Abstract
Bone morphogenetic protein-2 (BMP-2), a glycosylated protein, has been demonstrated to play a key role in osteoblast differentiation. However, the function of its glycosylation is incompletely understood. In this study, we examined the role that N-linked glycans (NLG) play in the secretion of BMP-2. Blocking the addition of NLGs or inhibiting initial glycan processing prevented the secretion of BMP-2. To identify the specific glycosylation sites, we abolished potential sites of N-linked glycosylation (Asn-Xaa-Ser/Thr) in BMP-2 by mutating the Asn residues to Gln individually or in combination, expressed the BMP-2 mutants in Chinese hamster ovary (CHO) and human embryonic kidney 293T (HEK293T) cells and determined their glycosylation state by using peptide:N-glycosidase F and endoglycosidase H digestion. We found that human BMP-2 contains three NLG on N135, N200 and N338. Elimination of N-glycosylation by mutation of N135 (N135Q) abolished the BMP-2 secretion from CHO cells. Overexpression of the BMP-2 mutant N135Q elicited endoplasmic reticulum (ER) stress and retention within the ER in CHO cells, indicating that N-glycosylation is required for folding of human BMP-2. Furthermore, we demonstrated that glycosylation at N135 was necessary for BMP-2-induced osteoblast differentiation in MC3T3-E1 cells. Taken together, these data provide further evidence of the critical role that individual NLG may play an important role in determining BMP-2 folding, secretion and function.