O-linked oligosaccharide on the 75-kDa neurotrophin receptor.
Four neurotrophic factors, important for survival and function of neurons, bind a common receptor, the 75-kDa neurotrophin receptor (NTR). An O-glycosylated peptide connects the ligand-binding domain of NTR to its transmembrane helix. This peptide, the transmembrane helix, and intracellular sequences are highly conserved in vertebrate evolution. To investigate the structure and function of O-glycosylation on NTR, we produced the extracellular domains by expression in mammalian cells. Addition during biosynthesis of O-linked glycans was evaluated, and structures were characterized by lectin blotting and glycosidase digestion. Effects of disialylation, deglycosylation, and lectin attachment on the equilibrium binding constant were measured. Addition of O-linked glycans during biosynthesis was found to have a large effect on NTR structure assessed by mobility in polyacrylamide gels. NTR O-linked glycans synthesized by cultured cells had the structure (NeuNAc)(1-2-) Gal beta 1-3GalNAc. Modification of the O-linked oligosaccharide produced small, possibly significant effects on the binding constant of NTR for nerve growth factor. The results are discussed in reference to a potential role for the stalk region in ligand binding and signaling.
Chapman
B S
BS
Department of Pharmaceutical Chemistry, University of California School of Pharmacy, San Francisco, USA.
Eckart
M R
MR
Kaufman
S E
SE
Lapointe
G R
GR
D000818:Animals; D001710:Biotin; D016466:CHO Cells / Q000737:chemistry; D002241:Carbohydrates / Q000737:chemistry; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008970:Molecular Weight; D009844:Oligosaccharides / Q000032:analysis*; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism; D017475:Receptors, Nerve Growth Factor / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology
D002241:Carbohydrates; D009844:Oligosaccharides; D011134:Polysaccharides; D017475:Receptors, Nerve Growth Factor; D001710:Biotin