SECRETION OF CYSTEINE PROTEINASE ACTIVITY BY THE ZOONOTIC HOOKWORM ANCYLOSTOMA-CANINUM

The zoonotic hookworm, Ancylostoma caninum, probably induces human eos inophilic enteritis by inducing allergic responses to its secretions. This species is already known to secrete metalloproteinases, but in ot her parasites, cysteine proteinases are involved in pathogenesis. We s tudied somatic extracts of A. caninum adults and infective larvae and adult excretory/secretory (ES) antigens for cysteine proteinase activi ty using fluorogenic peptide substrates and by gelatin and fluorogenic substrate polyacrylamide gel electrophoresis. Proteolytic activity wa s observed against the cathepsins L and B-specific substrate Z-phe-arg -AMC, against the plasmin substrate Boc-val-leu-lys-AMC, and against g elatin. The Z-phe-arg-AMC-hydrolyzing activity in ES antigens and in a dult extracts was enhanced up to 15-fold by the reducing agent dithiot hreitol (DTT), but was totally blocked by specific inhibitors of cyste ine proteinases, including the peptidyl diazomethyl ketone Z-phe-ala-C HN2, E-64, leupeptin, and N-ethylmaleimide. In a similar fashion, gela tinolytic activity in ES antigens detected using substrate Z- els was enhanced by the addition of reducing agents and inhibited by Z-phe-ala -CHN2 and E-64. The DTT-enhanced, Z-phe-arg-AMC-hydrolyzing activity i n ES antigens was active over a wide pH range (pH 5-9). Similar cystei ne proteinase activity to that detected in ES antigens was present in extracts of adult and infective larvae of A. caninum. Because the subs trate Z-phe-arg-AMC was specifically hydrolyzed, and because this hydr olysis was totally blocked by cysteine proteinase-specific inhibitors, ES antigens and tissue extracts of A. caninum clearly possess cystein e proteinase activity. Furthermore, since the specific activity of Z-p he-arg-AMC-cleaving enzyme was up to five-fold higher in ES antigens t han in soluble extracts of parasites, the cysteine proteinase is activ ely secreted by adult hookworms. This is the first demonstration of cy steine proteinases from hookworms and enlarges the range of potential allergens that may induce human eosinophilic enteritis.