|KINSELLA, JANE - UNIV. COLLEGE OF DUBLIN
|RYAN, MICHAEL - UNIV. COLLEGE OF DUBLIN
Submitted to: Veterinary Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/7/2001
Publication Date: 1/30/2002
Citation: KINSELLA, J.L., LICHTENFELS, J.R., RYAN, M.F. A PRELIMINARY ANALYSIS OF PROTEOLYTIC ACTIVITY OF EXCRETORY-SECRETORY PRODUCTS FROM CYATHAOSTOMINEA. VETERINARY PARASITOLOGY. 107: 73-83. 2002.
Interpretive Summary: Nematodes of the subfamily Cyathostominae are now regarded as the principal parasitic pathogen of the horse. The marked decline in prevalence of large strongyle infections has not dramatically reduced the occurrence of strongyle-associated disease, and clinical attention is now focusing on the pathogenicity of cyathostomins. There is a paucity of information surroundi ithe detailed biology and pathogenic mechanisms of the cyathostomins. In particular, there seems to be no quantification of proteolytic activities i Cyathostominae and their role in pathogenicity. This study presents a preliminary analysis of proteolytic activity in the excretory-secretory product (ESP) of Cyathostominae. The excretory-secretory product (ESP) derived from Cyathostominae in vitro was assessed in terms of subunit composition, and proteolytic activity. Sodium dodecyl sulphate-polyacrylami gel electrophoresis resolved 13 subunits, and the presence of the protein cysteine proteinase activator dithiothreitol (DTT) revealed 21 subunits. S data indicate the presence of cysteine-class proteinases in the ESP of adul Cyathostominae. Further understanding of the Cyathostominae ESP may offer n leads for use in immunological or chemical control of these parasites. The results will be used by researchers worldwide working to control these economically important nematodes of horses.
Technical Abstract: The excretory-secretory product (ESP) derived from Cyathostominea in vitro was assessed in terms of subunit composition, and proteolytic activity using as substrates azocasein and two synthetic fluorogenic peptides. Sodium dodecyl suphate-polyacrylamide gel electrophoresis resolved 13 subunits, and the presence of the protein cysteine proteinase activator dithiothreitol (DTT) revealed 21 subunits. DTT also enhanced azocaseinolysis, and hydrolysis of carbobenzoxy-phenylalanyl-arginine-7- amido-4-methylcoumarin (Z-Phe-Arg-NHMec) and carbobenzoxy-arginyl-arginine- 7-amido-4-methylcoumarin (Z-Arg-Arg-NHMec). At the optimum pH of 5.5, hydrolysis of Z-Phe-Arg-NHMec was 3-fold greater than that of Z-Arg-Arg- NHMec suggesting that the proteolytic specificities of the ESP are more like those of papain or cthepsin L, rather than cathepsin B. In SDS-PAGE gelatin gels, DTT was a requirement for proteolysis by the ESP. Optimum resolutio was at pH 5.5, resolving 6 bands ranging from 114-20 kDa. Cysteine proteinase inhibitors abolished all gelatinolytic activity at the pH values tested. Such data indicate the presence of cysteine-class proteinases in the ESP of Cyathostominea.