IL-1-converting enzyme requires aspartic acid residues for processing of the IL-1 beta precursor at two distinct sites and does not cleave 31-kDa IL-1 alpha.
IL-1 converting enzyme (ICE) specifically cleaves the human IL-1 beta precursor at two sequence-related sites: Asp27-Gly28 (site 1) and Asp116-Ala117 (site 2). Cleavage at Asp116-Ala117 results in the generation of mature, biologically active IL-1 beta. ICE is unusual in that preferred cleavage at Asp-X bonds (where X is a small hydrophobic residue), has not been described for any other eukaryotic protease. To further examine the substrate specificity of ICE, proteins that contain Asp-X linkages including transferrin, actin, complement factor 9, the murine IL-1 beta precursor, and human and murine IL-1 alpha precursors, were assayed for cleavage by 500-fold purified ICE. The human and murine IL-1 beta precursors were the only proteins cleaved by ICE, demonstrating that ICE is an IL-1 beta convertase. Analysis of human IL-1 beta precursor mutants containing amino acid substitutions or deletions within each processing site demonstrated that omission or replacement of Asp at site 1 or site 2 prevented cleavage by ICE. To quantitatively assess the substrate requirements of ICE, a peptide-based cleavage assay was established using a 14-mer spanning site 2. Cleavage between Asp [P1] and Ala [P1']2 was demonstrated. Replacement of Asp with Ala, Glu, or Asn resulted in a greater than 100-fold reduction in cleavage activity. The rank order in position P1' was Gly greater than Ala much greater than Leu greater than Lys greater than Glu. Substitutions at P2'-P4' and P6' had relatively little effect on cleavage activity. These results show that ICE is a highly specific IL-1 beta convertase with absolute requirements for Asp in P1 and a small hydrophobic amino acid in P1'.
Department of Biochemical and Molecular Pathology, Merck Sharp and Dohme Research Laboratories, Rahway, NJ 07065.
A D
AD
Howard
M J
MJ
Kostura
N
N
Thornberry
G J
GJ
Ding
G
G
Limjuco
J
J
Weidner
J P
JP
Salley
K A
KA
Hogquist
D D
DD
Chaplin
R A
RA
Mumford
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000378:metabolism; D020170:Caspase 1; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007375:Interleukin-1 / Q000737:chemistry / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000378:metabolism*; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D007375:Interleukin-1; D011498:Protein Precursors; D001224:Aspartic Acid; D010450:Endopeptidases; D020170:Caspase 1; D008666:Metalloendopeptidases