title: Hydroxyapatite induces autolytic degradation and inactivation of matrix metalloproteinase-1 and -3.
abstract: In the course of studies to identify a protease capable of producing a long-lived 50 kDa fragment of bone acidic glycoprotein-75 (BAG-75), it was observed that incubation of matrix metalloproteinase (MMP)-3 (stromelysin 1) with preparations of BAG-75 led to inactivation of proteolytic function, e.g., an inability to fragment 125I-labeled BAG-75 added subsequently. MMP-1 (interstitial collagenase) was also inactivated by exposure to BAG-75 preparations. Investigation of the mechanism revealed that BAG-75 preparations contained millimolar levels of inorganic phosphate which formed hydroxyapatite crystals under digestion conditions. Hydroxyapatite crystals alone and in BAG-75-hydroxyapatite complexes induced the autolytic degradation of both active and precursor forms of MMP-1 and MMP-3. Autolytic degradation in the presence of hydroxyapatite was demonstrated by a loss in catalytic function assayed with peptide and/or protein substrates, and, by fragmentation into polypeptides of <10 kDa. The fate of MMP-3 incubated with hydroxyapatite depends upon the time of incubation, the free calcium concentration, and the concentration of crystals. Specifically, hydroxyapatite-induced autolysis requires a near physiological free calcium concentration of 0.5-1.0 mM. Autolysis was maximal in the presence of 150 microg/ml hydroxyapatite where MMP-3 was only partially bound to crystals. However, autolysis also occurred at higher crystal concentrations where all input MMP-3 was bound (>1000 microg/ml), suggesting that autolysis may be mediated by bound enzyme. The effect of hydroxyapatite appears to be specific for MMP-1 and MMP-3 since the catalytic activity of chymotrypsin, trypsin, papain, and thermolysin remained unchanged after exposure to hydroxyapatite. These results document for the first time a novel catalytic role for hydroxyapatite crystals in vitro and provide an initial biochemical characterization of the intermolecular, autolytic, calcium ion-dependent, matrix metalloproteinase-specific degradative mechanism.
authors.affiliation: Division of Molecular Biology and Biochemistry, University of Missouri-Kansas City, Kansas City, Missouri 64110, USA.
authors.forename: E A
authors.initials: EA
authors.lastname: Kremer
authors.forename: Y
authors.initials: Y
authors.lastname: Chen
authors.forename: K
authors.initials: K
authors.lastname: Suzuki
authors.forename: H
authors.initials: H
authors.lastname: Nagase
authors.forename: J P
authors.initials: JP
authors.lastname: Gorski
mesh_terms: D000818:Animals; D001329:Autolysis; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D002478:Cells, Cultured; D017364:Collagenases / Q000378:metabolism*; D003460:Crystallization; D017886:Durapatite / Q000494:pharmacology*; D004792:Enzyme Precursors / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D058574:Integrin-Binding Sialoprotein; D020781:Matrix Metalloproteinase 1; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000378:metabolism; D053495:Osteopontin; D010446:Peptide Fragments / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D051381:Rats; D012795:Sialoglycoproteins / Q000378:metabolism
chemical_list: D004792:Enzyme Precursors; D006023:Glycoproteins; C546871:IBSP protein, human; C546873:Ibsp protein, rat; D058574:Integrin-Binding Sialoprotein; D010446:Peptide Fragments; D010710:Phosphates; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; C506897:Spp1 protein, rat; C076105:bone acidic glycoprotein-75, human; C512787:bone acidic glycoprotein-75, rat; D053495:Osteopontin; D017886:Durapatite; D017364:Collagenases; D008666:Metalloendopeptidases; C048047:prostromelysin; D019278:Matrix Metalloproteinase 3; D020781:Matrix Metalloproteinase 1; D002118:Calcium