Schistosoma japonicum cathepsin D aspartic protease cleaves human IgG and other serum components.

Recombinant cathepsin D aspartic protease of Schistosoma japonicum cleaved human IgG in vitro in a time and dose-dependent manner. Optimal cleavage was seen at pH 3.6-4.5; modest cleavage remained at pH 5.0, and no cleavage was detected above pH 5.0. Amino terminal sequencing of the major cleavage fragments of human IgG identified a Fab fragment from the VH1 domain, and 2 cleavage sites in the CH2 domain below the hinge region. The P1 and P1' residues at the 2 CH2 cleavage sites were Phe254-Leu255 and Leu325-Thr326, indicating a preference by the schistosome protease for bulky hydrophobic residues flanking the scissile bond. No cleavage of the immunoglobulin light chain was detected. In addition, the recombinant schistosome protease indiscriminately degraded the human serum proteins complement C3 and serum albumin into numerous small fragments. These results demonstrate specific cleavage of human IgG by the recombinant schistosome aspartic protease, and highlight the broad range digestive specificity of the enzyme which may play a role in the degradation of host serum proteins ingested as part of the schistosome bloodmeal.

Vaccine efficacy of recombinant cathepsin D aspartic protease from Schistosoma japonicum.

Mice were vaccinated with recombinant Schistosoma japonicum cathepsin D aspartic protease, expressed in both insect cells and bacteria, in order to evaluate the vaccine efficacy of the schistosome protease. Mean total worm burdens were significantly reduced in vaccinated mice by 21-38%, and significant reductions in female worm burdens were also recorded (22-40%). Vaccination did not reduce fecundity; rather, we recorded increased egg output per female worm in vaccinated animals, suggesting a crowding effect. Vaccinated mice developed high levels of antibodies (predominantly IgG1, IgG2a and IgG2b isotypes), but there was no correlation between antibody levels and protective efficacy. Immune sera from vaccinated mice did not inhibit the in vitro degradation of human haemoglobin by the recombinant protease, and passive transfer of serum or antibodies from vaccinated animals, before and after parasite challenge, did not significantly reduce worm or egg burdens in recipient animals. These results suggest that antibodies may not play a key role in the protective effect elicited, and that protection may be due to a combination of humoral and cell-mediated responses

Gulliver, a long terminal repeat retrotransposon from the genome of the oriental blood fluke Schistosoma japonicum.

We characterized the consensus sequence and structure of a long terminal repeat (LTR) retrotransposon from the genome of the human blood fluke, Schistosoma japonicum, and have named this element, Gulliver. The full length, consensus Gulliver LTR retrotransposon was 4788 bp, and it was flanked at its 5'- and 3'-ends by LTRs of 259 bp. Each LTR included RNA polymerase II promoter sequences, a CAAT signal and a TATA box. Gulliver exhibited features characteristic of a functional LTR retrotransposon including two read through (termination) ORFs encoding retroviral gag and pol proteins of 312 and 1071 amino acid residues, respectively. The gag ORF encoded motifs conserved in nucleic acid binding proteins, while the pol ORF encoded conserved domains of aspartic protease, reverse transcriptase (RT), RNaseH and integrase, in that order, a pol pattern conserved in the gypsy lineage of LTR retrotransposons. Whereas the sequence and structure of Gulliver was similar to that of gypsy, phylogenetic analysis revealed that Gulliver did not group particularly closely with the gypsy family. Rather, its closest relatives were a LTR retrotransposon from Caenorhabditis elegans, mag from Bombyx mori and, to a lesser extent, easel from the salmon Oncorhynchus keta. Dot blot hybridizations indicated that Gulliver was present at between 100 and several thousand copies in the S. japonicum genome, and Southern hybridization analysis suggested its probable presence in the genome of Schistosoma mansoni. Transcripts encoding the RT domain of Gulliver were detected by RT-PCR in larval and adult stages of S. japonicum, indicating that (at least) the RT domain of Gulliver is transcribed. This is the first report of the sequence and structure of an LTR retrotransposon from any schistosome or indeed from any species belonging to the phylum Platyhelminthes.

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.

Molecular modeling and substrate specificity of discrete cruzipain-like and cathepsin L-like cysteine proteinases of the human blood fluke Schistosoma mansoni.

Adult Schistosoma mansoni blood flukes express two discrete cysteine proteinases, SmCL1 and SmCL2, both of which are related to the cathepsin L-like enzymes of the C1 family of peptidases. Our previous phylogenetic analysis indicated that SmCL1 is more closely related to cruzipain from the parasitic protozoa Trypanosoma cruzi than to human cathepsin L, whereas the converse situation applies with SmCL2. To characterize their catalytic subsites and substrate specificities, we have now developed three-dimensional (3D) homology models of SmCL1 and SmCL2 using the structure of cruzipain and cathepsin L. Eisenberg analysis of the 3D models revealed self-compatibility scores of 90.1 and 96.1 out of a possible score of 97.6 for SmCL1 and SmCL2, respectively, verifying the accuracy and utility of the models. Substrate preferences of recombinant SmCL1 and SmCL2 at positions P3, P2, and P1 conformed to the substrate specificity predicted by the models. In particular, SmCL1 and SmCL2 both exhibited high affinity (k(cat)/K(m)) for substrates with hydrophobic residues at P2 including Z-Leu-Arg-NHMec (773.4 and 548.5 mM(-1) s(-1), respectively), Boc-Val-Leu-Lys-NHMec (116.8 and 306.5 mM(-1) s(-1)), and Z-Phe-Arg-NHMec (38.9 and 113.4 mM(-1) s(-1)). SmCL1 exhibited only a low affinity for the cathepsin B diagnostic substrate Z-Arg-Arg-NHMec while SmCL2 failed to cleave this substrate. The substrate specificities of SmCL1 and SmCL2 were clearly differentiated with H-Leu-Val-Tyr-NHMec and Suc-Leu-Tyr-NHMec since SmCL1 cleaved both efficiently (k(cat)/K(m) values of 51.9 and 41.1 mM(-1) s(-1), respectively), whereas SmCL2 cleaved neither. The 3D models revealed that this difference in specificity was due to restrictions imposed on the S3 subsite of SmCL2 as a result of insertion of two amino acids vicinal to residue 60.

Developmental expression of cathepsin D aspartic protease in Schistosoma japonicum.

Schistosomes utilise haemoglobin from ingested host erythrocytes as their main source of amino acids. Using reverse-transcriptase PCR, we detected transcripts encoding cathepsin D in eggs, miracidia, and adult male, female and mixed-sex Schistosoma japonicum. Using the synthetic fluorogenic peptide, o-aminobenzoyl-isoleucyl-glutamyl-phenylalanyl-p-nitro-phenylalanyl-a rgi nyl-leucine-NH2, and human haemoglobin as substrates, we detected cathepsin D-like aspartic protease activity at pH 3.6 in extracts of these developmental stages which was completely inhibited by the addition of l0 microM pepstatin. Using immunoblotting with rabbit antibodies raised against recombinant S. japonicum cathepsin D, we detected the aspartic protease in extracts of all developmental stages examined, although it appeared to be expressed at higher levels in the adult female schistosome. These results indicate that (almost) all stages of S. japonicum, express an aspartic protease. Moreover, they are consistent with the hypothesis that this enzyme plays a key role in maturing and adult schistosomes in the proteolysis of host haemoglobin from ingested erythrocytes.