BONE ACIDIC GLYCOPROTEIN-75 SELF-ASSOCIATES TO FORM MACROMOLECULAR COMPLEXES IN-VITRO AND IN-VIVO WITH THE POTENTIAL TO SEQUESTER PHOSPHATEIONS

Monoclonal antibody HTP IV-#1 specifically recognizes a complexation-d ependent neoepitope on bone acidic glycoprotein-75 (BAG-75) and a Mr = 50 kDa fragment. Complexes of BAG-75 exist in situ, as shown by immun ofluorescent staining of the primary spongiosa of rat tibial metaphysi s and osteosarcoma cell micromass cultures with monoclonal antibody HT P IV-#1. Incorporation of BAG-75 into complexes by newborn growth plat e and calvarial tissues was confirmed with a second, anti-BAG-75 pepti de antibody (#503). Newly synthesized BAG-75 immunoprecipitated from m ineralizing explant cultures of bone was present entirely in large mac romolecular complexes, while immunoprecipitates from monolayer culture s of osteoblastic cells were previously shown to contain only monomeri c Mr = 75 kDa BAG-75 and a 50 kDa fragment. Purified BAG-75 self-assoc iated in vitro to form large spherical aggregate structures composed o f a meshwork of 10 nm diameter fibrils. These structures have the capa city to sequester large amounts of phosphate ions as evidenced by X-ra y microanalysis and by the fact that purified BAG-75 preparations, eve n after extensive dialysis against water, retained phosphate ions in c oncentrations more than 1,000-fold higher than can be accounted for by exchange calculations or by electrostatic binding. The ultrastructura l distribution of immunogold-labeled BAG-75 in the primary spongiosa u nderlying the rat growth plate is distinct from that for other acidic phosphoproteins, osteopontin and bone sialoprotein. We conclude that B AG-75 self-associates in vitro and in vivo into microfibrillar complex es which are specifically recognized by monoclonal antibody HTP IV-#1. This propensity to self-associate into macromolecular complexes is no t shared with acidic phosphoproteins osteopontin and bone sialoprotein . We hypothesize that an extracellular electronegative network of macr omolecular BAG-75 complexes could serve an organizational role in form ing bone or as a barrier restricting local diffusion of phosphate ions . (C) 1997 Wiley-Liss, Inc.