Peptide substrate specificities and protein cleavage sites of human endometase/matrilysin-2/matrix metalloproteinase-26

Abstract

Human endometase/matrilysin-2/matrix metalloproteinase-26 (MMP-26) is a novel epithelial and cancer-specific metalloproteinase. Peptide libraries were used to profile the substrate specificity of MMP-26 from the P4-P4′ sites. The optimal cleavage motifs for MMP-26 were Lys-Pro-Ile/Leu-Ser(P1)-Leu/Met(P1′)-Ile/Thr-Ser/Ala-Ser. The strongest preference was observed at the P1′ and P2 sites where hydrophobic residues were favored. Proline was preferred at P3, and Serine was preferred at P1. The overall specificity was similar to that of other MMPs with the exception that more flexibility was observed at P1, P2′, and P3′. Accordingly, synthetic inhibitors of gelatinases and collagenases inhibited MMP-26 with similar efficacy. A pair of stereoisomers had only a 40-fold difference in K iapp values against MMP-26 compared with a 250-fold difference against neutrophil collagenase, indicating that MMP-26 is less stereoselective for its inhibitors. MMP-26 autodigested itself during the folding process. Two of the major autolytic sites were Leu 49-Thr 50 and Ala 75-Leu 76, which still left the cysteine switch sequence (PHC 82GVPD) intact. This suggests that Cys 82 may not play a role in the latency of the zymogen. Interestingly, inhibitor titration studies revealed that only ∼5% of the total MMP-26 molecules was catalytically active, indicating that the thiol groups of Cys 82 in the active molecules may be dissociated or removed from the active site zinc ions. MMP-26 cleaved Phe 352-Leu 353 and Pro 357-Met 358 in the reactive loop of α 1-proteinase inhibitor and His 140-Val 141 in insulin-like growth factor-binding protein-1, probably rendering these substrates inactive. Among the fluorescent peptide substrates analyzed, Mca-Pro-Leu-Ala-Nva-Dpa-Ala-Arg-NH 2 displayed the highest specificity constant (30,000/molar second) with MMP-26. This report proposes a working model for the future studies of pro-MMP-26 activation, the design of inhibitors, and the identification of optimal physiological and pathological substrates of MMP-26 in vivo.