 80 fmol/mg of protein, and was modulated by ions (Ca2+, Mg2+) and some pharmacological tools such as caffeine. Ryanodine was able to accelerate the rate of 45Ca2+ release from actively loaded vesicles, and also to induce a transient contraction of the whole worm. We conclude that ryanodine-sensitive Ca2+ release channels are present in S. mansoni, with properties very similar to the ones present in higher animals.

Traditionally, the family Campulidae has been associated either with the family Fasciolidae, parasites of ruminants, or the Acanthocolpidae, parasites of fishes, based on morphological similarities. Since morphology does not seem to resolve clearly the problem of the relationships of campulids, we have used the sequences of the 18S rRNA gene of the campulids Zalophotrema hepaticum, Campula oblonga and Nasitrema globicephalae, the fasciolid Fasciola hepatica, the acanthocolpid Stephanostomum baccatum and the outgroup Schistosoma mansoni to infer a phylogeny. Maximum parsimony and neighbour-joining methods were applied. Both methods indicated that campulids are closer to acanthocolpids than fasciolids. In order to confirm this relationship, we generated a second phylogeny using all the partial sequences of the 18S published for trematodes: Lobatostoma manteri, Echinostoma caproni, Calicophoron calicophorum, Tetracerasta blepta, Gyliauchen sp. and Opistorchis viverrini, plus those mentioned above, and Dicrocoelium dendriticum. The aspidogastrean L. manteri was used as the outgroup. Results were identical to the first analysis. According to this and the most recent Digenean phylogeny, which considers campulids and acanthocolpids as sister groups, we suggest that a common origin for these 2 groups would imply a host-switching process. The life-cycle of acanthocolpids includes marine gastropods as first intermediate hosts, and fishes as second intermediate and definitive hosts. In this context, the hypothesis would be that trematodes whose cycle ended in fishes were able to switch to mammalian hosts.

Catecholamines such as dopamine and noradrenaline play important roles as neuromuscular transmitters and modulators in all parasitic helminths, including the human parasite, Schistosoma mansoni. We have cloned a novel S. mansoni tyrosine hydroxylase (SmTH) cDNA that shows high homology to mammalian tyrosine hydroxylase, the enzyme that catalyzes the first and rate-limiting step in the biosynthesis of catecholamines. Two subsets of SmTH transcripts were identified, one of which carries the S. mansoni spliced-leader (SL) sequence at its 5' end, whereas the other does not appear to be trans-spliced to the S. mansoni SL. The two types of SmTH transcripts encode the same protein of 465 amino acids and a predicted size of 54 kDa. Expression of SmTH as an N-terminal histidine fusion protein in Escherichia coli produced an active enzyme that was purified approximately 52-fold to apparent homogeneity and had a final specific activity of 0.78 micromol/min/mg. The purified enzyme was found to have the same absolute requirement for a tetrahydrobiopterin cofactor and the same sensitivity to inhibition by high concentrations of the substrate, tyrosine, as the mammalian enzyme. Purified SmTH also showed characteristic inhibition by catecholamine products, although the sensitivity to product inhibition was lower than that of the mammalian enzyme. This evidence indicates that SmTH encodes a functional tyrosine hydroxylase that has catalytic properties similar to those of the mammalian host's enzyme but may differ in its properties of regulation. This first demonstration of tyrosine hydroxylase in a parasitic helminth further suggests that the parasites have the enzymatic capacity to synthesize catecholamines endogenously.

An enzyme presenting kallikrein-like activity (designated sK1) was purified from the supernatant of Schistosoma mansoni adult worm homogenate. The enzyme cleaves bradykinin from purified rat plasma kininogen. Activity was optimal at pH 9.0 and the enzyme showed amidolytic activity, since it hydrolysed the kallikrein synthetic substrate D-Pro-Phe-Arg-p-nitroanilide. The activity of sK1 upon rat plasma kininogen was strongly inhibited by the serine proteinase inhibitors phenylmethanesulfonyl fluoride, aprotinin or soybean trypsin inhibitor, but not by ethylenediaminetetraacetic acid or sodium tetrathionate. The molecular mass of sK1, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, was 66 kDa and the pI value, estimated by analytical chromatofocusing, was 4.2. Physical and chemical properties suggest that sK1 is a serine proteinase of the kallikrein family. Evidence is presented which suggests that sK1 is a component of the tegumental surface of the parasite and the levels of its activity in the male adult worm are approximately 21 times higher than those in the female adult worm. The intravenous injection of 3 micrograms of sK1 into an anaesthetized rat induced a drastic reduction in the arterial blood pressure of the animal. This effect lasted for about 1 min, and was followed by a progressive recovery of the arterial pressure. Neither bradycardia nor cardiac arrhythmias were noticed, suggesting a peripheral vasodilation effect. The presence of sK1 on the surface of adult male worms could play an important role in the wandering capacity of coupled worms into the visceral vasculature of the host.

The lipid metabolism of schistosomes is characterized by several intriguing adaptations to a parasitic way of living. The surface of the parasite consists of two closely apposed phospholipid bilayers, a structure unique to blood flukes. Schistosomes do not synthesize fatty acids de novo, but are able to modify fatty acids, which they obtain from the host, by chain elongation. Here we present evidence that schistosomes are capable of another type of fatty acid modification, resulting in the formation of 5-octadecenoic acid [C18:1(5)]. This highly unusual fatty acid, which is absent in the blood of the host, was shown to be almost exclusively located in the outer membrane complex of the schistosome. Within these membranes, it was almost exclusively present in one molecular phospholipid species, 1-palmitoyl-2,5-octadecenoyl phosphatidylcholine [C16:0-18:1(5)PtdCho]. Apart from dipalmitoyl phosphatidylcholine, this was the most abundant phosphatidylcholine species in the outer membrane complex. The specific synthesis by the schistosome of C18:1(5) and the highly specific localization of this fatty acid to the tegumental membranes suggest an important tegument-mediated role for this lipid.

Polymerase chain reaction and cDNA library screening approaches were employed to identify a putative member of the highly conserved family of ATP-binding cassette transport proteins from Fasciola hepatica. At the predicted protein level, the F. hepatica sequence identified in the present study shares 43% and 36% identity with the Schistosoma mansoni SMDR2 and human MDR1 ATP-binding cassette transport sequences, respectively. Northern blot and reverse transcriptase-PCR analyses have demonstrated that expression of the F. hepatica ABC-transporter homologue is confined to immature parasites. The biochemical basis for the stage-specific expression of the ATP-binding cassette transporter homologue within F. hepatica remains to be determined.

The chemical synthesis of beta-D-GalpNAc-(1-->4)-beta-D-GlcpNAc- (1-->3)-alpha-D-Galp-(1-->O)-(CH2)5NH2 is described. This structure represents the nonfucosylated backbone trisaccharide of the glycocalyx glycan of the cercarial stage of the parasite Schistosoma mansoni. Synthesis of the trisaccharide was achieved via a stepwise coupling approach. 5-Azidopentyl 4-O-acetyl-2,6-di-O-benzyl-alpha-D-galactopyranoside was condensed with ethyl 6-O-benzyl-2-deoxy-3,4-di-O-dimethylisopropylsilyl- 2-phthalimido-1-thio-beta-D-glucopyranoside, using N-iodosuccinimide and silver trifluoromethanesulfonate as a catalyst system, followed by the removal of the silyl ether groups to afford a disaccharide acceptor. Coupling of ethyl 4,6-di-O-acetyl-3-O- allyloxycarbonyl-2-deoxy-2-phthalimido-1-thio-beta-D-galactopyrano side to the disaccharide acceptor, using methylsulfenyl bromide and silver trifluoromethanesulfonate as a catalyst system, gave a protected trisaccharide. Deprotection of this compound yielded the target structure.

During antibody screening of a Taenia saginata oncosphere cDNA library a clone (R-Tso2) sharing a high degree of homology at both the DNA and amino acid levels with the small heat-shock protein (shsp) family was identified. The R-Tso2 clone was a full-length sequence (1162 bp) with an open reading frame of 945 bp and 314 amino acids, corresponding to a deduced molecular mass of 35.6 kDa and isoelectric point of 5.6. R-Tso2 had the highest degree of homology with the Schistosoma mansoni major egg antigens, showing the characteristic shsp 100 amino-acid sequence motif duplicated. The R-Tso2 expression product was not immuno-precipitated by any serum from a panel of serum samples obtained from bovine, porcine and human hosts suffering from either T. saginata or T. solium cysticercosis.

A number of HPLC chromatographic procedures can be used to separate intact molecular species of phosphatidylcholine (PC), but on-line quantification has remained problematic due to insensitivity of UV-detection for saturated species. Here, a new method is presented, separating all major PC molecular species from a variety of biological samples in intact form using a single, short and isocratic run. Species were separated on two RP18 reverse-phase columns in series and all species displayed an exponential relation between retention time and the percentage of acetonitrile or triethylamine in the mobile phase, allowing optimization of the mobile phase on a theoretical base, rather than on time-consuming test-runs. The use of triethylamine as a volatile additive instead of choline chloride allowed the use of light scattering detection. On a molar base, the response of the detector was invariant between species and allowed quantification of as little as 50 pmoles. The method was tested using phosphatidylcholines with widely different molecular species patterns, such a PC from rat liver, porcine pulmonary surfactant, bovine heart, boar sperm cells, and the parasite Schistosoma mansoni. As only volatile components are present in the solvents, individual molecular species can easily be recovered in pure form from the column effluent, enabling their further analysis (e.g., scintillation counting).

Sodium dodecyl sulphate- poly acrylamide gel electronphoresis (SDS-PAGE) fractionation of tegumental surface antigens (STEG-Ags.) of 7-day cultured normal and irradiated schistosomula showed no obvious qualitative differences. The observed polypeptide bands of both normal irradiated STEG-Ags. were almost identical and have similar corresponding molecular weights. The immunoblotting assay, using different types of mouse sera, revealed similarity between the bands of both normal and irradiated STEG-Ags. recognized by each type of mouse serum. No qualitative rather than quantitative differences have been observed. The quantitative differences were reflected in intensively staining of some bands from normal STEG-Ag. rather than their corresponding bands of the same molecular weights from irradiated STEG-Ag.

Biotinylation is proposed for the identification of surface proteins in Schistosoma mansoni using the streptavidin-HRP conjugate for the detection of labeled polypeptides. However, control samples also showed several endogenous biotinylated polypeptides. In an attempt to determine the possibility of nonspecific binding between the streptavidin-HRP conjugate and polypeptides from S. mansoni, the conjugate was blocked with biotinamidecaproate-N-hydroxysuccinimide ester (BcapNHS) before biotin-streptavidin blotting. No bands were detected on the nitrocellulose sheet, demonstrating the specific recognition of biotin by the streptavidin present in the conjugate. Whole cercariae and cercarial bodies and tails showed several endogenous biotinylated polypeptides. The biotin concentration was 13 micrograms/190,000 cercariae. Adult worms presented less endogenous biotinylated polypeptides than cercariae. These results may be due to changes in the environment from aerobic to anaerobic conditions when cercarial bodies (schistosomula) are transformed into adult worms and a decrease in CO2 production may occur. Cercariae, cercarial bodies and adult male worms were examined by transmission electron microscopy employing an avidin-colloidal gold conjugate for the detection of endogenous biotin. Gold particles were distributed mainly on the muscle fibers, but dispersed granules were observed in the tegument, mitochondria and cytosol. The discovery of endogenous biotin in S. mansoni should be investigated in order to clarify the function of this vitamin in the parasite.

DNA encoding the full-length Schistosoma mansoni epidermal growth factor receptor, SER, was obtained by combining partial cDNA clones. Three different anti-SER antibody preparations, specific either to the SER amino-terminus or the predicted ligand binding domain were generated with recombinant or synthetic peptides and purified by antigen affinity. Recombinant SER was expressed within insect cells using the baculovirus expression system and detected by each of the anti-SER antibodies as well as anti-phosphotyrosine antibodies. By in vitro phosphorylation of immunoprecipitated recombinant SER, the protein has been shown to be capable of tyrosine autophosphorylation but this activity is not affected by human epidermal growth factor. Native SER is detected as a 170 kDa protein in Western blots of S. mansoni adult worm membrane preparations. Adult worm sections, labeled with anti-SER antibodies, localize SER predominantly to the muscle of adult male and female worms. These results confirm a place for SER in the EGFR family of tyrosine kinases and strongly suggest that it participates in schistosome signal transduction, perhaps related to muscle development or function.

The A subunit of NF-Y nuclear factor from Schistosoma mansoni was expressed in E. coli fused to a histidine tag and purified by affinity chromatography using a Ni(2+)-Agarose matrix. Antibodies against the recombinant protein were prepared and used for Western blot and immunolocalization. The presence of SMNF-YA in all stages of the parasite life-cycle was determined by RT-PCR and Western blot analysis. The immunolocalization of SMNF-YA showed the presence of this factor in a parenchymal cell population of cercariae and adult worms and in embryos within eggs. The expression of SMNF-YA was demonstrated to decrease in maturating spermatozoites whereas an accumulation of this factor was observed in the nucleus from oocytes during their maturation processes.

The tegument of Schistosoma mansoni contains a number of proteins that presumably function in its maintenance and/or repair against damage incurred from host-mediated humoral immune responses. Here, we show that the schistosome antigen identified by monoclonal antibody 709A2/2 is a cytoplasmic dynein light chain. Dynein light chains are components of dynein, an enzyme complex involved in various aspects of microtubule-based motility. Monoclonal antibody 709A2/2 recognizes two polypeptides, one of 8.9 kDa and a second of 7.6 kDa, as determined by SDS-polyacrylamide gel electrophoresis. We find that expression of S. mansoni dynein light chain is developmentally regulated and localized to the tegument in the schistosomula, lung stage worms, and adult worms, but is not present in the cercariae or ciliated miracidia. By Northern blot analysis of adult worm RNA, S. mansoni dynein light chain is encoded by a single message of approximately 600 base pairs. A cDNA encoding this polypeptide contains an open reading frame of 89 amino acids with a deduced molecular mass of 10.4 kDa. Coprecipitation of an apparent 18.4-kDa antigen with S. mansoni dynein light chain by monoclonal antibody 709A2/2 illustrates that this molecule has an affinity for other proteins. Such interactions may play a role in S. mansoni dynein light chain participation in organelle trafficking in S. mansoni.

DNA is the essential substrate for the polymerase chain reaction (PCR). Standard protocols include a DNA purification step, but this is laborious if a large number of DNA preparations have to be performed, although a variety of simple methods exist for the isolation of crude DNA for PCR. For microorganisms, PCR protocols exist that allow the amplification of sequences directly from the organism without DNA purification. The results introduced in this paper demonstrate that a direct PCR approach also works with fruitflies and blood flukes as examples for intact multicellular organisms.

Schistosoma mansoni possesses two isoforms of ferritin, soma and yolk ferritin. The soma ferritin occurs at a low level in most cells of both genders, whereas the yolk ferritin is a female-specific gene product that is expressed at high level in the vitellarium. In higher animals, ferritin mRNA is regulated by iron via the interaction of cytoplasmic binding proteins (IRPs) with a specific sequence element in the 5' untranslated region (UTR) referred to as the iron-responsive element (IRE). Sequence studies of the 5' UTRs, gel retardation assays, and hybridization experiments show that neither ferritin mRNAs of S. mansoni is regulated by an IRE/IRP mechanism. It is suggested that ferritins in schistosomes are controlled only at the transcriptional level.

Utilizing a Biomphalaria alexandrina-derived lectin (BaSII) of proven specificity to a Schistosoma mansoni-associated fucosyllactose [(Fuc alpha 1-2)Gal beta 1-4 Glc] determinant, a 37-kDa determinant-bearing glycoprotein (Sm 37) was identified selectively on adult male schistosomes. Sm 37 was purified to homogeneity from extracts of adult male worms metabolically radiolabeled with [35S] methionine by BaSII affinity chromatography followed by separation on an HPLC column. Treatments with endoglycosidases, alkaline borohydride, as well as serial lectin affinity chromatography and analysis on 2-dimensional gels indicated that Sm 37 is synthesized as a 33-kDa polypeptide backbone that expresses the fucosyllactose determinant on the outer chain of a single N-linked complex-type glycan unit of either the biantennary or, to a lesser extent, the tri- or tetra-antennary types. The distinct structures of the complex oligosaccharides accounted for the expression of 2 isomorphs of Sm 37. the glycoprotein lacks other conventional high mannose-type or O-linked oligosaccharides and, as deduced from the N-terminal amino acid sequence, the Sm 37 polypeptide may be distinct from other schistosome polypeptides of known sequence. Based on the structural relatedness of the Sm 37-associated fucosyllactose determinant to the antigenic blood group H trisaccharide, these observations may have implications for mechanisms of these host-parasite interactions.

The properties of 4 fluorescent lipid compounds in the surface membrane of adult male Schistosoma mansoni worms were examined by fluorescent microscopy and fluorescent recovery after photobleaching (FRAP). The data suggest that the probes N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s- indacene-3-pentanoyl) sphingosine (BODIPY FL ceramide) and PKH2 pass through the outer membrane and enter structures in or below the membrane. In contrast 5-(N-octadecanoyl)aminofluorescein (AF18) and N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s- indacene-3-pentanoyl) sphingosylphosphocholine (BODIPY FL sphingomyelin) insert into the outer monolayer. The DL values of these latter 2 compounds, 8:83 +/- 2.35 x 10(-9)cm(2)s(-1) and 2.76 +/- 0.53 x 10(-9)cm(2)s(-1), respectively, suggest that they enter different domains. Furthermore, it was observed that both BODIPY FL ceramide and BODIPY FL sphingomyelin entered particular structures in or under the surface membrane. The possible nature of these particles is discussed.

The 100000Xg supernatant parasite platyhelminth Schistosoma mansoni exhibits a glutathione peroxidase activity with the substrate phosphatidylcholine hydroperoxide. Purification yielded a protein of 20 kDa molecular mass both on gel filtration column chromatography and SDS/PAGE, thus suggesting that S. mansoni expresses a protein similar to the mammalian selenoenzynic phospholipid-hydroperoxide glutathione peroxidase. Kinetic analysis and substrate specificity corroborated this assumption, the second-order rate constants for the oxidation of the ground-state enzyme (k+1) being higher with phosphatidylcholine hydroperoxide than with other peroxide substrates, such as cumene liydroperoxide or H2O2, and quantitatively similar to those of mammalian phospholipid-hydroperoxide glutathione peroxidase. Partial sequencing of the protein and selenium measurement by neutron activation analysis established that the purified peroxidase corresponded to the product of the S. mansoni gene previously reported and supposed to encode a selenium-containing glutathione peroxidase [Roche, C., Williams, D. L., Khalife, J., LePresle, T., Capron, A. & Pierce, R. J. (1994) Cloning and characterization of gene encoding Schistosoma mansoni glutathione peroxidase, Gene 138, 149 - 152]. S. mansoni thus contains a scienoperoxidase sharing molecular mass, catalytic efficiency and substrate specificity with phospholipid-hydroperoxide glutathione peroxidase, dismantling the concept that those enzymes are unique to vertebrate organisms.

Carbohydrates associated with the tegumental surface of Schistosoma mansoni primary sporocyst may serve as potential receptors for mediating recognition by the internal defence system of the molluscan host, Biomphalaria glabrata. Therefore, a combination of SDS-PAGE and lectin probe analyses were carried out on biotin-labelled tegumental glycopeptides as a first step to defining the carbohydrates expressed at the sporocyst surface. The majority of surface polypeptides, ranging in relative molecular masses from 27 to 113 kDa, reacted with horseradish peroxidase-labelled Canavalia ensiformis (Con A), Erythrina corallodendron (ECA), Glycine max (SBA) and Triticum vulgaris (WGA) lectins indicating that most, if not all, tegumental proteins are glycosylated. However, differences in the binding of some lectins to individual glycopeptides suggest a degree of heterogeneity in the structure/composition of sugar moieties comprising these surface glycoconjugates. This notion is supported by the finding that the fucose-specific Tetragonolobus purpureas (TPA) lectin only reacted with approximately 50% of glycopeptides identified at the tegumental surface. Experiments employing biotin-labelled plasma (cell-free haemolymph) from S. mansoni-susceptible and -resistant B. glabrata snails as probes, further demonstrated that many of the identified surface glycoproteins also serve as plasma-binding sites for both snail strains. Binding interactions between plasma and sporocyst surface glycoproteins appeared to be, at least in part, mediated by carbohydrates since periodate treatment of sporocyst proteins or pre-incubation of plasma with the glycoproteins, fetuin or mucin, resulted in a decrease in plasma reactivity to blotted larval proteins.