ENZYMATIC-ACTIVITY OF 2 CASPASES RELATED TO INTERLEUKIN-1-BETA-CONVERTING ENZYME

Interleukin-1 beta-converting enzyme is a member of a family of human cysteine proteases with specificity for aspartic acid, which have been named caspases. Within this family of enzymes, transcript X (TX) and transcript Y (TY) (caspases 4 and 5, respectively) are very similar to ICE (caspase 1) and form the ICE subfamily. Given the high degree of conservation in the sequences of these proteases (more than 50% amino acid identity in the mature enzymes), it was of interest to examine wh ether they shared similar substrate specificities. The three enzymes, ICE, TX and TY, were therefore expressed in boculovirus-infected insec t cells, as 30-kDa proteins lacking the propeptide. Automaturation int o p20 and p10 subunits occured within the cells. Active ICE, TX and TY were collected in the cell culture supernatants. In addition, their p roduction induced the activation of an endogenous 32-kDa putative cyst eine protease (CPP32) like caspase. T7-tagged ICE, TX and TY were puri fied by immunoaffinity and tested for their catalytic efficiency on YV AD-containing synthetic substrates and an the ICE natural substrate, p ro-interleukin-1 beta. TX cleaved the same synthetic substrates as ICE (K-m of 90 mu M and K-cat of 0.4 s(-1) for Suc-YVAD-NH-Mec, where Suc represents succinyl and NH-Mec represents amino-1-methylcoumarin) and could cleave pro-interleukin-1 beta into the same peptides as ICE but less efficiently. On the other hand, TY showed very little efficacy o n the different ICE substrates (K-m of 860 mu M for Suc-YVAD-NH-Mec). These results show that the ICE/TX/TY subfamily has functional heterog eneity and that ICE remains the preferred enzyme for pro-interleukin-1 beta cleavage.