IDENTIFICATION OF CALCIUM-ACTIVATED NEUTRAL PROTEASE AS A PROCESSING ENZYME OF HUMAN INTERLEUKIN-1-ALPHA

We describe here the involvement of calcium-activated neutral protease (CANP or calpain, EC 3.4.22.17) in calcium-dependent proteolytic processing of the precursor of human interleukin la (IL-1α) into mature IL-1α. Calcium ionophore ionomycin enhanced proteolytic processing of pre-IL-1α and the release of mature IL-1α either from lipopolysaccharide (LPS)-activated human adherent mononuclear cells or from a human bladder carcinoma cell line (HTB9 5637) that constitutively produces human IL-1α and -β. The proteolytic processing of pre-IL-1α was completely inhibited by EGTA. Similar calcium-dependent proteolytic processing of pre-IL-1α was also observed with lysates of either LPS-activated human adherent mononuclear cells or HTB9 5637 cells. Since the optimal pH for processing was between 7 and 8, and E-64 (a cysteine protease inhibitor) and leupeptin (a serine and cysteine protease inhibitor) both inhibited this processing by cell lysates, we hypothesized that a calcium-activated neutral protease, CANP, might be responsible for this processing. This hypothesis was supported by data showing that the specific CANP inhibitor peptide inhibited this proteolysis in cell lysates in a dose-dependent fashion (IC50 = 0.05 μM) and that treatment of pre-IL-1α with purified CANP yielded the 17-kDa mature form of IL-1α, which has an amino terminus identical with that reported for mature human IL-1α. Taken together, these findings indicate that calcium-dependent proteolytic processing of pre-IL-1α is selectively mediated by CANP.