PLAP-1/asporin Antagonizes BMP-2 Function Through LRR-5

Objectives: We previously reported that PLAP-1/asporin, which we have newly discovered as a novel periodontal ligament (PDL) specific molecule, is one of negative regulators for the cytodifferentiation and mineralization of PDL cells. We also demonstrated that PLAP-1/asporin negatively regulates BMP-2-induced alkaline phosphatase activity in PDL cells. However, direct evidence showing molecular mechanisms of PLAP-1/asporin function is limited. Thus, the aim of this study is to dissect more details of PLAP-1/asporin function especially against BMP-2 in PDL cells. Methods: We generated recombinant protein of PLAP-1/asporin and assessed direct molecular interaction between PLAP-1/asporin and BMP-2 by co-immunoprecipitation assay. We then examined whether PLAP-1/asporin interfered with binding of BMP-2 to the type IB receptor (BMPR-IB) by co-immunoprecipitation assay. We introduced single nucleotide substitution into leucine-rich repeat 5 (LRR-5) of PLAP-1/asporin and transfected mutant PLAP-1/asporin expressing vector into PDL cells. After transfection, we assessed the BMP-2-induced alkaline phosphatase activities of the transfectants. We finally synthesized a small peptide of the LRR-5 region and measured ALPase activities of BMP-2-stimulated normal PDL cells cultured in the presence of the LRR-5 peptide. Results: Co-immunoprecipitation assays revealed that PLAP-1/asporin directly interacted with BMP-2 and inhibited the binding of BMP-2 to BMPR-IB. Single nucleotide substitution in LRR5 of PLAP-1/asporin resulted in cancellation of suppressing BMP-2-induced ALPase activities in PDL cells. Furthermore, LRR-5 peptide inhibited BMP-2-induced ALPase activity in normal PDL cells. Conclusion: Present study demonstrates that PLAP-1/asporin antagonizes BMP-2 function through LRR-5 and suggestes that this antagonistic action is one of molecular mechanisms of homeostasis of PDL tissues to prevent ossification in vivo.