Purification of a Type V Collagen Degrading Metalloproteinase from Rabbit Alveolar Macrophages

Mainardi, Carlo L.; Hibbs, Margaret S.; Hasty, Karen A.; Seyer, Jerome M.

doi:10.1016/s0174-173x(84)80014-x

Cited by 90 publications

(50 citation statements)

References 30 publications

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“…The 92 kDa enzyme was originally recognized as a gelatinolytic enzyme in polymorphonuclear leucocytes (Sopata & Dancemicz, 1974) and purified from human (Hibbs et al, 1985) and pig (Murphy et al, 1989a) neutrophils. Macrophages secrete a metalloproteinase that digests gelatins and type-V collagen (Mainardi et al, 1984). These enzymes have now been shown by immunochemical criteria to be identical with the 92 kDa form of gelatinase/type-IV collagenase from various transformed cells (Hibbs et al, 1987;Murphy et al, 1989b;Wilhelm et al, 1989;Moll et al, 1990).…”

Section: Introductionmentioning

confidence: 99%

Purification and characterization of matrix metalloproteinase 9 from U937 monocytic leukaemia and HT1080 fibrosarcoma cells

Morodomi

Osuga

Sasaguri

et al. 1992

Biochemical Journal

185

126

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603The precursor of matrix metalloproteinase 9 (proMMP-9), also known as '92 kDa progelatinase/type IV procollagenase', was purified from the conditioned medium of U937 monocytic leukaemia and HT1080 fibrosarcoma cell lines stimulated with phorbol 12-myristate 13-acetate. ProMMP-9 in these culture media is non-covalently complexed with the 29 kDa tissue inhibitor of metalloproteinases (TIMP), but free proMMP-9 was separated from the TIMP-proMMP-9 complex by chromatography on Green A Dyematrex gel. The final product was homogeneous on SDS/PAGE, with a molecular mass of 88 kDa without reduction and 92 kDa with reduction. Treatment of proMMP-9 with 4-aminophenylmercuric acetate converted the 88 kDa precursor into 80 kDa and 68 kDa forms. Gelatin-containing zymographic analysis showed zones of lysis associated with all three species. However, only the 68 kDa species was shown to be catalytically active by its ability to bind to a2-macroglobulin. In the presence of an equimolar amount of TIMP, only the 80 kDa species was generated by treatment with 4-aminophenylmercuric acetate, but no enzyme activity was detected. This indicates that TIMP binds to the 80 kDa intermediate and inhibits the generation of the active 68 kDa species. Eight endopeptidases (trypsin, chymotrypsin, plasmin, plasma kallikrein, thrombin, cathepsin G, neutrophil elastase and thermolysin) were tested for their ability to activate proMMP-9. Of them, trypsin was the most effective activator of proMMP-9. Only partial activation (10-30%) was observed with plasmin, cathepsin G and chymotrypsin. The active forms generated by trypsin were identified as 80 kDa, 74 kDa and 66 kDa by their abilities to bind to a2-macroglobulin. In the presence of an equimolar amount of TIMP, proMMP-9 was also converted into the same molecular-mass species by trypsin, but they were not proteolytically active. This suggests activated MMP-9 is inhibited by TIMP. Activated MMP-9 digested gelatin, type-V collagen, reduced carboxymethylated transferrin and, to a lesser extent, type-IV collagen and laminin A chain. The specific activity against gelatin was estimated to be 15000 units/mg (1 unit = 1 ,ug of gelatin degraded/min at 37°C) by titration with a2-macroglobulin. Comparative studies on digestion of gelatin and collagen types IV and V by MMP-9 and MMP-2 indicated that both enzymes degrade these substrates into similar fragments. However, the susceptibilities of laminin, fibronectin and reduced carboxymethylated transferrin to these two MMPs were sufficiently different to indicate differences in substrate specificities between these two closely related proteinases.

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Section: Introductionmentioning

confidence: 99%

Purification and characterization of matrix metalloproteinase 9 from U937 monocytic leukaemia and HT1080 fibrosarcoma cells

Morodomi

Osuga

Sasaguri

et al. 1992

Biochemical Journal

185

126

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“…However, the production of interstitial collagenase by human monocytes and macrophages has recently been documented (4,5,21,22). Furthermore, both human (23) and rabbit (24) macrophages have been reported to secrete a gelatinolytic enzyme of 92 kD, which also can readily cleave native types IV and V collagens. These findings suggest that human mononuclear phagocytes may have substantial capacity to degrade extracellular matrix via secreted metalloproteinases.…”

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confidence: 99%

Neutral metalloproteinases produced by human mononuclear phagocytes. Enzyme profile, regulation, and expression during cellular development.

Welgus¹,

Campbell²,

Cury³

et al. 1990

J. Clin. Invest.

418

211

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Mononuclear phagocytes are developmentally and functionally complex cells that play critical roles in extracellular matrix remodeling. We hypothesized that differentiated mononuclear phagocytes, typified by alveolar macrophages, use a spectrum of metalloproteinases to degrade various matrix macromolecules. To test this hypothesis, we have evaluated synthesis and secretion of four metalloproteinases (interstitial collagenase, stromelysin, 72-kD type IV collagenase, and 92-kD type IV collagenase) by human mononuclear phagocytes with regard to (a) the effect of cellular differentiation, (b) regulation of secretion, and (c) comparisons/contrasts with a prototype metalloproteinase-secretory cell, the human fibroblast. We found that regulated secretion of greater quantities and a wider spectrum of metalloenzymes correlated with a more differentiated cellular phenotype. As extreme examples, the 92-kD type IV collagenase was released by peripheral blood monocytes and uninduced U937 monocyte-like cells, whereas stromelysin was secreted only by lipopolysaccharide-stimulated alveolar macrophages. Macrophage production of interstitial collagenase, stromelysin, and 72-kD type IV collagenase was approximately 20%, 10%, and 1-2%, respectively, of that from equal numbers of fibroblasts; secretion of the 92-kD type IV collagenase was not shared by fibroblasts. This work confirms the potential of macrophages to directly degrade extracellular matrix via secreted metalloproteinases in a manner that differs both qualitatively and quantitatively from that of fibroblasts.Moreover, varying regulation of metalloenzyme synthesis, evidenced by distinct patterns of basal and stimulated secretion during differentiation, can be studied at a molecular level in this model system. (J. Clin. Invest. 1990Invest. . 86:1496Invest. -1502

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“…Gelatinase degrades native type V collagen and is a latent, neutral metalloproteinase (34)(35)(36). Rabbit bones (37), fibroblasts (21), and macrophages (34) secrete gelatinases into culture medium.…”

Section: Discussionmentioning

confidence: 99%

Septic arthritis

Schurman

1990

Arthritis & Rheumatism

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We report herein that cartilage proteolytic activity increased in bovine and rabbit articular cartilage after treatment with a purified staphylococcal culture medium or intraarticular infection with Staphylococcus aureus. Staphylococcal culture medium increased the release of gelatinolytic, collagenolytic, and caseinolytic activity into the medium of isolated chondrocytes or cartilage organ culture. The proteolytic activities were determined in assays using radiolabeled substrate and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Staphylococcal culture medium was proteolytically inactive by both assay techniques. RNA synthesis of isolated chondrocytes was unaffected by staphylococcal culture medium, whereas overall protein synthesis was inhibited by 84%. An analysis of extracts of Staphylococcus aureus-infected rabbit knee cartilage by substrate gels showed increased gelatinolytic and caseinolytic activity compared with extracts of uninfected knee cartilage. Our data suggest that the rapid loss of proteoglycan and persistent degradation of cartilage in staphylococcal septic arthritis is due to the production and activation of chondrocyte proteases.Septic arthritis continues to be a serious problem in urban medical centers (I ,2) and is recognized as a complicating factor in rheumatoid arthritis and other chronic arthritic conditions (3). Although most joint infections are acquired hematogenously, direct infection can result from deep wounds, surgical procedures, and intraarticular joint injections (2). In nongonococcal septic arthritis, Staphylococcus aureus is the organism most frequently found by culture (64%); in 25% of the patients positive for S aureus, the bacteria are methicillin resistant (1). Sterile "postinfectious" arthritis provides the potential for residual joint damage (43).In an animal model of staphylococcal infectious arthritis, cartilage degradation was shown to continue, despite early antibiotic treatment (5). Infection of rabbit knees with S aureus was shown to cause a rapid loss of cartilage proteoglycan (PG) (a), with a loss of as much as 40% of the total PG from the cartilage matrix within the first 48 hours after infection (8). Loss of collagen becomes significant between the second and third weeks after infection (8). Antibiotic treatment begun within 24 hours of infection decreases collagen loss but fails to prevent loss of PG from the matrix (5).In vitro, S aureus or S aureus-conditioned culture medium induces cartilage degradation in viable cartilage explants (9). Cartilage degradation is marked by a loss of partially degraded PG, which is consistent with increased proteolytic activity. PG release from cartilage by S aureus or staphylococcal culture medium is significantly reduced by freeze-thawing cartilage (9). Killed bacteria have no effect on cartilage degradation. Staphylococcus-induced PG release is dependent on chondrocyte metabolism and can be blocked by inhibitors of RNA and protein synthesis, i.e., actinomycin D and cycloheximide, respectively

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Purification of a Type V Collagen Degrading Metalloproteinase from Rabbit Alveolar Macrophages

Cited by 90 publications

References 30 publications

Purification and characterization of matrix metalloproteinase 9 from U937 monocytic leukaemia and HT1080 fibrosarcoma cells

Purification and characterization of matrix metalloproteinase 9 from U937 monocytic leukaemia and HT1080 fibrosarcoma cells

Neutral metalloproteinases produced by human mononuclear phagocytes. Enzyme profile, regulation, and expression during cellular development.

Septic arthritis

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