Mineralization-inhibiting effects of transglutaminase-crosslinked polymeric osteopontin

Abstract Osteopontin (OPN) belongs to the SIBLING family (Small, Integrin-Binding LIgand N-linked Glycoproteins) of mineral-binding matrix proteins found in bones and teeth. OPN is a well-known inhibitor of matrix mineralization, and enzymatic modification of OPN can affect this inhibitory function. In bone, OPN exists both as a monomer and as a high-molecular-weight polymer – the latter is formed by transglutaminase-mediated crosslinking of glutamine and lysine residues in OPN to create homotypic protein assemblies. OPN can be covalently crosslinked by transglutaminase 2 (TG2) and Factor XIII-A. Polymeric OPN has increased binding to collagen and promotes osteoblast adhesion, but despite these initial observations, its role in mineralization is not clear. In this study, we investigated the effect of polymerized OPN on mineralization using a hydroxyapatite crystal growth assay and mineralizing MC3T3-E1 osteoblast cultures. In the cultures, endogenous polymeric OPN was detected after mineralization occurred. In cell-free conditions, TG2 was used to crosslink bovine OPN into its polymeric form, and atomic force microscopy and dynamic light scattering revealed variably-sized, large branched aggregates ranging across hundreds of nanometers. These OPN polymers inhibited the growth of hydroxyapatite crystals in solution at concentrations similar to monomeric OPN, although the crosslinking slightly reduced its inhibitory potency. When added to MC3T3-E1 osteoblast cultures, this exogenous polymeric OPN essentially did not inhibit mineralization when given during the later mineralization stages of culture; however, cultures treated early and then continuously with polymeric OPN throughout both the matrix assembly and mineral deposition stages showed reduced mineralization. Immunoblotting of protein extracts from these continuously treated cultures revealed exogenous OPN polymers incorporated into mature matrix that had not yet mineralized. These results suggest that in bone, the increased size and branched structure of crosslinked inhibitory polymeric OPN near the mineralization front could hinder it from accessing focal mineralization sites in the dense collagen-rich matrix, suggesting that OPN-crosslinking into polymers may represent a way to fine-tune the inhibitory potency of OPN on bone mineralization.