Utilization of a Novel Recombinant Myoglobin Fusion Protein Expression System to Characterize the Tissue Inhibitor of Metalloproteinase (TIMP)-4 and TIMP-2 C-terminal Domain and Tails by Mutagenesis

Abstract

Tissue inhibitor of metalloproteinase (TIMP)-4 binds pro-matrix metalloproteinase (MMP)-2 and efficiently inhibits MT1-MMP, but unlike TIMP-2 neither forms a trimolecular complex nor supports pro-MMP-2 activation. To investigate the structural and functional differences between these two TIMPs, the C-terminal domains (C-TIMP-4 and C-TIMP-2) were expressed independently from their N domains and mutations were introduced into the C-terminal tails. Myoglobin was used as a novel recombinant fusion protein partner because spectroscopic measurement of the heme Soret absorbance at 408 nm readily enabled calculation of the molar equivalent of the red-colored recombinant protein, even in complex protein mixtures. Both C-TIMP-4 and C-TIMP-2 bound pro-MMP-2 and blocked concanavalin A-induced cellular activation of the enzyme. Measurement of k on rates revealed that the inhibition of MMP-2 by TIMP-4 is preceded by a C domain docking interaction, but in contrast to TIMP-2, this is not enhanced by a C-terminal tail interaction and so occurs at a slower rate. Indeed, the binding stability of C-TIMP-4 was unaltered by deletion of the C-terminal tail, but replacement with the tail of TIMP-2 increased its affinity for pro-MMP-2 by ϳ2-fold, as did substitution with the TIMP-2 C-terminal tail acidic residues in the tail of C-TIMP-4 (V193E/ Q194D). Conversely, substitution of the C-terminal tail of C-TIMP-2 with that of TIMP-4 reduced pro-MMP-2 binding by ϳ75%, as did reduction of its acidic character by mutation to the corresponding TIMP-4 amino acid residues (E192V/D193Q). Together, this shows the importance of Glu 192 and Asp 193 in TIMP-2 binding to pro-MMP-2; the lack of these acidic residues in the TIMP-4 C-terminal tail, which reduces the stability of complex formation with the MMP-2 hemopexin C domain, probably precludes TIMP-4 from supporting the activation of pro-MMP-2.Matrix metalloproteinases (MMPs) 1 are a family of important processing enzymes that can cleave and regulate the activity of an expanding degradome of bioactive molecules (1-5) as well as degrade extracellular matrix proteins in pathology (6). Gelatinase A (MMP-2) has been implicated in numerous biological processes, including the activation of cytokines such as tumor necrosis factor-␣ (7), transforming growth factor-␤1 (8), and interleukin-1␤ (9). MMP-2 has also been shown to have anti-inflammatory actions by converting monocyte chemokine agonists to antagonists (10, 11) and causes loss of protection of CD4 ϩ cells from human immunodeficiency virus-1 infection by processing stromal cell-derived factor-1␣ (12).2 MMP-2 cleavage of type IV collagen, a major component of basement membranes, is important for tumor cell metastasis and angiogenesis (14 -16). In view of these diverse and biologically important functions, it is not surprising that MMPs are under tight regulatory control, both at the transcriptional and post-transcriptional levels (17-19). Post-translational regulation is also pivotally important in regulating proteolytic activity in the pericellul...

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

“…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). A pro-MMP-2⅐TIMP-4 complex does not bind MT1-MMP (22), an inability that has been attributed to specific structural constrains in the C-terminal region of TIMP-4 (24,70,71). Although not examined in this study, the relative low affinity and the structural orientation of the pro-MMP-2⅐TIMP-4 complex may preclude formation of a ternary complex with MT3-MMP and therefore activation does not ensue.…”

“…The binding affinity and conformation of the pro-MMP-2⅐TIMP-2 complex allows the N-terminal region of TIMP-2 to dock into the active site of MT1-MMP yielding trimolecular complexes either in solution (22) or on the cell surface (14). Because the C-terminal tail of TIMP-3 has a net charge of zero, based on amino acid sequence (70), it has been suggested that the interaction of TIMP-3 with the positively charged hemopexin-like domain of pro-MMP-2 may be further weakened (70). In addition, studies using gelatin affinity chromatography suggested a lower affinity of pro-MMP-2 for TIMP-3 when compared with TIMP-2 (28).…”

“…In addition, studies using gelatin affinity chromatography suggested a lower affinity of pro-MMP-2 for TIMP-3 when compared with TIMP-2 (28). Therefore, the ability of TIMP-3 to support pro-MMP-2 activation via a ternary complex with MT-MMPs has been questioned (70). Although the affinities between TIMP-3/TIMP-2 and pro-MMP-2 in the presence and absence of MT3-MMP need to be measured directly in future studies, the results presented here could not have been predicted solely from the amino acid sequence and charge distribution within the C-terminal tail of TIMP-3 and the hemopexin-like domain of pro-MMP-2, which under physiological conditions may very well be fully solvated.…”

Characterization of the AB Loop Region of TIMP-2

“…Several reports have focused on the importance of the C-terminal domain of TIMP-2 for pro-MMP-2 activation (15,16,28). The results show that the charged residues Glu in the unstructured tail of TIMP-2 are crucial for the activation process.…”

“…TIMP-4 also binds to pro-MMP-2 through the hemopexin-like domain (13); however, it is unable to promote the activation of the zymogen by MT1-MMP (14,24,28), although it is an excellent inhibitor of the latter. Recent mutagenesis studies by several groups have identified residues Glu 192 and Asp 193 of the C-terminal "tail" of TIMP-2 as the key residues for binding to pro-MMP-2 (15,16). The lack of these residues in the TIMP-4 C-terminal tail probably reduces the stability of complex formation with the hemopexin-like domain of pro-MMP-2 leading to the inability of TIMP-4 to promote activation of pro-MMP-2.…”