Sequential Analysis of the N/O-Glycosylation of Heavily Glycosylated HIV-1 gp120 Using EThcD-sceHCD-MS/MS.

Yong Zhang,Yueqiu Liu,Yonghong Mao,Wanjun Zhao,Meng Gong,Wenjuan Zeng,Liqiang Hu,Shanshan Zheng,Wei Cao,Jingqiu Cheng,Younan Chen,Hao Yang

doi:10.3389/fimmu.2021.755568

Abstract

Deciphering the glycosylation of the viral envelope (Env) glycoprotein is critical for evaluating viral escape from the host’s immune response and developing vaccines and antiviral drugs. However, it is still challenging to precisely decode the site-specific glycosylation characteristics of the highly glycosylated Env proteins, although glycoproteomics have made significant advances in mass spectrometry techniques and data analysis tools. Here, we present a hybrid dissociation technique, EThcD-sceHCD, by combining electron transfer/higher-energy collisional dissociation (EThcD) and stepped collision energy/higher-energy collisional dissociation (sceHCD) into a sequential glycoproteomic workflow. Following this scheme, we characterized site-specific N/O-glycosylation of the human immunodeficiency virus type 1 (HIV-1) Env protein gp120. The EThcD-sceHCD method increased the number of identified glycopeptides when compared with EThcD, while producing more comprehensive fragment ions than sceHCD for site-specific glycosylation analysis, especially for accurate O-glycosite assignment. Finally, eighteen N-glycosites and five O-glycosites with attached glycans were assigned unambiguously from heavily glycosylated gp120. These results indicate that our workflow can achieve improved performance for analysis of the N/O-glycosylation of a highly glycosylated protein containing numerous potential glycosites in one process. Knowledge of the glycosylation landscape of the Env glycoprotein will be useful for understanding of HIV-1 infection and development of vaccines and drugs.

Highlights

Human immunodeficiency virus type 1 (HIV-1) is the major cause of the life-threatening disease known as acquired immune deficiency syndrome (AIDS) [1]
We present a sequential glycoproteomic workflow for the characterization of the intact N/Oglycopeptides of the human immunodeficiency virus type 1 (HIV-1) Env gp120 based on two mass spectrometry (MS) methods, stepped collision energy/higher-energy collisional dissociation (sceHCD)-MS/MS and electron transfer/higher-energy collisional dissociation (EThcD)-sceHCD-MS/MS
The HIV-1 envelope glycoprotein gp120 plays a critical role in inducing glycan-dependent HIV neutralizing antibodies and protective immunity [10, 39]

Summary

INTRODUCTION

Human immunodeficiency virus type 1 (HIV-1) is the major cause of the life-threatening disease known as acquired immune deficiency syndrome (AIDS) [1]. Cao et al sequentially treated peptides with two specific endoglycosidases End H and PNGase F to determine the degree of glycan occupancy and the proportion of highmannose and complex-type glycans at each glycosite of the HIV-1 Env using CID-MS/MS [21, 22]. These studies mainly focused on N-glycans, N-glycosites, and N-glycan site occupancy. The O-glycosylation profile of gp120 revealed some unreported O-glycosites and the O-glycans at these sites This method for obtaining detailed and high-quality information regarding the N/O-glycosylation of gp120 will assist our understanding of N/O-glycosylation on highly glycosylated proteins from different types of viruses, such as HIV, SARS-CoV, and SARS-CoV-2 [38]. Such information will provide guidance on how to assess immunogens with optimal glycosylation for vaccine development

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION

DATA AVAILABILITY STATEMENT