Secretion and processing of a novel multi-domain cystatin-like protein by intracellular stages of Trichinella spiralis.
The excretory-secretory (ES) proteins of nematode parasites are of major interest as they function at the host-parasite interface and are likely to have roles crucial for successful parasitism. Furthermore, the ES proteins of intracellular nematodes such as Trichinella spiralis may also function to regulate gene expression in the host cell. In a recent proteomic analysis we identified a novel secreted cystatin-like protein from T. spiralis L1 muscle larva. Here we show that the protein, MCD-1 (multi-cystatin-like domain protein 1), contains three repeating cystatin-like domains and analysis of the mcd-1 gene structure suggests that the repeated domains arose from duplication of an ancestral cystatin gene. Cystatins are a diverse group of cysteine protease inhibitors and those secreted by parasitic nematodes are important immuno-modulatory factors. The cystatin superfamily also includes cystatin-like proteins that have no cysteine protease inhibitory activity. A recombinant MCD-1 protein expressed as a GST-fusion protein in Escherichia coli failed to inhibit papain in vitro suggesting that the T. spiralis protein is a new member of the non-inhibitory cystatin-related proteins. MCD-1 secreted from T. spiralis exists as high- and low-molecular weight isoforms and we show that a recombinant MCD-1 protein secreted by HeLa cells undergoes pH-dependent processing that may result in the release of individual cystatin-like domains. Furthermore, we found that mcd-1 gene expression is largely restricted to intracellular stages with the highest levels of expression in the adult worms. It is likely that the major role of the protein is during the intestinal stage of T. spiralis infections.
Robinson
Mark W
MW
School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, Scotland, UK.
Massie
Diane H
DH
Connolly
Bernadette
B
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D015891:Cystatins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010206:Papain / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017160:Trichinella spiralis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D015891:Cystatins; D018076:DNA, Complementary; D011994:Recombinant Proteins; D010206:Papain