Matrix metalloproteinase 2 from human rheumatoid synovial fibroblasts

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 SDSjPAGE with M , = 72000 under reducing conditions. The NH2-terminal sequence of proMMP-2 is Ala-Pro-Ser-Pro-Ile-Ile-Lys-Phe-Pro-Gly-AspVal-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 M , = 67 000, and the new NH2-terminal generated is Tyr-Asn-Phe-Phe-Pro-Arg-Lys-Pro-Lys-Trp-Asp-Lys-Asn-GlnIle. 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,lO-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.Connective tissue cells in culture synthesize and secrete tissue collagenase (matrix matalloproteinase 1 ; EC 3.4.24.7) and at least two other matrix metalloproteinases (MMP-2 and MMP-3) that digest various components of the extracellular matrix. The deduced primary sequences of these three MMPs from their cDNA sequencing have indicated that they are structurally related [l -61, but the substrate specificities of these enzymes are distinct. MMP-1 digests collagen types I, I1 and I11 at specific sites and generates characteristic threequarter and one-quarter fragments of the native collagen molecules [7 -91. It has also been shown to digest collagen typesCorrespondence to H. Nagase,

An analysis of the conformational changes that accompany the activation and inhibition of gelatinase A

“…Once the active site is liberated there follows a series of autolytic cleavages to the proenzyme that serve to remove the propeptide. The initial cleavage is an intramolecular event because the rate of proenzyme disappearance is not dependent upon its own starting concentration [11][12]. This supports the concept that the addition of APMA must in the first instance generate an active MMP that still possesses a complete propeptide.…”

“…At the start of the incubation progelatinase A displayed < 0.1% of maximum activity and a molecular mass of 72 kDa. Full activity was achieved after approximately 2 h, at which point the proenzyme had been converted to a 66 kDa form that, according to previous studies, lacks the 80 amino acid N-terminal propeptide [12]. 50% of maximum activity was achieved at a time (-20 min) when approximately half of the proenzyme had been processed.…”

Efficacy of the MMP inhibitor MMI270 against lung metastasis following removal of orthotopically transplanted human colon cancer in rat

“…The sample was then centrifuged at 10,000g at 4°C for 20 min, and the supernatant was used for inhibitory assay against gelatinolytic activity of 1 lg of purified MMP-2 or MMP-9, as previously reported. 19,20 Statistical analysis Data were analyzed using the chi-square test for lung metastasis and Student's t-test for body weight, lung wet weight, MVD, PI and AI between the groups. The Kaplan-Meier method was used to calculate survival rates; significant difference in survival rates was assessed using the Logrank test.…”

Convenient fluorometric assay for matrix metalloproteinase activity and its application in biological media

“…The gelatinase zymogens pro-MMP-2 [14] [17,18] and was therefore considered suitable for the present study. Pro-MMP-2 was activated at 37°C for 2 h and pro-MMP-9 at 4°C for 18 h in buffer A (50 mM Tris (pH 7.6), 150 mM NaC1, 5 mM CaC12, 1 gM ZnC12, 0.01% Brij 35) plus 1 mM APMA, resulting in > 90% activation into the active enzymes (validated with gelatin zymography [19]).…”