title: Purification, biochemical and molecular analysis of a chymotrypsin protease with prophenoloxidase suppression activity from the entomopathogenic nematode Steinernema carpocapsae.
abstract: A chymotrypsin serine protease (designated Sc-CHYM) was purified by gel filtration and anion-exchange chromatography from excretory-secretory products of parasitic stage Steinernema carpocapsae. The purified protease had an apparent molecular mass of 30kDa and displayed a pI of 5.9. This protease demonstrated high activity against the chymotrypsin-specific substrate N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and was highly sensitive to the inhibitor aprotinin. This protease digested the chromogenic substrate N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide with K(m), V(max) and k(cat) values of 409microM/min, 0.389microM/min and 24.9s(-1), respectively. The protease was most active at pH 8.0 and 35 degrees C, and its proteolytic activity was almost completely reduced after incubation at 75 degrees C for 30min. In vitro, this enzyme suppressed prophenoloxidase activity. In vivo, demonstration of encapsulation and melanization by purified chymotrypsin imbibed beads showed it could prevent hemocyte encapsulation and melanization by 12 and 24h, respectively. Sequence comparison and evolutionary marker analysis showed that the putative protein was a chymotrypsin-like protease with potential degradative, developmental and fibrinolytic functions. Expression pattern analysis revealed that the gene expression of Sc-CHYM was up-regulated in the parasitic stage. Sc-CHYM was clustered with several insect chymotrypsins and formed an ancestral branch in the phylogenetic tree, suggesting that Sc-CHYM branched off at an early stage of cluster divergence. The results of this study suggest that Sc-CHYM is a new member of the chymotrypsin serine protease family and that it might act as a virulence factor in host-parasite interactions.
authors.affiliation: CIRN and Department of Biology, University of Azores, Azores, Portugal. natesanbala@uac.pt
authors.forename: Natesan
authors.initials: N
authors.lastname: Balasubramanian
authors.forename: You-Jin
authors.initials: YJ
authors.lastname: Hao
authors.forename: Duarte
authors.initials: D
authors.lastname: Toubarro
authors.forename: Gisela
authors.initials: G
authors.lastname: Nascimento
authors.forename: Nelson
authors.initials: N
authors.lastname: Simões
mesh_terms: D000818:Animals; D004156:Catechol Oxidase / Q000037:antagonists & inhibitors; D002918:Chymotrypsin / Q000235:genetics* / Q000378:metabolism; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D006790:Host-Parasite Interactions / Q000235:genetics*; D007313:Insecta / Q000469:parasitology*; D008969:Molecular Sequence Data; D009348:Nematoda / Q000235:genetics* / Q000378:metabolism; D010572:Pest Control, Biological; D057057:Serine Proteases / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D004792:Enzyme Precursors; C038451:pro-phenoloxidase; D004156:Catechol Oxidase; D057057:Serine Proteases; D002918:Chymotrypsin