Determination of the glycoprotein specificity of lectins on cell membranes through oxidative proteomics.

Yixuan Xie,Ying Sheng,Qiongyu Li,Joe Reyes,Seunghye Ju,Carlito B Lebrilla

doi:10.1039/d0sc04199h

Abstract

The cell membrane is composed of a network of glycoconjugates including glycoproteins and glycolipids that presents a dense matrix of carbohydrates playing critical roles in many biological processes. Lectin-based technology has been widely used to characterize glycoconjugates in tissues and cell lines. However, their specificity toward their putative glycan ligand and sensitivity in situ have been technologically difficult to study. Additionally, because they recognize primarily glycans, the underlying glycoprotein targets are generally not known. In this study, we employed lectin proximity oxidative labeling (Lectin PROXL) to identify cell surface glycoproteins that contain glycans that are recognized by lectins. Commonly used lectins were modified with a probe to produce hydroxide radicals in the proximity of the labeled lectins. The underlying polypeptides of the glycoproteins recognized by the lectins are oxidized and identified by the standard proteomic workflow. As a result, approximately 70% of identified glycoproteins were oxidized in situ by all the lectin probes, while only 5% of the total proteins were oxidized. The correlation between the glycosites and oxidation sites demonstrated the effectiveness of the lectin probes. The specificity and sensitivity of each lectin were determined using site-specific glycan information obtained through glycomic and glycoproteomic analyses. Notably, the sialic acid-binding lectins and the fucose-binding lectins had higher specificity and sensitivity compared to other lectins, while those that were specific to high mannose glycans have poor sensitivity and specificity. This method offers an unprecedented view of the interactions of lectins with specific glycoproteins as well as protein networks that are mediated by specific glycan types on cell membranes.

Highlights

The carbohydrate layer on the cell surface that is anchored by protein and lipid scaffolds is involved in a host of important and central cellular processes, including cellular adhesion, immune defense, and cell permeability.1–3 Extensive covalent and non-covalent interactions occur on the cell membrane that de nes the topology and availability of glycan-binding sites and antigens
We have previously found that 50–300 mM hydrogen peroxide concentrations and 30 minutes reaction time produce hydroxyl radicals within 20 Aof the probe, which was optimal for identifying proteins that were in the vicinity of the probe
We found that ITA2 was oxidized by Sambucus nigra agglutinin (SNA) lectin at T337, which was closer to the site of sialylation than mannosylation

Summary

Introduction

The carbohydrate layer on the cell surface that is anchored by protein and lipid scaffolds is involved in a host of important and central cellular processes, including cellular adhesion, immune defense, and cell permeability.1–3 Extensive covalent and non-covalent interactions occur on the cell membrane that de nes the topology and availability of glycan-binding sites and antigens. 70% of identi ed glycoproteins were oxidized by the lectin probe, while only 5% of the nonglycosylated proteins were oxidized (Fig. 2). The glycosylated proteins contained the target glycans, and these oxidized glycoproteins were quanti ed using Byologic so ware (Fig. S5†).

Results

Conclusion