title: Low density lipoprotein receptor class A repeats are O-glycosylated in linker regions.
abstract: 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.
authors.affiliation: From the Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark and.
authors.forename: Nis Borbye
authors.initials: NB
authors.lastname: Pedersen
authors.affiliation: From the Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark and.
authors.forename: Shengjun
authors.initials: S
authors.lastname: Wang
authors.affiliation: From the Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark and.
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Narimatsu
authors.affiliation: From the Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark and.
authors.forename: Zhang
authors.initials: Z
authors.lastname: Yang
authors.affiliation: From the Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark and.
authors.forename: Adnan
authors.initials: A
authors.lastname: Halim
authors.affiliation: From the Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark and.
authors.forename: Katrine Ter-Borch Gram
authors.initials: KT
authors.lastname: Schjoldager
authors.affiliation: From the Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark and.
authors.forename: Thomas Daugbjerg
authors.initials: TD
authors.lastname: Madsen
authors.affiliation: the Clinical Research Institute of Montreal, University of Montreal, Montreal, Quebec H2W 1R7, Canada.
authors.forename: Nabil G
authors.initials: NG
authors.lastname: Seidah
authors.affiliation: From the Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark and.
authors.forename: Eric Paul
authors.initials: EP
authors.lastname: Bennett
authors.affiliation: From the Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark and.
authors.forename: Steven B
authors.initials: SB
authors.lastname: Levery
authors.affiliation: From the Copenhagen Center for Glycomics, Departments of Cellular and Molecular Medicine and School of Dentistry, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark and hclau@sund.ku.dk.
authors.forename: Henrik
authors.initials: H
authors.lastname: Clausen
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D009865:Oocytes; D017434:Protein Structure, Tertiary; D011973:Receptors, LDL / Q000235:genetics / Q000378:metabolism*; D020449:Repetitive Sequences, Amino Acid; D012799:Sialyltransferases / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: C573412:LDLR protein, human; D011973:Receptors, LDL; D012799:Sialyltransferases
keywords: GALNT; Glycosylation; Glycosyltransferase; Lectin; Lipoprotein Receptor-related Protein (LPR); Low Density Lipoprotein (LDL); O-Glycan; Zinc Finger Nuclease