Matrix GLA Protein Inhibition of Hydroxyapatite Crystal Growth

Based on studies on genetically modified mice, the γ-carboxylated protein Matrix GLA protein (MGP) is believed to inhibit the calcification of vascular tissues. However, the effects of MGP on the formation of biomineral crystals has not been studied. Objectives: To determine whether MGP inhibits hydroxyapatite (HA) crystal growth and, if so, to identify the region of the protein responsible for this activity. Methods: The following peptides of human MGP were synthesized with and without γ-carboxylation and/or phosphorylation: YγEpSHEpSMEpSYELNP (1-14), FINRRNANTFISPQ (15-28), QRWRAKVQγERIRγER (29-42), SKPVHγELNRγEACDD (43-56), YRLCERYAMVYGYN (57-70) and AAYNRYFRKRRGTK (71-84). The effects of these peptides on HA growth were determined using a constant-composition/seeded growth assay. The above sequences were also used for molecular dynamics (MD) simulations of peptide adsorption to the {100} face of HA. Results: Peptides 15-28, 29-42, 57-70 and 71-84 had no significant effect on HA crystal growth. Peptides 43-56 was inhibitory only when γ-carboxylated. Peptide 1-14 was a very potent inhibitor, and had significant effect even in the absence of γ-carboxylation. Similar results were obtained by MD: phosphorylated peptide 1-14 and peptide 43-56 adsorbed rapidly to the {100} face of HA, while 29-42 exhibited some interaction. Lack of γ-carboxylation affected the adsorption of 29-42, but not 1-14. MD simulations of the interaction of full-length MGP with HA showed that the initial interaction was through the N-terminus of the protein, with a subsequent interaction involving the putative gla helix (corresponding to amino acids 37-52). Conclusions: These studies demonstrate that MGP affects HA growth by a direct interaction with the crystal surface. This interaction primarily involves the phosphorylated N-terminus of the protein. Interestingly, the 5 gla residues of human MGP play only a minor role in the crystal-inhibiting activity of the protein. Supported by the CIHR.