Article ID Journal Published Year Pages File Type
5915442 Molecular and Biochemical Parasitology 2014 8 Pages PDF
Abstract

•Fasciola gigantica expresses a 170 kDa multi-domain cystatin.•The protein carries a signal peptide and 12 cystatin-like domains.•It is post-translationally processed and present in the ES product.•It is localized in testis lobes and sperm in seminal vesicle and uterus.•Domains 1, 4, 6, 9 are inhibitors of cysteine proteases.

Cystatins are functional as intra- and extracellular inhibitors of cysteine proteases and are expressed as single or multi-domain proteins. We have previously described two single domain type 1 cystatins in the trematode Fasciola gigantica that are released into the parasite's intestinal tract and exhibit inhibitory activity against endogenous and host cathepsin L and B proteases. In contrast, the here presented 170 kDa multi-domain cystatin (FgMDC) comprises signal peptide and 12 tandem repeated cystatin-like domains with similarity to type 2 single domain cystatins. The domains show high sequence divergence with identity values often <20% and at only 26.8% between the highest matching domains 6 and 10. Several domains contain degenerated QVVAG core motifs and/or lack other important residues of active type 2 cystatins. Domain-specific antisera detected multiple forms of FgMDC ranging from <10 to >120 kDa molecular mass in immunoblots of parasite crude extracts and ES product with different banding patterns for each antiserum demonstrating complex processing of the proprotein. The four domains with the highest conserved QVVAG motifs were expressed in Escherichia coli and the refolded recombinant proteins blocked cysteine protease activity in the parasite's ES product. Strikingly, immunohistochemical analysis using seven domain-specific antisera localized FgMDC in testis lobes and sperm. It is speculated that the processed cystatin-like domains have function analogous to the mammalian group of male reproductive tissue-specific type 2 cystatins and are functional in spermiogenesis and fertilization.

Graphical abstractFasciola gigantica multi-domain cystatin is a post-translationally processed inhibitor of cysteine proteases localized in testis lobes and sperm in seminal vesicle and uterus.Download high-res image (234KB)Download full-size image

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