E. coli Expression of TIMP-4 and Comparative Kinetic Studies with TIMP-1 and TIMP-2:  Insights into the Interactions of TIMPs and Matrix Metalloproteinase 2 (Gelatinase A)

Abstract

The inhibitory properties of TIMP-4 for matrix metalloproteinases (MMPs) were compared to those of TIMP-1 and TIMP-2. Full-length human TIMP-4 was expressed in E. coli, folded from inclusion bodies, and the active component was purified by MMP-1 affinity chromatography. Progress curve analysis of MMP inhibition by TIMP-4 indicated that association rate constants (k(on)) and inhibition constants (K(i)) were similar to those for other TIMPs ( approximately 10(5) M(-)(1) s(-)(1) and 10(-)(9)-10(-)(12) M, respectively). Dissociation rate constants (k(off)) for MMP-1 and MMP-3 determined using alpha(2)-macroglobulin to capture MMP dissociating from MMP-TIMP complexes were in good agreement with values deduced from progress curves ( approximately 10(-)(4) s(-)(1)). K(i) and k(on) for the interactions of TIMP-1, -2, and -4 with MMP-1 and -3 were shown to be pH dependent. TIMP-4 retained higher reactivity with MMPs at more acidic conditions than either TIMP-1 or TIMP-2. Molecular interactions of TIMPs and MMPs investigated by IAsys biosensor analysis highlighted different modes of interaction between proMMP-2-TIMP-2 (or TIMP-4) and active MMP-2-TIMP-2 (or TIMP-4) complexes. The observation that both active MMP-2 and inactive MMP-2 (with the active site blocked either by the propeptide or a hydroxamate inhibitor) have essentially identical affinities for TIMP-2 suggests that there are two TIMP binding sites on the hemopexin domain of MMP-2: one with high affinity that is involved in proMMP-2 or hydroxamate-inhibited MMP-2; and the other with low affinity involved in formation of the complex of active MMP-2 and TIMP-2. Similar models of interaction may apply to TIMP-4. The latter low-affinity site functions in conjunction with the active site of MMP-2 to generate a tight enzyme-inhibitor complex.

Differential Inhibition of Membrane Type 3 (MT3)-Matrix Metalloproteinase (MMP) and MT1-MMP by Tissue Inhibitor of Metalloproteinase (TIMP)-2 and TIMP-3 Regulates Pro-MMP-2 Activation

“…TIMP-4 exhibited a comparable lower affinity for both MT3-MMP cat and MT1-MMP cat (K i ϭ 0.3 nM) ( Table I). The affinity of TIMP-4 for MT1-MMP cat is in good agreement to that previously reported by Troeberg et al (25). TIMP-1 interaction with MT1-MMP cat has been shown to be practically negligible (22,55) and, at concentrations up to 9 nM, showed no inhibition of MT3-MMP cat (data not shown).…”

“…Our data also showed that TIMP-4 is a potent inhibitor of MT3-MMP as it is of MT1-MMP (22,25,71) and MT2-MMP (23). However, despite its ability to bind pro-MMP-2 (24), albeit with lower affinity than TIMP-2 (25), TIMP-4 was unable to support pro-MMP-2 activation by MT3-(shown here), MT1-(22, 26), or MT2-MMP (23).…”

“…However, how pro-MMP-2 interacts with MT2-MMP in the absence of TIMPs is unknown. Although TIMP-4 is capable of binding pro-MMP-2 (24) and is an effective inhibitor of MT1-MMP and MT2-MMP (22,23,25), it cannot support pro-MMP-2 activation (22,26). Consistently, TIMP-4 has been shown to be unable to generate ternary complexes with these MT-MMPs (22).…”

Low Density Lipoprotein Receptor-related Protein Mediates Endocytic Clearance of Pro-MMP-2·TIMP-2 Complex through a Thrombospondin-independent Mechanism

“…Proteins and Antibodies-Recombinant human pro-MMP-2 and TIMP-2 were expressed in HEK-293 cells using the mammalian expression vector pCEP4 (Invitrogen) (33). Pro-MMP-2 was purified using gelatin-Sepharose affinity chromatography and gel filtration with S-200 (Amersham Biosciences) as described previously (34).…”

Metalloproteinases and their inhibitors in tumor angiogenesis

“…The activity of MMPs can be regulated by endogenous inhibitors, which include the TIMPs, 21 a 2 -macroglobulin 22 and the membrane-anchored glycoprotein RECK (for ''reversioninducing cysteine-rich protein with kazal motifs''). 23 There are 4 vertebrate TIMPs.…”