A unique substrate recognition profile for matrix metalloproteinase-2.

Emily I Chen,Eric W Howard,Steven J Kridel,Jeffrey W Smith,Weizhong Li,Adam Godzik

doi:10.1074/jbc.m109469200

Abstract

The catalytic domains of the matrix metalloproteinases (MMPs) are structurally homologous, raising questions as to the degree of distinction, or overlap, in substrate recognition. The primary objective of the present study was to define the substrate recognition profile of MMP-2, a protease that was historically referred to as gelatinase A. By cleaving a phage peptide library with recombinant MMP-2, four distinct sets of substrates were identified. The first set is structurally related to substrates previously reported for other MMPs. These substrates contain the PXX/X(Hy) consensus motif (where X(Hy) is a hydrophobic residue) and are not generally selective for MMP-2 over the other MMPs tested. Two other groups of substrates were selected from the phage library with similar frequency. Substrates in group II contain the L/IXX/X(Hy) consensus motif. Substrates in group III contain a consensus motif with a sequence of X(Hy)SX/L, and the fourth set of substrates contain the HXX/X(Hy) sequence. Substrates in Group II, III, and IV were found to be 8- to almost 200-fold more selective for MMP-2 over MMP-9. To gain an understanding of the structural basis for substrate selectivity, individual residues within substrates were mutated, revealing that the P(2) residue is a key element in conferring selectivity. These findings indicate that MMP-2 and MMP-9 exhibit different substrate recognition profiles and point to the P(2) subsite as a primary determinant in substrate distinction.

Highlights

The matrix metalloproteinases (MMPs)1 have important roles in several normal tissue remodeling events [1]
To gain an understanding of the structural basis for substrate selectivity, individual residues within substrates were mutated, revealing that the P2 residue is a key element in conferring selectivity. These findings indicate that MMP-2 and MMP-9 exhibit different substrate recognition profiles and point to the P2 subsite as a primary determinant in substrate distinction
Expression and Purification of the Catalytic Domain of Human MMP-2 and 9 —Recombinant MMP-2 was generated in a manner similar to that we have previously reported for MMP-9 [21]

Summary

Introduction

The matrix metalloproteinases (MMPs) have important roles in several normal tissue remodeling events [1]. Recent work counters the notion that MMPs have an overlapping substrate recognition profile Recent studies on both MMP-13 and MMP-9 show that a high degree of selectivity can be obtained for individual MMPs, even among substrates comprised of the PXX2XHy motif [20, 21]. Given these recent observations, we wondered whether very closely related MMPs would exhibit distinctions in substrate recognition. We focus on substrate recognition by MMP-2 and its closely related homolog, MMP-9 These two MMPs are unique in that they contain three type II fibronectin domains intercalated within their catalytic domains. Until now it was assumed that MMP-2 and MMP-9 have over-

Objectives

Results

Conclusion