Identification of a new metalloproteinase inhibitor that forms tight-binding complexes with collagenase.
Connective-tissue cells produce a family of metalloproteinases which, once activated, can degrade all the components of the extracellular matrix. These potent enzymes are all inhibited by the tissue inhibitor of metalloproteinases (TIMP), and it was thought that the levels of this inhibitor controlled the extracellular activity of these enzymes. We recently detected a new metalloproteinase inhibitor present in culture media of WI-38 fibroblasts. The inhibitor, named 'large inhibitor of metalloproteinases' (LIMP), can be separated from TIMP by gel filtration on Ultrogel AcA 44, where it is eluted with an apparent Mr of 76,000. A portion of this inhibitor-containing peak binds to concanavalin A-Sepharose, indicating that at least some of the inhibitor contains carbohydrate. LIMP inhibits collagenase (MMP-1), stromelysin (MMP-3) and gelatinase (MMP-2) in a dose-dependent fashion. Collagenase forms tight-binding complexes with LIMP, which can be separated from free collagenase on gel-filtration columns. The complex is eluted with Mr 81,600 (AcA 44) or Mr 60,000 (Superose 12). This complex is larger than that formed between collagenase and TIMP, which has Mr 52,800 (Aca 44) or 41,000 (Superose 12). Polyclonal antibody to TIMP does not recognize LIMP by immunoblotting, and will not block the inhibition of collagenase by LIMP, showing that LIMP is not a multimeric form of TIMP. The role of this new inhibitor in connective-tissue breakdown studies and its relationship to previously described inhibitors of metalloproteinases is discussed.
Cawston
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Rheumatology Research Unit, Addenbrooke's Hospital, Cambridge, U.K.
Curry
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Clark
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Hazleman
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D002460:Cell Line; D002850:Chromatography, Gel; D005347:Fibroblasts / Q000378:metabolism; D006023:Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D003012:Microbial Collagenase / Q000378:metabolism*; D008970:Molecular Weight; D019714:Tissue Inhibitor of Metalloproteinases
D006023:Glycoproteins; D019714:Tissue Inhibitor of Metalloproteinases; D008666:Metalloendopeptidases; D003012:Microbial Collagenase