Hydroxyapatite induces autolytic degradation and inactivation of matrix metalloproteinase-1 and -3.

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.

Bone acidic glycoprotein-75 self-associates to form macromolecular complexes in vitro and in vivo with the potential to sequester phosphate ions.

Monoclonal antibody HTP IV-#1 specifically recognizes a complexation-dependent neoepitope on bone acidic glycoprotein-75 (BAG-75) and a Mr = 50 kDa fragment. Complexes of BAG-75 exist in situ, as shown by immunofluorescent staining of the primary spongiosa of rat tibial metaphysis and osteosarcoma cell micromass cultures with monoclonal antibody HTP IV-#1. Incorporation of BAG-75 into complexes by newborn growth plate and calvarial tissues was confirmed with a second, anti-BAG-75 peptide antibody (#503). Newly synthesized BAG-75 immunoprecipitated from mineralizing explant cultures of bone was present entirely in large macromolecular complexes, while immunoprecipitates from monolayer cultures of osteoblastic cells were previously shown to contain only monomeric Mr = 75 kDa BAG-75 and a 50 kDa fragment. Purified BAG-75 self-associated in vitro to form large spherical aggregate structures composed of a meshwork of 10 nm diameter fibrils. These structures have the capacity to sequester large amounts of phosphate ions as evidenced by X-ray microanalysis and by the fact that purified BAG-75 preparations, even after extensive dialysis against water, retained phosphate ions in concentrations more than 1,000-fold higher than can be accounted for by exchange calculations or by electrostatic binding. The ultrastructural distribution of immunogold-labeled BAG-75 in the primary spongiosa underlying the rat growth plate is distinct from that for other acidic phosphoproteins, osteopontin and bone sialoprotein. We conclude that BAG-75 self-associates in vitro and in vivo into microfibrillar complexes which are specifically recognized by monoclonal antibody HTP IV-#1. This propensity to self-associate into macromolecular complexes is not shared with acidic phosphoproteins osteopontin and bone sialoprotein. We hypothesize that an extracellular electronegative network of macromolecular BAG-75 complexes could serve an organizational role in forming bone or as a barrier restricting local diffusion of phosphate ions.

Bone acidic glycoprotein-75 self-associates to form large macromolecular complexes.

Bone acidic glycoprotein-75 (BAG-75) displays a strong propensity to self-associate to form large fibrillar complexes above concentrations of 7 x 10(-8) M; acidic phosphoproteins osteopontin and bone sialoprotein do not form similar complexes. Although the majority of the data supporting this conclusion is derived from in vitro studies, the fact that similar sized complexes are observed in crude extracts of bone and calcified cartilage suggests that macromolecular BAG-75 complexes are also a component of mineralized matrices in vivo. An awareness of the existence of complexes in extracts from bone necessitates that these forms are accounted for in terms of the relative amounts of individual acidic phosphoproteins in bone matrix. We now estimate that the amount of BAG-75 in rat calvarial bone is equivalent to that of osteopontin. While BAG-75 is capable of binding up to 139 atoms of calcium/mole with an average affinity constant of 0.5-1.0 x 10(-3) M, millimolar concentrations of calcium are not required for self-association. Assuming macromolecular diffusion within osteoid is restricted, osteoblastic cells could control the extent of self-association through the rate at which BAG-75 is synthesized and secreted into the osteoid matrix. Based on these findings, we hypothesize that BAG-75 self-associates to form fibrillar complexes in vivo which function in a supportive mechanical role and/or as an electronegative ionic barrier. Electronegative BAG-75 barrier structures could play a role in concentrating phosphate ions within bone matrix, thus facilitating mineralization.