Extracellular matrix degradation occurs in chronic inflammatory diseases such as arthritis and disturbances in connective tissue homeostasis contribute to various lung, neurological and cardiovascular diseases and is pivotal in tumor metastasis. The matrix metalloproteinase (MMP) family of endoproteinases is implicated in these processes by a general ability to degrade the structural components of the extracellular matrix. To understand the biological role of MMP proteolysis in physiological and pathological processes, it is necessary to identify biologically relevant substrates. I initiated yeast two-hybrid screens to identify novel substrates of gelatinase A using the hemopexin C domain of the enzyme as bait. Initial screens of a cDNA library constructed from Concanavalin A-treated human gingival fibroblasts identified the chemokine monocyte chemoattractant protein (MCP)-3 as a hemopexin C domain binding protein. Incubation of MCP-3 with gelatinase A resulted in cleavage of MCP-3 at Gly⁴-lle⁵ a preferred scissile bond sequence for the enzyme. The turnover rate, k[sub cat]/K[sub m] was determined to be 8,000 M⁻¹s⁻¹, more efficient than gelatinase A cleavage of gelatin. Indeed, cleaved MCP-3 was identified in human rheumatoid arthritis synovial fluids. Gelatinase A (and other MMP)-mediated cleavage of MCP-3 resulted in conversion of chemokine receptor agonist activity to a general acting antagonist, impairing the activity of several related chemokines. The mechanistic importance of the hemopexin C domain in gelatinase A cleavage of MCP-3 was determined. The turnover rate was reduced to 500 M⁻¹s⁻¹ upon removal of the hemopexin C domain from the enzyme. Exogenous hemopexin C domain competed for cleavage whereas the collagen binding domain of gelatinase A did not. Specificity of MCP cleavage could be attributed to unique binding of both the gelatinase A and membrane-type (MT)-1 hemopexin C domains to only MCP-3 and not MCP-1, -2, or -4. Chemokine chimeras further demonstrated the importance of hemopexin C domain exosites in catalysis. My results provide evidence that MMP activity can contribute toward the reparative process in inflammation and that interactions of MMPs with chemokines provide a self-attenuating network to dissipate pro-inflammatory activities. I propose that MMP processing of chemokines is a new paradigm in chemokine and MMP biology in the regulation of inflammation.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat
