Initial Characterization of a Neutral Metalloproteinase,Active on Native 3/4-Collagen Fragments,Synthesized by ROS 17/2.8 Osteoblastic Cells,Periodontal Fibroblasts,and Identified in Gingival Crevicular Fluid

Abstract

Analysis of collagenolytic activity in gingival crevicular fluid (GCF) has revealed the presence of an enzyme capable of fragmenting native 3/4 and ¼-collagen cleavage products generated by collagenase. An enzyme with similar activity was also identified in media conditioned by fibroblasts from rat periodontal ligament and gingiva, and by rat osteoblastic cells (ROS 17/2.8, 17/2A, 17/2B). In culture, the enzyme was secreted in a latent form that could be activated by organomercurials. For further characterization of this novel enzyme (MMP-V), the osteoblast proteinase was partially purified. ROS 1712.8 conditioned medium was harvested daily and the 25% - 60% sat. ammonium sulfate fraction chromatographed on an AcA 54 gel filtration column. Latent forms of MMP-V (apparent Mr ~54 k) and collagenase (M _r ~54 k) were resolved from gelatinase (M _r ~76 k) and two collagenase inhibitors (M _r ~62 k, ~36 k). Activated MMP-V degraded native 3/4collagen fragments from collagen types I and II in a step-wise manner and was active on denatured collagen. MMP-V showed a divalent cation requirement, was active at neutral pH, and was inhibited by collagenase inhibitor and fetal bovine serum, but not by serine, thiol, or carboxyl proteinase inhibitors. These properties indicate that MMP-V is a member of the matrix-degrading, neutral-metalloproteinase family of enzymes which include collagenase, gelatinase, stromelysin, and telopeptidase. The enzyme may function in the degradation of collagen fibrils by cleaving proteinase-resistant 3/4-collagen fragments that are stabilized by association with neighboring collagen molecules.

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