Characterization of the Monomeric and Dimeric Forms of Latent and Active Matrix Metalloproteinase-9: DIFFERENTIAL RATES FOR ACTIVATION BY STROMELYSIN 1

Matthew W Olson,M.Margarida Bernardo,Martin Pietila,David C Gervasi,Marta Toth,Lakshmi P Kotra,Irina Massova,Shahriar Mobashery,Rafael Fridman

doi:10.1074/jbc.275.4.2661

Abstract

Matrix metalloproteinase-9 (MMP-9) is a member of the MMP family that has been associated with degradation of the extracellular matrix in normal and pathological conditions. A unique characteristic of MMP-9 is its ability to exist in a monomeric and a disulfide-bonded dimeric form. However, there exists a paucity of information on the properties of the latent (pro-MMP-9) and active MMP-9 dimer. Here we report the purification to homogeneity of the monomer and dimer forms of pro-MMP-9 and the characterization of their biochemical properties and interactions with tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. Gel filtration and surface plasmon resonance analyses demonstrated that the pro-MMP-9 monomeric and dimeric forms bind TIMP-1 with similar affinities. In contrast, TIMP-2 binds only to the active forms. After activation, the two enzyme forms exhibited equal catalytic competence in the turnover of a synthetic peptide substrate with comparable kinetic parameters for the onset of inhibition with TIMPs and for dissociation of the inhibited complexes. Kinetic analyses of the activation of monomeric and dimeric pro-MMP-9 by stromelysin 1 revealed K(m) values in the nanomolar range and relative low k(cat) values (1.9 x 10(-3) and 4.1 x 10(-4) s(-1), for the monomer and dimer, respectively) consistent with a faster rate (1 order of magnitude) of activation of the monomeric form by stromelysin 1. This suggests that the rate-limiting event in the activation of pro-MMP-9 may be a requisite slow unfolding of pro-MMP-9 near the site of the hydrolytic cleavage by stromelysin 1.

Highlights

Matrix metalloproteinase-9 (MMP-9) is a member of the MMP family that has been associated with degradation of the extracellular matrix in normal and pathological conditions
Matrix metalloproteinase-9 (MMP-9),1 known as gelatinase B, is a member of the MMP family of zinc-dependent endopeptidases known for their ability to degrade many extra
Pro-MMP-9 is unique among the members of the MMP family in that it forms dimers consisting of covalently tethered monomers via a disulfide bond that can be found in tissues

Summary

EXPERIMENTAL PROCEDURES

Buffers—Buffer C (50 mM HEPES (pH 7.5), 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij-35); buffer B (10 mM sodium acetate (pH 4.5)); buffer W (7.8 mM NaH2PO4, 8 mM Na2HPO4 (pH 7.2), 137 mM NaCl, 0.1 mM CaCl2, 3 mM KCl, 1.5 mM KH2PO4, and 0.02% Tween 20); buffer R (50 mM HEPES (pH 7.5), 150 mM NaCl, 5 mM CaCl2, 0.01% Brij-35, and 1% (v/v) Me2SO); buffer D (50 mM Tris (pH 7.4), 150 mM NaCl, 5 mM CaCl2, and 0.02% Brij-35) and lysis buffer (25 mM Tris-HCl (pH 7.5), 1% Nonidet P-40, 100 mM NaCl, 5 mM EDTA, 20 mM N-ethylmaleimide, 10 ␮g/ml aprotinin, 1 ␮g/ml pepstatin A, 1 ␮g/ml leupeptin, 2 mM benzamidine, and 1 mM phenylmethylsulfonyl fluoride). Activation of pro-MMP-9M or pro-MMP-9D was monitored in reaction mixtures containing 2–120 nM of either substrate and 0.5 nM stromelysin 1 in 70 ␮l of buffer D at 37 °C. The MMP-9 (monomer or dimer) concentrations were calculated using the Michaelis-Menten equation and the kcat and Km values for the reaction of the enzyme (monomer and dimer) with the fluorogenic substrate, as described above. Initial velocities of proMMP-9M or pro-MMP-9D activation were determined from the linear increase in MMP-9 concentration as a function of time. TIMP-1 (0 –30 nM) or TIMP-2 (0 – 60 nM) were added to the fluorogenic substrate solution, and the assay was initiated by addition of enzyme to give a final concentration of 1 nM for MMP-9M and 0.5 nM for MMP-9D. The graphical analysis of the resulting structure was performed using Sybyl software version 6.4

RESULTS

Km nM

Ki nM

DISCUSSION