title: Reduced expression of the oligosaccharyltransferase exacerbates protein hypoglycosylation in cells lacking the fully assembled oligosaccharide donor.
abstract: A defect in the assembly of the oligosaccharide donor (Dol-PP-GlcNAc(2)Man(9)Glc(3)) for N-linked glycosylation causes hypoglycosylation of proteins by the oligosaccharyltransferase (OST). Mammalian cells express two OST complexes that have different catalytic subunits (STT3A or STT3B). We monitored glycosylation of proteins in asparagine-linked glycosylation 6 (ALG6) deficient cell lines that assemble Dol-PP-GlcNAc(2)Man(9) as the largest oligosaccharide donor. Based upon pulse labeling experiments, 30-40% of STT3A-dependent glycosylation sites and 20% of STT3B-dependent sites are skipped in ALG6-congenital disorders of glycosylation fibroblasts supporting previous evidence that the STT3B complex has a relaxed preference for the fully assembled oligosaccharide donor. Glycosylation of STT3B-dependent sites was more severely reduced in the ALG6 deficient MI8-5 cell line. Protein immunoblot analysis and RT-PCR revealed that MI8-5 cells express 2-fold lower levels of STT3B than the parental Chinese hamster ovary cells. The combination of reduced expression of STT3B and the lack of the optimal Dol-PP-GlcNAc(2)Man(9)Glc(3) donor synergize to cause very severe hypoglycosylation of proteins in MI8-5 cells. Thus, differences in OST subunit expression can modify the severity of hypoglycosylation displayed by cells with a primary defect in the dolichol oligosaccharide assembly pathway.
authors: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors: Shiteshu
authors: S
authors: Shrimal
authors: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA reid.gilmore@umassmed.edu.
authors: Reid
authors: R
authors: Gilmore
mesh_terms: D002460:Cell Line; D006031:Glycosylation; D006602:Hexosyltransferases / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D009844:Oligosaccharides; D011506:Proteins; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase
keywords: asparagine-linked glycosylation; congenital disorders of glycosylation; lipid-linked oligosaccharide; oligosaccharyltransferase