Isolation of putative cysteine protease genes of Ostertagia ostertagi.

Recombinant phage containing putative Ostertagia ostertagi cysteine protease genes have been isolated from a lambda EMBL-3:genomic DNA library using a Haemonchus contortus cathepsin B-like cysteine protease cDNA as hybridization probe. Restriction enzyme maps of the phages suggest that they identify at least 3 genes, 2 of which appear to be linked in tandem. The complete nucleotide sequence of one gene, CP-1, was determined. The CP-1 gene appears to be organized into 12 exons than span 4.5 kb of DNA. The number and sizes of the exons are essentially identical to those in the H. contortus AC-2 cysteine protease gene. Partial nucleotide sequences obtained for a second O. ostertagi gene, CP-3, revealed a similar organization for exons 8-12 in this gene. Like other cathepsin B-like cysteine proteases, CP-1 appears to be synthesized initially as a preproprotein that is proteolytically processed to its mature form. The amino acid identity between the presumptive CP-1 and CP-3 proteins is 66%, which is similar to the level of homology between the presumed mature protein regions of CP-1 and AC-2. Amino acid identity between CP-1 and AC-2 is greatest in the mature protein region and lowest in the signal sequence and propeptide regions. The CP-3 protein appears to be most closely related to the H. contortus AC-5 protein. CP-1 and CP-3 display significantly greater homology to H. contortus cysteine proteases than they do to human cathepsin B or the Sm31 cysteine protease of Schistosoma mansoni (about 40% identity with each).     

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.     

Single nucleotide polymorphisms identification in expressed genes of Schistosoma mansoni

Single nucleotide polymorphism (SNP) markers have been shown to be useful in genetic investigations of medically important parasites and their hosts. In this paper, we describe the prediction and validation of SNPs in ESTs of Schistosoma mansoni. We used 107,417 public sequences of S. mansoni and identified 15,614 high-quality candidate SNPs in 12,184 contigs. The presence of predicted SNPs was observed in well characterized antigens and vaccine candidates such as those coding for myosin; Sm14 and Sm23; cathepsin B and triosephosphate isomerase (TPI). Additionally, SNPs were experimentally validated for the cathepsin B. A comparative model of the S. mansoni cathepsin B was built for predicting the possible consequences of amino acid substitutions on the protein structure. An analysis of the substitutions indicated that the amino acids were mostly located on the surface of the molecule, and we found no evidence for a significant conformational change of the enzyme. However, at least one of the substitutions could result in a structural modification of an epitope.     

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.     

Cloning and expression of a gene encoding Sm16, an anti-inflammatory protein from Schistosoma mansoni

The gene encoding Sm16, an anti-inflammatory, immunomodulatory protein present abundantly in secretions of the infective stages of Schistosoma mansoni was cloned and partially characterized. A data base analysis showed sequence homology to an earlier reported schistosomular stathmin-like gene sequences reported in dbEST and Genbank. The putative gene coding for Sm16 is of 500 bp with an open reading frame of 117 aa that included an N-terminal signal peptide sequence of 18 aa. There are three potential sites for phosphorylation (two serine and one tyrosine residue) but no glycosylation sites in the sequence. The coding region of Sm16 was amplified from cercarial cDNA, cloned and expressed in bacterial and insect expression systems. The purified recombinant protein showed strong immunoreactivity with a polyclonal rabbit anti-Sm16 antibody raised against the native anti-inflammatory protein Sm16. Contrary to earlier report, this gene appears to be not stage-specific. Metabolic labeling studies suggested that Sm16 is phosphorylated and is synthesized by both cercariae and schistosomula of S. mansoni. Sequence homology with human stathmin, a cell cycle regulatory phospho protein, was 30%. However, when probed with specific antibodies, no cross reactivity was observed between Sm16 and human stathmin.     

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.     

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.     

Nucleotide sequences of the helper component-proteinase genes of aphid transmissible and non-transmissible isolates of turnip mosaic virus

Summary We have compared nucleotide sequences of the helper component-proteinase (HC-Pro) coding region of aphid transmissible (isolate 1) and non-transmissible (isolate 31) isolates of turnip mosaic virus (TuMV). HC-Pro coding regions of both TuMV isolates 1 and 31 were 1,374 nucleotide long. The nucleotide sequence homology between these isolates was 93.5%, with 89 nucleotides substitution. The nucleotides of HC-Pro regions of two isolates of TuMV genomes encoded 458 amino acids of M_r 51,746 (isolate 1) and M_r 51,764 (isolate 31). The deduced amino acid sequence homology between these isolates was 98.7% with six different amino acids. These amino acids appeared to regulate the activity of HC-Pro needed for aphid transmissibility of TuMV.     

Characterization of the repetitive DNA elements in the genome of fish lymphocystis disease viruses

The complete DNA nucleotide sequence of the repetitive DNA elements in the genome of fish lymphocystis disease virus (FLDV) isolated from two different species (flounder and dab) was determined. The size of these repetitive DNA elements was found to be 1413 bp which corresponds to the DNA sequences of the 5' terminus of the EcoRI DNA fragment B (0.034 to 0.052 m.u.) and to the EcoRI DNA fragment M (0.718 to 0.736 m.u.) of the FLDV genome causing lymphocystis disease in flounder and plaice. The degree of DNA nucleotide homology between both regions was found to be 99%. The repetitive DNA element in the genome of FLDV isolated from other fish species (dab) was identified and is located within the EcoRI DNA fragment B and J of the viral genome. The DNA nucleotide sequence of one duplicate of this repetition (EcoRI DNA fragment J) was determined (1410 bp) and compared to the DNA nucleotide sequences of the repetitive DNA elements of the genome of FLDV isolated from flounder. It was found that the repetitive DNA elements of the genome of FLDV derived from two different fish species are highly conserved and possess a degree of DNA sequence homology of 94%. The DNA sequences of each strand of the individual repetitive element possess one open reading frame.     

Structural homology of tropomyosins from the human trematode Schistosoma mansoni and its intermediate host Biomphalaria glabrata

Molecular mimicry has been considered as a possible way for parasites to escape host immune responses. This work concerns the characterization of protein determinants shared by Schistosoma mansoni and its intermediate host Biomphalaria glabrata. Parasite (Sm39) and mollusc (Bg 39) cross-reactive proteins were identified and shown to induce in rabbit and mouse, antibodies specific for invertebrate determinants. Ultrastructural studies demonstrated that antibodies to Sm39 specifically bound to muscular structures of parasite and mollusc. Molecular cloning and sequencing indicated that Bg39 corresponded to a muscular isoform of tropomyosin. The mollusc sequence showed a 51-65% homology with seven different muscular tropomyosins from vertebrate and invertebrate species. The highest score of homology was observed with S. mansoni tropomyosin, suggesting that cross-reactive determinants could be specific for the trematode and its intermediate host. In miracidia, Sm39 epitopes were also shown to be contained in the vesicles present in epidermal ridges and cellular bodies. Such vesicles are involved in the formation of a protective tegument around sporocysts, suggesting a possible role of cross-reactive tropomyosins in miracidia and/or sporocyst-snail interactions.     

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.     

Cloning and sequence comparisons of four distinct cysteine proteases expressed by Haemonchus contortus adult worms.

Three new members of a developmentally regulated cysteine protease gene family of the parasitic nematode Haemonchus contortus have been isolated and characterized. One of the new genes, AC-3, was found to be linked in tandem to the previously characterized AC-2 gene. Nucleotide sequence analyses revealed that the first 90 amino acids of AC-3 are organized into four exons, similar to the situation in AC-2. A cDNA that appears to be a near full-length copy of the AC-3 gene was isolated using the polymerase chain reaction (PCR) technique to amplify cDNAs from adult worm poly(A)+ mRNAs. In addition to AC-3, a distinct cysteine protease cDNA, AC-4, was amplified by the same oligonucleotide primers. cDNAs encoding a fifth cysteine protease, AC-5, were isolated from an adult worm cDNA expression library using specific rabbit antisera and by PCR. Comparison of the predicted amino acid sequences of AC-3, AC-4 and AC-5 reveal that they share 64-77% identity with one another and with the previously reported AC-1 and AC-2 sequences. The amino acids surrounding the active site cysteine are highly conserved, as are the positions of other cysteine residues in the mature protein sequences. The H. contortus proteases are more similar to one another than they are to human cathepsin B (38-44% amino acid identity) or to the Sm31 cysteine protease of Schistosoma mansoni (36-40% identity). Our studies indicate that H. contortus adult worms express mRNAs for several distinct cysteine proteases. The significant primary sequence differences between the proteases suggest that they differ in their substrate specificities and precise physiological functions.     

Rapid detection of genomic variations in different strains of hantaviruses by polymerase chain reaction techniques and nucleotide sequence analysis

The polymerase chain reaction (PCR) with subsequent nucleotide sequence analysis was employed to rapidly detect genomic variations among different Hantavirus strains. Using synthetic oligonucleotide primers derived from the M and S segment RNAs of nephropathia epidemica virus strain H?lln?s B1 (NEV) we succeeded in amplifying the corresponding sequences of Hantaan and Puumala viruses. The nucleotide sequences of the cDNAs derived from the Puumala M and S RNA segments were analyzed. It was found that the particular nucleotide sequences of Puumala M and S segments were 81% and 82% homologous to the corresponding genomic segments of NEV, respectively. The amino acid homology was 94% for both segments. In contrast, the degree of homology to the corresponding Hantaan M and S genomic RNA segments was 63% at the nucleotide level for both segments and 53 and 55% at the deduced amino acid level, respectively. This demonstrates that Puumala virus is very similar to NEV and significantly different from Hantaan virus at both the nucleotide and protein level.     

Characterisation of Sm20, a 20-kilodalton calcium-binding protein of Schistosoma mansoni

A cDNA clone encoding part of a 20-kDa antigen of Schistosoma mansoni (Sm20) has been isolated. The amino acid sequence of this antigen, as predicted from the sequence of the cDNA, has significant homology to the family of calcium binding proteins which include calmodulin, troponin C and the light chain of myosin. Although we have been unable to show any immunological cross-reactivity between Sm20 and calmodulins from a range of other species, we have verified that Sm20 is a functional calcium binding protein. Sm20 is encoded by a small multigene family and is expressed in schistosomula and adult worms but not in eggs. The 20-kDa nascent polypeptide appears to be post-translationally modified to give a 38-kDa species. Sm20 is present in preparations of tegumental membranes and is easily removed from intact schistosomula by detergent treatment, suggesting that it is associated with the tegument. However, the cloned portion does not appear to be exposed on the surface.     

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.     

RNA segment 5 of broadhaven virus, a tick-borne orbivirus, shows sequence homology with segment 5 of bluetongue virus

The sequence of Broadhaven (BRD) virus segment 5, the major genetic determinant of serotype, is 1658 nucleotides in length and contains a single open reading frame (ORF) having the coding capacity for a protein of Mr 52.5K. Comparison of the ORF of segment 5 of BRD virus with published sequences of bluetongue virus (BTV) revealed 30% nucleotide homology and 31% amino acid homology with the protein encoded by segment 5 of BTV serotype 10. Significant homology was not shown with segment 2 of BTV, the major genetic determinant of the BTV serotype. The sequences at the 3' and 5' ends determined for BRD segment 5 were similar to the respective 3' and 5' regions of BTV. The sequence data provide evidence of an evolutionary relationship between two ecologically distinct groups of orbiviruses and demonstrate changes that have occurred in the functions of genetically related genomic segments.     

Substrate specificity of schistosome versus human legumain determined by P1-P3 peptide libraries

Asparaginyl endopeptidases, or 'legumains' have been identified and characterized in plants, the blood fluke parasite Schistosoma, and mammals. The legumains are a novel family of cysteine proteases and display restricted specificity for peptide hydrolysis on the carboxyl side of asparagine residues. Two forms of recombinant asparaginyl endopeptidase from Schistosoma mansoni (C197 Sm32 and N197C Sm32), expressed in Pichia pastoris, have been analyzed for substrate specificity using a positional-scanning synthetic combinatorial library (PS-SCL). We first screened Sm32 using a P1-diverse library. This library demonstrated the absolute specificity of Sm32 for asparagine at P1. To determine the P2-P3 preferences of Sm32, we constructed a library with asparagine fixed at P1, and the P2-P3 positions randomized. The library was screened using the two forms of Sm32, human asparaginyl endopeptidase, and to confirm its diversity, cruzain from Trypanosoma cruzi. The schistosome legumain showed a preference for P3: Thr>Ala>Val>Ile, and P2: Ala>Thr>Val>Asn, with an overall broader specificity at P3 than at P2. Both human and schistosome legumain can accommodate Thr and Ala at P2 and P3. However, optimal substrate sequences differ, with Sm32 preferring Thr-Ala-Asn, and human legumain preferring Pro-Thr-Asn. Predictions of substrate specificity from the library screen were confirmed using single peptide substrates for kinetic assays.