Characterization and expression of a novel cystatin gene from Schistosoma japonicum

Cystatins are a family of cysteine protease inhibitors that play a crucial role in the immune evasion from their host and in the adaptation to host defence. Here, we isolated a full-length cDNA sequence inferred to encode a novel cystatin gene from a blood fluke, Schistosoma japonicum. The cDNA, designated SjCystatin, comprised an open reading frame (ORF) of 306 bp, and encoded 101 amino acids with a predicted molecular weight of 11.3 kDa. This predicted protein shared a significant degree of sequence identity with the type I cystatin (stefin) of Schistosoma mansoni and Homo sapiens. These proteins exhibited a typical cystatin topology, including the absence of disulfide bonds and three conserved catalytic motifs, Gly at the N-terminus (Gly(6)), Gln-X-Val-X-Gly motif (Q(49)VVAG(53)) and an LP pair at the C-terminus (L(76)P(77)). The SjCystatin gene spanned 376 bp and contained three exons. The positions of two introns were conserved between the cystatin genes of trematodes and their vertebrate hosts. Reverse transcription polymerase chain reaction confirmed the transcription of SjCystatin in the egg, schistosomula and adult stages of S. japonicum. The encoding ORF region was cloned into pET-28a (+) prokaryotic expression vector. After purification, the recombinant protein SjCystatin (recSjCystatin), expressed in Escherichia coli, was used to immunize animals and produce its specific polyclonal antibody. Western blot analysis revealed that the native SjCystatin was expressed in the egg and adult stages. The enzyme activity assay of the recSjCystatin showed that it inhibited the proteolytic activity of papain. SjCystatin protein was mainly localized on the miracidium within eggs. Immunohistochemistry revealed that SjCystatin mainly localized in the epithelial cells lining the gut as well as the tegument on the surface of adult worms. The conserved genomic DNA structure among cystatin homologues of trematode and their vertebrate host emphasized the characteristics of compatibility between parasites and their hosts. This study provides the first insight into the gene and protein of S. japonicum cystatin and a basis for a further understanding the functions of this gene.