title: Asparagine-linked glycosylation of bone morphogenetic protein-2 is required for secretion and osteoblast differentiation.
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.
authors: The Jiangsu Province Key Laboratory of Neuroregeneration, Nantong University, and.
authors: Qinglei
authors: Q
authors: Hang
authors: Ying
authors: Y
authors: Zhou
authors: Sicong
authors: S
authors: Hou
authors: Dongmei
authors: D
authors: Zhang
authors: Xiaojing
authors: X
authors: Yang
authors: Jianping
authors: J
authors: Chen
authors: Zhiyun
authors: Z
authors: Ben
authors: Chun
authors: C
authors: Cheng
authors: Aiguo
authors: A
authors: Shen
mesh_terms: D000818:Animals; D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D055396:Bone Morphogenetic Protein 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D009154:Mutation; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D010012:Osteogenesis; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D021381:Protein Transport
chemical_list: C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D001216:Asparagine
keywords: CHO cells; MC3T3-E1 cells; N-glycosylation; bone morphogenetic protein-2; osteoblast differentiation