Binding of latent and high Mr active forms of stromelysin to collagen is mediated by the C-terminal domain.
A specific high-titre polyclonal antiserum to recombinant human prostromelysin was raised in a sheep and shown by immunoblotting to detect latent prostromelysin, high and low Mr active forms and the C-terminal domain. This antiserum was used to demonstrate by indirect immunofluorescence that latent and active high Mr prostromelysin bind to reconstituted collagen fibrils, and to other extracellular matrix components in tissues ex vivo but that active low Mr stromelysin does not. Isolation of the C-terminal domain was carried out to demonstrate that stromelysin binding was through this domain. By use of an antiserum to the tissue inhibitor of metalloproteinases (TIMP) it was shown that TIMP is unable to bind to reconstituted collagen fibrils. TIMP, however, will bind when active high Mr stromelysin is present but not if latent prostromelysin is bound. We conclude that stromelysin has different binding specificities from those previously documented for collagenase; only active collagenase binds to reconstituted collagen fibrils. However, TIMP binds to the active forms of both stromelysin and collagenase when these are bound to the collagen fibrils. These results have important implications for the interpretation of immunolocalization data in establishing the roles of metalloproteinases and their inhibitors in vivo.
Allan
J A
JA
Cell & Molecular Biology Department, Strangeways Research Laboratory, Cambridge, UK.
Hembry
R M
RM
Angal
S
S
Reynolds
J J
JJ
Murphy
G
G
D000595:Amino Acid Sequence; D000818:Animals; D002358:Cartilage, Articular / Q000737:chemistry / Q000378:metabolism; D004792:Enzyme Precursors / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D005109:Extracellular Matrix / Q000737:chemistry / Q000378:metabolism*; D005347:Fibroblasts / Q000737:chemistry; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D007106:Immune Sera; D015151:Immunoblotting; D019278:Matrix Metalloproteinase 3; D008666:Metalloendopeptidases / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013552:Swine; D013583:Synovial Membrane / Q000737:chemistry / Q000378:metabolism; D019714:Tissue Inhibitor of Metalloproteinases
D004792:Enzyme Precursors; D006023:Glycoproteins; D007106:Immune Sera; D011994:Recombinant Proteins; D019714:Tissue Inhibitor of Metalloproteinases; D008666:Metalloendopeptidases; C048047:prostromelysin; D019278:Matrix Metalloproteinase 3