Abstract

Glycoconjugates at the cell surface are crucial for cells to communicate with each other and the extracellular microenvironment. While it is generally accepted that glycans are vectorial biopolymers, their information content is unclear. This report provides evidence that distinct N-glycan structures influence the spatial arrangement of two integral membrane glycoproteins, Kv3.1 and E-cadherin, at the adherent membrane which in turn alter cellular properties. Distinct N-glycan structures were generated by heterologous expression of these glycoproteins in parental and glycosylation mutant Chinese hamster ovary cell lines. Unlike the N-linked glycans, the O-linked glycans of the mutant cell lines are similar to those of the parental cell line. Western and lectin blots of total membranes and GFP immunopurified samples, combined with glycosidase digestion reactions, were employed to verify the glycoproteins had predominantly complex, oligomannose, and bisecting type N-glycans from Pro-5, Lec1, and Lec10B cell lines, respectively. Based on total internal reflection fluorescence and differential interference contrast microscopy techniques, and cellular assays of live parental and glycosylation mutant CHO cells, we propose that glycoproteins with complex, oligomannose or bisecting type N-glycans relay information for localization of glycoproteins to various regions of the plasma membrane in both a glycan-specific and protein-specific manner, and furthermore cell-cell interactions are required for deciphering much of this information. These distinct spatial arrangements also impact cell adhesion and migration. Our findings provide direct evidence that N-glycan structures of glycoproteins contribute significantly to the information content of cells.

Highlights

  • IntroductionLike proteins and nucleic acids, are vectorial biomolecules

  • Glycans, like proteins and nucleic acids, are vectorial biomolecules

  • To examine whether N-glycan occupancy and structure impacts spatial arrangements of Kv3.1 and E-cadherin in the plasma membrane, wild type Kv3.1, N220Q/N229Q Kv3.1, and E-cadherin proteins tagged with EGFP were heterologously expressed in a parental CHO cell line (Pro-5) and two Nglycosylation mutant CHO cell lines (Lec1 and LEC10B)

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Summary

Introduction

Like proteins and nucleic acids, are vectorial biomolecules. Unlike proteins and nucleic acids, the information content of polysaccharides encoded by the sequence of monosaccharides is unclear. N-Glycosylation of newly synthesized membrane proteins is the most ubiquitous protein co-translational modification in the lumen of the endoplasmic reticulum [1]. An important physiochemical property conferred by membrane proteins is their lateral heterogeneity in biological membranes. Since the majority of membrane proteins are N-glycosylated, it is of considerable interest whether the glycans of N-glycosylated membrane proteins contain information about their association, clustering, and distribution in the cell membrane. The information of the N-glycans of glycoproteins has been shown to affect important physiochemical properties such as conformation, stability, protease resistance, charge, and water binding capacity [2]. Establishing the cellular information content of N-glycans would tremendously assist in understanding their role in the development and maintenance of an organism

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