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 1 alpha (IL-1 alpha) into mature IL-1 alpha. Calcium ionophore ionomycin enhanced proteolytic processing of pre-IL-1 alpha and the release of mature IL-1 alpha either from lipopolysaccharide (LPS)-activated human adherent mononuclear cells or from a human bladder carcinoma cell line (HTB9 5637) that constitutively produces human IL-1 alpha and -beta. The proteolytic processing of pre-IL-1 alpha was completely inhibited by EGTA. Similar calcium-dependent proteolytic processing of pre-IL-1 alpha 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 microM) and that treatment of pre-IL-1 alpha with purified CANP yielded the 17-kDa mature form of IL-1 alpha, which has an amino terminus identical with that reported for mature human IL-1 alpha. Taken together, these findings indicate that calcium-dependent proteolytic processing of pre-IL-1 alpha is selectively mediated by CANP.
Kobayashi
Y
Y
Laboratory of Molecular Immunoregulation, National Cancer Institute, Frederick Cancer Research Facility, MD 21701-1013.
Yamamoto
K
K
Saido
T
T
Kawasaki
H
H
Oppenheim
J J
JJ
Matsushima
K
K
D002118:Calcium / Q000502:physiology; D002154:Calpain / Q000378:metabolism*; D002460:Cell Line; D004533:Egtazic Acid / Q000494:pharmacology; D006801:Humans; D007375:Interleukin-1 / Q000096:biosynthesis / Q000235:genetics*; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D008715:Methionine / Q000378:metabolism; D009000:Monocytes / Q000187:drug effects / Q000276:immunology; D011499:Protein Processing, Post-Translational; D014407:Tumor Cells, Cultured / Q000276:immunology; D001749:Urinary Bladder Neoplasms
D007375:Interleukin-1; D008070:Lipopolysaccharides; D004533:Egtazic Acid; D015759:Ionomycin; D008715:Methionine; D002154:Calpain; D002118:Calcium