Characterization of Ascaris suum larval excretory-secretory proteins: Protease activity and glycosylation

Stephen Ray Morris, Purdue University

Abstract

The excretory-secretory products of the infective larvae of Ascaris suum have been examined for proteolytic activity and glycosylation. The larval stages of this nematode, a direct infection parasite of swine, undergo liver-lung migration before becoming established as adults in the intestine. During this migration, the infective L2 larva develops successively to the L3 and L4 stages, which are separated by molting events. Two peaks of activity have been observed at acid and neutral pH. Upon further characterization, three major protease activities belonging to three different families have been identified. A cysteine endoprotease active at pH 4.5 was detected which may be responsible for the collagenase activity also observed at this pH. An aminopeptidase, belonging to the metalloprotease family, and a serine protease were detected at neutral pH. The identification of these activities as members of one particular protease family or another was based on their experimental inhibition when assayed at optimal pH with specific synthetic substrates. The cysteine protease, assayed at pH 4.5 with the substrate CBZ-Gly-Pro-pNA, was strongly inhibited by E-64, N-ethylmalimide, p-hydroxymercuribenzoic acid, and iodoacetic acid. The serine protease, assayed at pH 7.0 with the substrate CBZ-Gly-Pro-Arg-pNA, was most inhibited by the compounds PMSF and benzamidine. The other neutral activity, that of the aminopeptidase assayed at pH 7.0 with CBZ-Leu-pNA, was inhibited by l,10-phenanthroline, EDTA, and DTT. It appears that there is stage-specific expression of these proteases during the development of the larvae in vitro. When proteins recovered from medium conditioned by larvae at the stages of hatching, molting, or growth were assayed for proteases, different patterns of specific activity were observed. Based on these patterns, it is suggested that the acid cysteine proteinase is involved with molting. The neutral aminopeptidase may also be involved with molting, or, along with the serine proteinase, tissue penetration. Other methods of protein characterization have indicated that the ES material of Ascaris suum infective larvae contains proteins which are modified by the attachment of complex carbohydrate chains. These methods included the enzymatic and chemical removal of carbohydrates from proteins transferred to nitrocellulose, allowing the examination of separated proteins without the alteration of their molecular weights.

Degree

Ph.D.

Advisors

Kazacos, Purdue University.

Subject Area

Biochemistry|Veterinary services|Biology

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