title: Matrix metalloproteinase 2 from human rheumatoid synovial fibroblasts. Purification and activation of the precursor and enzymic properties.
abstract: Human rheumatoid synovial cells in culture secrete at least three related metalloproteinases that digest extracellular matrix macromolecules. One of them, termed matrix metalloproteinase 2 (MMP-2), has been purified as an inactive zymogen (proMMP-2). The final product is homogeneous on SDS/PAGE with Mr = 72,000 under reducing conditions. The NH2-terminal sequence of proMMP-2 is Ala-Pro-Ser-Pro-Ile-Ile-Lys-Phe-Pro-Gly-Asp-Val-Ala-Pro-Lys-Thr, which is identical to that of the so-called '72-kDa type IV collagenase/gelatinase'. The zymogen can be rapidly activated by 4-aminophenylmercuric acetate to an active form of MMP-2 with Mr = 67,000, and the new NH2-terminal generated is Tyr-Asn-Phe-Phe-Pro-Arg-Lys-Pro-Lys-Trp-Asp-Lys-Asn-Gln-Ile. However, following 4-aminophenylmercuric acetate activation, MMP-2 is gradually inactivated by autolysis. Nine endopeptidases (trypsin, chymotrypsin, plasmin, plasma kallikrein, thrombin, neutrophil elastase, cathepsin G, matrix metalloproteinase 3, and thermolysin) were tested for their abilities to activate proMMP-2, but none had this ability. This contrasts with the proteolytic activation of proMMP-1 (procollagenase) and proMMP-3 (prostromelysin). The optimal activity of MMP-2 against azocoll is around pH 8.5, but about 50% of activity is retained at pH 6.5. Enzymic activity is inhibited by EDTA, 1,10-phenanthroline or tissue inhibitor of metalloproteinases, but not by inhibitors of serine, cysteine or aspartic proteinases. MMP-2 digests gelatin, fibronectin, laminin, and collagen type V, and to a lesser extent type IV collagen, cartilage proteoglycan and elastin. Comparative studies on digestion of collagen types IV and V by MMP-2 and MMP-3 (stromelysin) indicate that MMP-3 degrades type IV collagen more readily than MMP-2, while MMP-2 digests type V collagen effectively. Biosynthetic studies of MMPs using cultured human rheumatoid synovial fibroblasts indicated that the production of both proMMP-1 and proMMP-3 is negligible but it is greatly enhanced by the treatment with rabbit-macrophage-conditioned medium, whereas the synthesis of proMMP-2 is constitutively expressed by these cells and is not significantly affected by the treatment. This suggests that the physiological and/or pathological role of MMP-2 and its site of action may be different from those of MMP-1 and MMP-3.
authors: Department of Pathology, School of Medicine, University of Kanazawa, Japan.
authors: Y
authors: Y
authors: Okada
authors: T
authors: T
authors: Morodomi
authors: J J
authors: JJ
authors: Enghild
authors: K
authors: K
authors: Suzuki
authors: A
authors: A
authors: Yasui
authors: I
authors: I
authors: Nakanishi
authors: G
authors: G
authors: Salvesen
authors: H
authors: H
authors: Nagase
mesh_terms: D000595:Amino Acid Sequence; D001172:Arthritis, Rheumatoid / Q000201:enzymology*; D015153:Blotting, Western; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D004789:Enzyme Activation; D016326:Extracellular Matrix Proteins / Q000302:isolation & purification* / Q000378:metabolism; D005347:Fibroblasts / Q000201:enzymology; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007797:Laminin / Q000378:metabolism; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D013583:Synovial Membrane / Q000201:enzymology*
chemical_list: D016326:Extracellular Matrix Proteins; D005353:Fibronectins; D007797:Laminin; D003094:Collagen; D010450:Endopeptidases; D008666:Metalloendopeptidases