IL-1-converting enzyme requires aspartic acid residues for processing of the IL-1β precursor at two distinct sites and does not cleave 31-kDa IL-1α

IL-1 converting enzyme (ICE) specifically cleaves the human IL-1β 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-1β. 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-1β precursor, and human and murine IL-1α precursors, were assayed for cleavage by 500-fold purified ICE. The human and murine IL-1β precursors were the only proteins cleaved by ICE, demonstrating that ICE is an IL-1β convertase. Analysis of human IL-1β 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 > Ala ≫ Leu > Lys > Glu. Substitutions at P2′-P4′ and P6′ had relatively little effect on cleavage activity. These results show that ICE is a highly specific IL-1β convertase with absolute requirements for Asp in P1 and a small hydrophobic amino acid in P1′.