Methods: Glycosylation sites in TWSG1 were predicted using the EnsemblGly software and confirmed by site-directed mutagenesis and enzymatic deglycosylation, followed by western blotting. Interaction of BMPs with TWSG1 was assayed by immunoprecipitation with wild type TWSG1 and TWSG1 that had mutated or absent glycosylation sites. Non-glycosylated and glycosylated recombinant TWSG1 proteins were generated in bacterial and insect cell expression systems, respectively, and assayed quantitatively for BMP binding using surface plasmon resonance analysis. A mandibular explant culture system was used to examine the effect of these TWSG1 proteins on the expression of the BMP target gene Msx2.
Results: TWSG1 in mice has two glycosylation sites, which are both encoded by the fourth exon. Deletion of the entire exon 4 or mutation of both glycosylation sites abolishes glycosylation of mTWSG1. Constructs with mutated glycosylation sites have significantly reduced BMP binding activity. A non-glycosylated form of the protein binds to BMPs with approximately 10-fold reduced affinity compared to the glycosylated form. The non-glycosylated form of TWSG1 is unable to suppress Msx2 expression in mandibular explants, while glycosylated forms do suppress Msx2.
Conclusions: We report that glycosylation is essential for normal action of TWSG1 and hence may represent an important variable in the regulation of BMP signaling.
Keywords: Bone, Bone repair, Embryology, Gene expression and Growth & development
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