Schistosoma Japonicum: The Molecular Characteristics Of Cystatin And It’s Immune Regulation Effects On Mice

At present, we have very few knowledge about the immune evasion mechanisms of S. japonicum. Recently, it has been reported that cysteine protease inhibitors （cystatins） of parasite play an important role in suppressing the immune response of host. It may be an important molecule involved in immune evasion. In this study, we have obtained a novel cystatin gene from Schistosoma japonicum by the ways of bioinformatics and molecular biology, which is named SjCystatin. By the research of the structure and function of this gene and its recombinant protein, especially the immune regulatory function for the host, we will aim to clarify the role of S. japonicum’s cystatin in the immune evasion preliminarily.First of all, by the partial sequences of S. japonicum’s cystatin searched through the database of NCBI and the genomic databases of S. japonicum, we obtained the full-length cDNA and genomic DNA sequences of SjCystatin by use of PCR and 3 ’RACE techniques. Using bioinformatics to analyze this gene, we found that the cDNA of SjCystatin comprised an open reading frame （ORF） of 306 bp, and encoded for 101 amino acids with a predicted molecular weight of 11.3 kDa and pI of 6.95. The full-length genomic DNA sequences of SjCystatin were 376 bp, including the two introns and three exons. By the analysis of comparative genomics, genomic structure of SjCystatin was similar with stefins family and cystatins family of vertebrate. We speculated that they might be evolved from a common ancestral gene. Phylogenetic analysis showed that SjCystatin protein had a high homology with cystatin of Schistosoma mansoni （Genebank No. XP₀02572115） and the similarity was 77%. Bioinformatics analysis showed that SjCystatin didn’t have signal peptide and transmembrane region, which was suggesting that the protein might be intracellular and play a biological role in the cell. By the analysis of GeneDoc, we found that SjCystatin exhibited a typical cystatin topology, including the absence of the disulfide bonds and three conserved catalytic motifs, Gly at the N-terminus （Gly6）, Gln-X-Val-X-Gly motif （Q49VVAG53） and LP pair at the C-terminus （L76P77）. SjCystatin shared the same conserved motifs with stefins family and cystatins family of vertebrate. According to the above characteristics, we thought that SjCystatin should be a new member of stefins family.The prokaryotic expression vector of SjCystatin was successfully constructed. And the recombinant protein SjCystatin was efficiently expressed and was purified by a Ni-NTA agarose column. As a result of the His tag, the putative molecular weight of rSjCystatin was 12.1 kD. The specific polyclonal antibody of rSjCystatin could identify with the protein of Mw 11.3 in AWA and SEA from S. japonicum. The bacterially expressed rSjCystatin showed inhibitions of the papain proteolytic activity. rSjCystatin of 10μg could inhibit papain activity unit of 0.0517 per minute under the conditions of 37℃and pH 7.4. Reverse transcriptase polymerase chain reaction revealed Sj-cystatin could be detected in egg, schistosomula and adult. Immunohistochemistry and confocal laser scanning microscopy analysis showed SjCystatin mainly localized in the miracidium of eggs and in the epithelial cells lining the gut as well as the tegument on the surface of adult worms. The experiment of immune protection revealed the worm reduction rates and the egg reduction rates of immunized with rSjCystatin group had no significant difference compared with PBS control group. That indicated rSjCystatin had no significant protective effect on mice. We observed that rSjCystatin could go into DC cells by confocal laser scanning microscopy. And the rSjCystatin only entered the cytoplasm of DC cell. We proved rSjCystatin could inhibit activity and metabolic state of DC cell by MTS experiment. The average fluorescence intensity of MHC-Ⅱmolecules on the surface of DC cell was detected by Flow CytoMeter. And it was indirectly confirmed that rSjCystatin could reduced antigen presenting function of DC cell. The experiment of rSjCystatin inducing proliferation of CD4+CD25+ Foxp3+ T cell of mice in vivo showed that rSjCystatin could significantly increase the ratio of CD4+CD25+ Foxp3+ regulatory T cell in infected mice. These results suggested that rSjCystatin had the roles of inhibiting antigen’s presentation and inducing of immune suppression. So it might be an immunosuppressive factor from schistosome and played a role in immune evasion of schistosome.