Domain Of Gelatinase Research Articles

Discovery of chemokine substrates for matrix metalloproteinases by exosite scanning: a new tool for degradomics.

Increasingly it is being recognized that matrix metalloproteinases (MMPs) are important processing enzymes that regulate cellular behaviour and immune cell function by selective proteolysis of cell surface receptors and adhesion molecules, cytokines and growth factors. These functions will likely prove to be as important in vivo as the proposed roles of MMPs in pathological matrix degradation. To screen for new protease substrates we have reported a novel 'exosite scanning' strategy that utilizes protease substrate-binding exosite domains as yeast two-hybrid baits. We discovered that the chemokine monocyte chemoattractant protein-3 (MCP-3) binds the hemopexin C domain of gelatinase A (MMP-2) leading to its efficient cleavage, converting an agonist to a potent receptor antagonist. We have now found that other MMPs cleave MCP-1, MCP-2, MCP-3, MCP-4, SDF-lalpha and SDF-1beta indicating that the intersection between the chemokine and MMP families is broad with important implications for the control of inflammatory and immune processes. Use of engineered substrates with altered exosite binding affinities further revealed the power of exosites in dictating proteolytic specificity - either directing cleavage of non-preferred sites or in other cases virtually eliminating proteolysis of readily accessible scissile bonds. Hence, bioinformatic searches for protease substrates based on scissile bond preference will only reveal a subset of substrates unless the influence of exosites is considered.

Domain Interactions in the Gelatinase A·TIMP-2·MT1-MMP Activation Complex: THE ECTODOMAIN OF THE 44-kDa FORM OF MEMBRANE TYPE-1 MATRIX METALLOPROTEINASE DOES NOT MODULATE GELATINASE A ACTIVATION

On the cell surface, the 59-kDa membrane type 1-matrix metalloproteinase (MT1-MMP) activates the 72-kDa progelatinase A (MMP-2) after binding the tissue inhibitor of metalloproteinases (TIMP)-2. A 44-kDa remnant of MT1-MMP, with an N terminus at Gly(285), is also present on the cell after autolytic shedding of the catalytic domain from the hemopexin carboxyl (C) domain, but its role in gelatinase A activation is unknown. We investigated intermolecular interactions in the gelatinase A activation complex using recombinant proteins, domains, and peptides, yeast two-hybrid analysis, solid- and solution-phase assays, cell culture, and immunocytochemistry. A strong interaction between the TIMP-2 C domain (Glu(153)-Pro(221)) and the gelatinase A hemopexin C domain (Gly(446)-Cys(660)) was demonstrated by the yeast two-hybrid system. Epitope masking studies showed that the anionic TIMP-2 C tail lost immunoreactivity after binding, indicating that the tail was buried in the complex. Using recombinant MT1-MMP hemopexin C domain (Gly(285)-Cys(508)), no direct role for the 44-kDa form of MT1-MMP in cell surface activation of progelatinase A was found. Exogenous hemopexin C domain of gelatinase A, but not that of MT1-MMP, blocked the cleavage of the 68-kDa gelatinase A activation intermediate to the fully active 66-kDa enzyme by concanavalin A-stimulated cells. The MT1-MMP hemopexin C domain did not form homodimers nor did it bind the gelatinase A hemopexin C domain, the C tail of TIMP-2, or full-length TIMP-2. Hence, the ectodomain of the remnant 44-kDa form of MT1-MMP appears to play little if any role in the activation of gelatinase A favoring the hypothesis that it accumulates on the cell surface as an inactive, stable degradation product.

The gelatin-binding site of the second type-II domain of gelatinase A/MMP-2.

We have shown previously that all three fibronectin type-II modules of gelatinase A contribute to its gelatin affinity. In the present work the second type-II module was subjected to site-directed mutagenesis in order to localize its gelatin-binding site. The functional integrity of mutant proteins was assessed by their affinity for gelatin using gelatin-Sepharose affinity chromatography. The structural integrity of the mutant proteins, i.e. their resistance to thermal and chaotropic agent-induced denaturation, was characterized by CD spectroscopy. Our studies show that, in the case of mutants R19L, R38L, K50G, K50R and R19L/R38L, the mutations had no significant effect on the structure and gelatin affinity of the type-II module, excluding the direct involvement of these residues in ligand binding. In the case of mutants Y25A, Y46A, D49A and Y52A, the mutations yielded proteins that were devoid of gelatin affinity. Structural characterization of these proteins, however, indicated that they had also lost their ability to fold into the native structure characteristic of the wild-type domain. In the case of mutant Y37A, the structure and stability of the mutant protein is similar to the wild-type module. However, its gelatin affinity was severely impaired compared with the wild-type protein. The fact that the Y37A mutation impairs ligand binding without detectable distortion of the module's architecture suggests that Y37 is directly involved in ligand binding. Homology modeling based on the three-dimensional structure of the second type-II module of PDC-109 places Y37 on the right-hand rim of a hydrophobic pocket that includes residues F20, W39, Y46, Y52 and F54, and thus provides proof for the involvement of this pocket in ligand binding.