Primary structures of Sm31/32 diagnostic proteins of Schistosoma mansoni and their identification as proteases

We have constructed cDNA clones containing the complete nucleotide sequences coding for two highly antigenic Schistosoma mansoni adult worm proteins, Sm31 and Sm32. The predicted amino acid sequence of Sm31 shows significant homology to mouse, rat and human cathepsin B. The nucleotide sequence of Sm32 is identical to that reported by others for S. mansoni "haemoglobinase'. The different nucleotide sequences demonstrate the existence of two different proteolytic enzymes, both of which are synthesised in the form of precursor molecules. Structural homology of the schistosome cathepsin B to the mammalian ones indicates that the mature protein is processed from a propeptide. The calculated molecular weight of haemoglobinase of 47,000 suggests that post-translational processing is also involved in generating an active protease.     

Comparison of the cloned genes of the 26- and 28-kilodalton glutathione S-transferases of Schistosoma japonicum and Schistosoma mansoni.

Both Schistosoma japonicum and S. mansoni contain 28- and 26-kDa glutathione S-transferases (GSTs). Despite their immunological cross-reactivity using rabbit antisera, the S. japonicum 28-kDa GST (Sj28) is weakly immunogenic relative to the S. mansoni protein (Sm28) in mouse immunization experiments using GSTs purified from adult worms. The difference in immunogenicity is also observed during schistosome infection in mice. Using surface-labeled living S. japonicum worms, evidence was obtained for a surface location of Sj28 comparable to that reported for the S. mansoni molecule. The nucleotide and deduced amino acid sequences of cDNA clones corresponding to Sj28 and Sm28 were compared. Despite obvious homology (77% identity), differences were found in regions known to contain T epitopes in the S. mansoni protein which may be an explanation for the striking differences in immunogenicity in regard to antibody production in mice. The 26-kDa GSTs of these two parasites (Sj26 and Sm26) are also closely related on the basis of nucleotide and deduced amino acid sequences, there being 82% identity in the putative coding regions. When the amino acid sequences of Sj28 and Sm28 were compared with those of Sj26 and Sm26, the overall sequence identity was approximately 20%. However, a relatively conserved region was identified in otherwise structurally different molecules which may participate in common properties of these enzymes.     

Schistosoma mansoni and its intermediate host Biomphalaria glabrata express a common 39 kilodalton acidic protein

Two Schistosoma mansoni proteins of 43 and 39 kDa (Sm43 and Sm39) were shown to react with rabbit antibodies produced against Biomphalaria glabrata proteins. Two-dimensional gel electrophoresis of miracidial proteins indicated that Sm43 and Sm39 were acidic proteins (pI 4.8 and 4.9 respectively) and were in vitro translated from miracidial messenger RNA in the same molecular forms. Sm43 and Sm39 were expressed by all parasite stages of S. mansoni. Using anti-Sm43 and anti-Sm39 mouse sera, we demonstrated that both parasite proteins were antigenically related and cross-reacted with a unique 39 kDa (pI 4.9) protein from B. glabrata (Bg39). Cross-reactive components were found in fresh water and land snails but not in vertebrate tissues, suggesting that the 39 kDa protein was specific for invertebrates.     

Immunological similarity between Schistosoma and bovine cathepsin D

IgG antibodies from sera of rabbits immunized with a mixture of three synthetic peptides of highly conserved surface-exposed sequences between Schistosoma japonicum and S. mansoni cathepsin D, and a rabbit anti-bovine cathepsin D serum strongly recognized a 45 kDa molecule on immunoblots of adult S. mansoni worm saline extracts (AWSE). This recognition was abolished by immunoadsorption with two of the three selected peptides. The anti-peptide antibodies fixed onto Protein A-Sepharose specifically immunoprecipitated a S. mansoni AWSE component that was able to degrade bovine hemoglobin at pH 3.8. This reaction was inhibited by 7 microM pepstatin A, a classical aspartyl protease inhibitor, suggesting that the parasite cathepsin D was immunoprecipitated. The anti-peptide antibodies also recognized on a dot-blot assay a purified, commercially obtained bovine cathepsin D preparation but not the purified human counterpart. On the other hand, the anti-bovine cathepsin D serum recognized the two above-mentioned schistosome peptides. In addition, S. mansoni-infected patient sera recognized on immunoblots the bovine but not the human cathepsin D. These results, together with a comparative analysis of the selected peptide sequence regions between the schistosome and the two mammal enzymes, allowed us to pinpoint to one amino acid the cross-reactivity between parasite and bovine cathepsin D and the lack of it with human cathepsin D. This difference might be of relevance for immunodiagnosis.     

Candidate vaccine antigens identified by antibodies from mice vaccinated with 15- or 50-kilorad-irradiated cercariae of Schistosoma mansoni.

In murine schistosomiasis, the highest levels of resistance to cercarial challenge are obtained by vaccination with radiation-attenuated cercariae. To identify candidate vaccine antigens relevant to the vaccine model, we examined parasite antigens recognized by antibodies from mice vaccinated with irradiated cercariae of Schistosoma mansoni. To optimize recognition of a wide spectrum of antigens, several factors that influence the level of protection in this model were varied; specifically, we examined the effect of (i) single versus multiple vaccinations with irradiated cercariae, (ii) the dose of irradiation (15 or 50 kilorads) administered to the cercariae, and (iii) the genetic background of mouse strains, high-responder (C57BL/6J) versus moderate-responder (CBA/J) mice. We found that the number of vaccinations did not alter antibody specificity but modified the relative antibody titers against particular antigens. The dose of irradiation used to attenuate the immunizing cercariae had a similar effect on antibody titers but in addition influenced antibody specificity. Only mice that had been vaccinated with moderately irradiated cercariae recognized cathepsin B (Sm31) and Sm32. Interestingly, when vaccinated mice of the two strains, C57BL/6J and CBA/J, were compared, differences in antibody responses to particular antigens were observed. Both strains recognized the integral membrane protein Sm23, glutathione S-transferase, and cathepsin B, whereas Sm32 and paramyosin were recognized only by CBA/J mice, and heat shock protein 70 was recognized exclusively by C57BL/6J mice. In this study, we conclusively identified six distinct antigens that are specifically recognized by the humoral immune response of vaccinated mice.     

Molecular cloning and tissue distribution of a 26-kilodalton Schistosoma mansoni glutathione S-transferase

A Schistosoma mansoni cDNA library was constructed from the mRNA of adult worms in the expression vector lambda gt11 and screened with a rabbit antiserum raised against the 26-kDa S. mansoni glutathione S-transferase isoforms (Sm GST 26). Two clones were selected and the nucleotide sequences deduced. The predicted amino acid sequence, specified by these cDNAs, shows strong homology with a Schistosoma japonicum 26 kDa glutathione S-transferase and a lower level of homology with mammalian glutathione S-transferase class mu isoenzymes (EC 2.5.1.18). No significant homology score was found with a 28-kDa S. mansoni glutathione S-transferase (Sm GST 28). A study of the tissue distribution of the cloned Sm GST 26 by immunoelectron microscopy shows similarities to Sm GST 28 in that they are present in the tegument and in subtegumentary parenchymal cells. However, a major difference exists in the protonephridial region in which Sm GST 26 is present in the cytoplasmic digitations localized in the apical chamber delineated by the flame cell body, suggesting that Sm GST 26 may be actively excreted by adult worms.     

Characterization of the cathepsin-like cysteine proteinases of Schistosoma mansoni.

Adult Schistosoma mansoni parasites synthesize and secrete both cathepsin L and cathepsin B cysteine proteinases. These cysteine proteinase activities, believed to be involved in hemoglobin digestion by adult schistosomes, were characterized by using specific fluorogenic peptide substrates and zymography. Both cathepsin L- and B-like activities with pH optima of 5.2 and 6.2, respectively, predominated in soluble extracts of worms, and both these activities were secreted by adult worms into the culture medium. The specific activity of cathepsin L was about double that of cathepsin B when each was assayed at its pH optimum, and moreover, the specific activities of cathepsins L and B in extracts of female schistosomes were 50 to 100% higher than in extracts of male schistosomes. Analysis of the primary structure of two cloned S. mansoni cathepsins L, here termed cathepsin L1 and cathepsin L2, revealed that they are only 44% similar and that cathepsin L2 showed more identity (52%) with human cathepsin L than with schistosome cathepsin L1. Moreover, differences in their active site, propeptide region, and potential for glycosylation suggest separate functions for schistosome cathepsin L1 and cathepsin L2.     

SmCB2, a novel tegumental cathepsin B from adult Schistosoma mansoni

Papain-like cysteine endopeptidases have been recognized as potential targets for chemotherapy and serodiagnostic reagents in infections with the human parasitic helminth Schistosoma. A novel cathepsin B endopeptidase from adult S. mansoni has been isolated and characterized. The enzyme is termed SmCB2 to distinguish it from the first recorded schistosome cathepsin B, SmCB1, also known as Sm31. A rapid and convenient protocol involving anion exchange and affinity chromatography is described for the isolation of SmCB1 and SmCB2 from the same parasite starting material. SmCB2 has been functionally expressed in and purified from Pichia pastoris. Both native and recombinant SmCB2 migrate similarly (33 kDa) by SDS-PAGE. Both display strict acidic pH activity profiles and similar K(m) and k(cat) for dipeptidyl amidomethylcoumarin substrates. We conclude that the recombinant enzyme is properly folded. The S(2) subsite specificity of recombinant SmCB2 exhibits the preferences Phe>Leu>Val>Arg. By immunoblotting with anti-SmCB2 IgG, a 33 kDa protein was identified in soluble extracts of male schistosomes. By immunohistochemistry, SmCB2 was localized in the tegumental tubercles and parenchyma of males with less product being visualized in the parenchyma of females. The enzyme may be lysosomal and function at the host parasite-interface.     

Proteolysis of human hemoglobin by schistosome cathepsin D

Schistosomes feed on human blood. They employ proteases to degrade hemoglobin from ingested erythrocytes, using the residues released for amino acid metabolism. However, the identity and the role of the participating protease(s) are unclear and controversial. Confocal microscopy localized schistosomal cathepsin D to the parasite gastrodermis, and revealed elevated protease expression in females. At sub-cellular level, cathepsin D was localized to superficial digestive vacuoles of the gut and to cisternae of the gastrodermal rough endoplasmic reticulum. Schistosome cathepsin D, expressed in insect cells, autoactivated at pH 3.6 to a approximately 40 kDa form that cleaved the substrates o-aminobenzoyl-Ile-Glu-Phe-nitroPhe-Arg-leu-NH(2) and hemoglobin. The NH(2)-terminal residues of mature cathepsin D of Schistosoma japonicum and Schistosoma mansoni were Asn1 and Gly1, respectively, revealing that the proregion peptide was comprised of 35 residues. The proteases cleaved hemoglobin at pH 2.5--4.6, releasing numerous fragments. S. Japonicum cathepsin D cleaved at 13 sites, S. mansoni cathepsin D at 15 sites. Early cleavage sites were alpha Phe33-Leu34 and beta Phe41-Phe42, while others included alpha Leu109-Ala-110 and beta Leu14-Trp15, demonstrating a preference for bulky hydrophobic residues at P1 and P1'. Most of the schistosomal cathepsin D cleavage sites were discrete from those of human cathepsin D. The gastrodermal location, elevated expression in females, acidic pH optima, similar substrate preferences in two species, and the discrete substrate preferences compared with human cathepsin D together provide compelling support for the hypothesis that schistosomal cathepsin D plays an integral role in hemoglobin proteolysis, and might be selectively targeted by drugs based on protease inhibition.     

Cathepsin B-like activity predominates over cathepsin L-like activity in adult Schistosoma mansoni and S. japonicum

Both cathepsin B-like and cathepsin L-like endopeptidase activities have been described in schistosomes, but their relative contribution to proteinolysis remains controversial. In an attempt to clarify which type of activity predominates, the selective mammalian cathepsin B inhibitor CA-074 was tested under standardized assay conditions with different preparations from Schistosoma mansoni and S. japonicum. CA-074 (0.94 μM) inhibited at least 92% and 80% of proteinolytic activity, respectively, for these species: completely inhibited bovine-spleen cathepsin B activity; but showed only marginal inhibition (4%) of rat-liver cathepsin L activity. We discuss the results with respect to previous studies and conclude that schistosome cathepsin B-like, not L-like, activity predominates. Received: 13 November 1996 / Accepted: 13 January 1997     

Functional expression and characterization of Schistosoma mansoni cathepsin B and its trans-activation by an endogenous asparaginyl endopeptidase

Peptidases are essential for the establishment and survival of the medically important parasite, Schistosoma mansoni. This helminth expresses a number of gut-associated peptidases that degrade host blood proteins, including hemoglobin, as a means of nutrition. Using irreversible affinity probes, we demonstrate that S. mansoni cathepsin B-like endopeptidase 1 (SmCB1) is the most abundant papain family cysteine peptidase in both the parasite gut and somatic extracts. SmCB1 zymogen (SmCB1pm) was functionally expressed in Pichia pastoris (4-11mgl(-1)). Monospecific and immunoselected antibodies raised against SmCB1pm localized the enzyme exclusively to the gut lumen and surrounding gastrodermis of adult worms. Recombinant SmCB1pm was unable to catalyze its activation, even at low pH. However, recombinant S. mansoni asparaginyl endopeptidase (SmAE), another gut-associated cysteine peptidase, processed and activated SmCB1pm in trans. Consistent with the known specificity of AEs, processing occurred on the carboxyl side of an asparagine residue, two residues upstream of the start of the mature SmCB1 sequence. The remaining pro-region dipeptide was removed by rat cathepsin C (dipeptidyl-peptidase I)-an action conceivably performed by an endogenous cathepsin C in vivo. The activated recombinant SmCB1 is biochemically identical to the native enzyme with respect to dipeptidyl substrate kinetics and pH profiles. Also, the serum proteins, hemoglobin, serum albumin, IgG, and alpha-2 macroglobulin were efficiently degraded. Further, a novel application of an assay to measure the peptidyl carboxypeptidase activity of SmCB1 and other cathepsins B was developed using the synthetic substrate benzoyl-glycinyl-histidinyl-leucine (Bz-Gly-His-Leu). This study characterizes the major digestive cysteine peptidase in schistosomes and defines novel trans-processing events required to activate the SmCB1 zymogen in vitro which may facilitate the digestive process in vivo.     

Paramyosin is the Schistosoma mansoni (Trematoda) homologue of antigen B from Taenia solium (Cestoda)

Antigen B, a major antigen of the cestode parasite Taenia solium, has been purified and a portion of amino acid sequence obtained. Paramyosin of the trematode parasite Schistosoma mansoni, an immunogenic protein that has shown promise as a vaccine candidate, has several biochemical and immunological properties in common with antigen B. A full-length cDNA clone of S. mansoni paramyosin has been obtained and the predicted translation product contains a sequence that is highly homologous to the sequence obtained for antigen B. The predicted amino acid composition and isolectric point of paramyosin are nearly identical to those established for antigen B. Recombinant S. mansoni paramyosin, expressed in Escherichia coli as a fusion protein with beta-galactosidase, was recognized by antisera against T. solium antigen B. We conclude from these results that S. mansoni paramyosin and T. solium antigen B are homologous proteins. Since S. mansoni paramyosin is thought to be a muscle protein and T. solium antigen B a secreted glycoprotein with anti-complement activity, this conclusion raises some interesting questions regarding the role of this class of proteins in the host-parasite relationship.     

T-Helper-Cell Determinants in Protein Antigens are Preferentially Located in Cysteine-Rich Antigen Segments Resistant to Proteolytic Cleavage by Cathepsin B, L, and D

We report on a computer algorithm capable of predicting the location of T-helper-cell epitopes in protein antigen (Ag) by analysing the Ag amino acid sequence. The algorithm was constructed with the aim of identifying segments in Ag which are resistant to proteolytic degradation by the enzymes cathepsin B, L, and D. These are prominent enzymes in the endocytic pathway through which soluble protein Ag enter APC, and resistant segments in Ag may, therefore, be expected to contain more T-cell determinants than susceptible segments. From information available in the literature on the substrate specificity of the three enzymes, it is clear that a cysteine is not accepted in any of the S2, S1, S1', and S2' subsites of cathepsin B and L, and not in the S1 and S1' subsites of cathepsin D. Moreover, we have noticed that cysteine-containing T-cell determinants in a number of protein Ag are particularly rich in the amino acids alanine, glycine, lysine, leucine, serine, threonine, and valine. By searching protein Ag for clusters of amino acids containing cysteine and two of the other amino acids we were able to predict 17 out of 23 empirically known T-cell determinants in the Ag with a relatively low number of false (positive) predictions. Furthermore, we present a new principle for searching Ag for potential amphipatic alpha-helical protein segments. Such segments accord well with empirically known T-cell determinants and our algorithm produces a lower number of false positive predictions than the principle based on discrete Fourier transformations previously described.     

GM-CSF and TNF-alpha synergize to increase in vitro granuloma size of PBMC from humans induced by Schistosoma mansoni recombinant 28-kDa GST

The 28-kDa Glutathione S-transferase of Schistosoma mansoni (Sm28 GST) was described as a protective antigen capable of reducing female fecundity and the number of eggs in mice hepatic tissues. The role of GM-CSF and TNF-alpha in the in vitro granuloma reaction of peripheral blood mononuclear cells (PBMC) from chronic intestinal schistosomiasis patients before and after chemotherapy treatment to S. mansoni recombinant Sm28 GST was evaluated. Treatment of PBMC with recombinant Sm28 GST caused a significant increase in granuloma formation when compared to SEA or SWAP. Contrary to SEA or SWAP, Sm28 GST was not capable of inducing significant cellular proliferation. Moreover, recombinant Sm28 GST promoted a significant elevation in GM-CSF and TNF-alpha levels. However, we did not detect any significant IL-10 production. When Sm28 GST was applied in the presence of anti-GM-CSF or anti-TNF-alpha antibodies in cultures, we observed a significant decrease in granuloma size. Indeed, our results demonstrated that Sm28 GST was capable of promoting high in vitro granuloma index, and this event was associated with the balance of GM-CSF and TNF-alpha. These evidences suggest a role for GM-CSF as a major mediator in increasing granuloma reaction in human schistosomiasis. This event may contribute to exacerbate the pathology resulting from egg deposition in host tissues.     

A new superfamily of lymphoid and melanoma cell proteins with extensive homology to Schistosoma mansoni antigen Sm23.

A novel cDNA clone termed R2 was isolated by subtractive hybridization of a cDNA library of phytohemagglutinin (PHA)/phorbol myristate acetate-stimulated Jurkat cells and by rescreening a cDNA library of PHA-stimulated peripheral blood lymphocytes. It hybridizes to a single mRNA species of about 2.2 kb, which is inducible in lymphoid cells and codes for a protein of 267 amino acids which contains four potential transmembrane domains. A computer-aided comparison showed strong homology to four other membrane proteins, the pan B cell marker CD37, the pan leukocyte marker CD53, the melanoma antigen ME491 and, surprisingly, the Schistosoma mansoni antigen Sm23. The four human proteins share a number of additional similarities in their overall structure. These include identical spacing of the transmembrane domains, similar hydrophobicity plots, possible N-linked glycosylation sites of similar number and position as well as similar distribution of the cysteine residues. The majority of these characteristics are still conserved in the evolutionary most distant member of this family, the Schistosoma mansoni antigen Sm23. Here we introduce this new protein superfamily and characterize the inducible, lymphoid-specific member R2.     

Schistosome asparaginyl endopeptidase (legumain) is not essential for cathepsin B1 activation in vivo

Schistosomes are parasitic platyhelminths that constitute an important public health problem. Adult parasites live in the vasculature of their vertebrate hosts where they consume blood. Ingested blood proteins are degraded by a proteolytic cascade. One of the best characterized schistosome proteases is cathepsin B1 (SmCB1 or Sm31). This protein is synthesized as a large 38 kDa precursor form which is proteolytically cleaved to yield a mature, active 31 kDa enzyme. A second schistosome protease--the asparaginyl endopeptidase SmAE (also known as Sm32, or schistosome legumain), has been proposed to proteolytically convert the 38 kDa precursor SmCB1 into its mature form. Recombinant activated SmAE has been shown to trans-process SmCB1 into the mature, catalytic form in vitro. In the present study, our aim was to test the hypothesis that in vivo SmAE likewise processes SmCB1 into its active form. To do this, expression of the SmAE gene was suppressed in adult Schistosoma mansoni using RNA interference (RNAi). The results of these experiments show that, even in the absence of detectable SmAE protein, SmCB1 is fully processed and active and support the assertion that SmAE is not essential to activate SmCB1 in vivo. The data indicate that our original hypothesis is incorrect and that SmAE is not pivotal in the in vivo conversion of cathepsin B1 into its mature, active form.     

Lupus autoantibodies discriminate between the highly homologous Sm polypeptides B/B' and SmN by binding an epitope restricted to B/B'.

The ubiquitous Sm polypeptides B/B' (28 and 29 kD) and the highly homologous tissue-specific Sm N polypeptide (29 kD) share several autoepitopes recognized by systemic lupus erythematosus (SLE) sera. Previous studies on the antigenicity of nuclear antigens recognized by human autoantibodies have not discriminated between ubiquitous and tissue-specific forms. We set out to examine whether a tissue-specific nuclear antigen, Sm N, is autoantigenic in SLE by comparing the immunoreactivity of the most unique sequences in this polypeptide. Synthetic peptides from the two regions of least sequence homology that occur between Sm N and Sm B/B', a dodecamer (amino acid residues 179-190 containing five substitutions) and an undecamer (residues 203-213 containing four substitutions) were coupled to a carrier protein. These conjugates were used to quantify IgG anti-peptide antibodies in sera from patients with SLE. Of 43 sera with anti-Sm specificity, six bound to the B/B' 179-190 peptide but not to the N version. None of 17 anti-Sm-negative SLE sera bound these peptides. The second region of least sequence homology between N and B/B' (203-213) was not antigenic. Our data suggest that a subset of SLE patients with anti-Sm reactivity have IgG autoantibodies capable of discriminating between Sm N and SmB/B' polypeptides by binding a previously unreported SmB/B'-specific autoepitope. The data also indicate that brain and heart-specific anti-Sm antibodies do not exist in SLE sera, suggesting that these tissues do not participate in the induction or maintenance of the autoimmune anti-Sm response.