Pedersen, Nis Borbye6; Wang, Shengjun6; Narimatsu, Yoshiki6; Yang, Zhang6; Halim, Adnan6; Schjoldager, Katrine Ter-Borch Gram6; Madsen, Thomas Daugbjerg6; Seidah, Nabil G4; Bennett, Eric Paul7; Levery, Steven B6; Clausen, Henrik6
1 Section III. Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, Københavns Universitet2 Glycomics Program, Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, Københavns Universitet3 Section 03 - Radiology, Department of Odontology, Faculty of Health and Medical Sciences, Københavns Universitet4 the Clinical Research Institute of Montreal, University of Montreal, Montreal, Quebec H2W 1R7, Canada.5 Section 01 - Prosthetics, Department of Odontology, Faculty of Health and Medical Sciences, Københavns Universitet6 Section III. Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, Københavns Universitet7 Section 01 - Prosthetics, Department of Odontology, Faculty of Health and Medical Sciences, Københavns Universitet
The low density lipoprotein receptor (LDLR) is crucial for cholesterol homeostasis and deficiency in LDLR functions cause hypercholesterolemia. LDLR is a type I transmembrane protein that requires O-glycosylation for stable expression at the cell surface. It has previously been suggested that LDLR O-glycosylation is found N-terminal to the juxtamembrane region. Recently we identified O-glycosylation sites in the linker regions between the characteristic LDLR class A repeats in several LDLR-related receptors using the "SimpleCell" O-glycoproteome shotgun strategy. Herein, we have systematically characterized O-glycosylation sites on recombinant LDLR shed from HEK293 SimpleCells and CHO wild-type cells. We find that the short linker regions between LDLR class A repeats contain an evolutionarily conserved O-glycosylation site at position -1 of the first cysteine residue of most repeats, which in wild-type CHO cells is glycosylated with the typical sialylated core 1 structure. The glycosites in linker regions of LDLR class A repeats are conserved in LDLR from man to Xenopus and found in other homologous receptors. O-Glycosylation is controlled by a large family of polypeptide GalNAc transferases. Probing into which isoform(s) contributed to glycosylation of the linker regions of the LDLR class A repeats by in vitro enzyme assays suggested a major role of GalNAc-T11. This was supported by expression of LDLR in HEK293 cells, where knock-out of the GalNAc-T11 isoform resulted in the loss of glycosylation of three of four linker regions.
Journal of Biological Chemistry, 2014, Vol 289, Issue 25, p. 17312-17324