Pedersen Nis Borbye, Wang Shengjun, Narimatsu Yoshiki, Yang Zhang, Halim Adnan, Schjoldager Katrine Ter-Borch Gram, Madsen Thomas Daugbjerg, Seidah Nabil G, Bennett Eric Paul, Levery Steven B, Clausen Henrik
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.
the Clinical Research Institute of Montreal, University of Montreal, Montreal, Quebec H2W 1R7, Canada.
J Biol Chem. 2014 Jun 20;289(25):17312-24. doi: 10.1074/jbc.M113.545053. Epub 2014 May 5.
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.
低密度脂蛋白受体（LDLR）对胆固醇稳态至关重要，LDLR功能缺陷会导致高胆固醇血症。LDLR是一种I型跨膜蛋白，其在细胞表面的稳定表达需要O-糖基化。此前有研究表明，LDLR的O-糖基化位于近膜区域的N端。最近，我们使用“SimpleCell”O-糖蛋白组鸟枪法策略，在几种LDLR相关受体的特征性LDLR A类重复序列之间的连接区中鉴定出了O-糖基化位点。在此，我们系统地表征了从HEK293 SimpleCells和CHO野生型细胞中释放的重组LDLR上的O-糖基化位点。我们发现，LDLR A类重复序列之间的短连接区在大多数重复序列的第一个半胱氨酸残基的-1位含有一个进化保守的O-糖基化位点，在野生型CHO细胞中，该位点被典型的唾液酸化核心1结构糖基化。LDLR A类重复序列连接区中的糖基化位点在从人到非洲爪蟾的LDLR中保守，并在其他同源受体中也有发现。O-糖基化由一大类多肽N-乙酰半乳糖胺转移酶控制。通过体外酶分析探究哪种同工型有助于LDLR A类重复序列连接区的糖基化，结果表明GalNAc-T11起主要作用。HEK293细胞中LDLR的表达也支持了这一点，在该细胞中，GalNAc-T11同工型的敲除导致四个连接区中的三个失去糖基化。
