CLONING AND EXPRESSION OF CATHEPSIN L-LIKE PROTEINASES IN THE HEPATOPANCREAS OF THE SHRIMP PENAEUS-VANNAMEI DURING THE INTERMOLT CYCLE

Cysteine protease activities have been characterized with benzyloxycar bonyl-lysine p-nitrophenyl ester as a synthetic substrate and E64 as a specific inhibitor in the hepatopancreas of the shrimp Penaeus vannam ei. An optimum pH of 5.1 has been measured. To characterize these cyst eine proteases, a hepatopancreas cDNA library was screened by hybridiz ation to a Norway lobster cysteine protease cDNA fragment. Two cDNAs e ncoding P. vannamei cysteine protease precursors have been cloned and sequenced. The encoded polypeptides have 326 and 322 amino acid residu es, respectively, each consisting of partial signal sequences (15 and 10 residues), a pro-region (93 and 94 residues), and a mature enzyme p olypeptide (218 residues). Cys(25), His(159) and Asn(175) form the cat alytic triad in the putative active site of the mature enzymes. Compar ed with invertebrate cysteine proteases (Homarus and Fasciola), each o f the two shrimp enzymes shows 70 and 52% amino acid sequence identity , respectively; 63% identity is shown with rat cathepsin L. Northern h ybridization analysis showed the same size for the different cysteine protease transcripts in hepatopancreas tissue (approximately 1.1 kb). During intermolt cycles, variations in cysteine protease activity were correlated with the variations in the levels of specific mRNA.