Novel glycation patterns help understand Omicron immune escape

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019 to trigger the coronavirus disease 2019 (COVID-19) pandemic, devastating human health, social interactions, and economic transactions.

Several variants have been identified since then, some of which have been capable of immune escape and/or increased transmissibility. The latest variant of concern has been the Omicron variant, with numerous spike mutations that confer antibody escape capability as well as higher infectiousness, compared to the Delta variant.

Study: Distinct Core Glycan and O-Glycoform Utilization of SARS-CoV-2 Omicron Variant Spike Protein RBD Revealed by Top-Down Mass Spectrometry. Image Credit: Naeblys/ShutterstockStudy: Distinct Core Glycan and O-Glycoform Utilization of SARS-CoV-2 Omicron Variant Spike Protein RBD Revealed by Top-Down Mass Spectrometry. Image Credit: Naeblys/Shutterstock

A new preprint discusses changes in the sugar structure of the Omicron variant, which may shed light on its immunologic escape and transmissibility characteristics.

Introduction

Omicron has the largest number of mutations of all known variants of SARS-CoV-2 so far, with 15 in the spike protein receptor-binding domain (S-RBD) alone, compared to the wildtype virus. Since this is the immunodominant antigen, as well as the prime target of neutralizing antibodies, this has aroused considerable concern about this variant, which is already known to be more infectious and antibody-resistant, causing a high rate of breakthrough infections even after a third booster dose of a nucleic acid vaccine.

The current study deals with the glycation of the spike protein of the Delta and Omicron variants, since this is a feature that is key to receptor-binding, cell entry and immune evasion characteristics. Both N- and O-glycans are known to be present, but the latter is extremely diverse in structure. This has led to its being left out of most glycation studies.

In this study, posted to the bioRxiv* preprint server, the researchers used a proprietary hybrid top-down mass spectrometry (MS) platform. The advantages include being able to examine multiple aspects, such as the molecular structure, specificity of site binding, and the relative abundance of different glycofoms, at the same time. In addition, they were able to look at the post-translational modification (PTM) compositions of the various different protein isoforms present at the same time to produce a proteoform-resolved analysis.

Findings

This is the first time that the Delta and Omicron variants have been studied in detail for their O-glycoform structures and sites using this platform. This showed that the greatest difference in terms of mutational change between the Delta/Omicron variants vs the wildtype virus was in the RBD.

They first removed the N-glycans, and then separated the S-RBD O-glycoforms using a ultra-high resolution tool, the Fourier transform ion cyclotron (FTICR)-MS). The various isotopes were resolved, showing different stoichiometric ratios and relative abundance between the O-glycoforms. The Omicron had the most heterogeneous O-glycoform structure.

Using another technique called trapped ion mobility spectrometry (TIMS)-MS, they isolated each of the O-glycoforms of the RBD. They found many RBD glycoforms with Core 1 and Core 2 structures in the three variants. Microheterogeneity was greatest for O-glycans in Omicron.

The abundance of Core 2 O-glycan structures in Omicron was markedly higher, especially structures with multiple sialylated and fucosylated structures. Core 1 was in a ratio of 71:29 to Core 2 for Omicron, and the most abundant O-glycoform was the Core 1 GalNAcGal(NeuAc)2, with almost 70% relative abundance. In fact, this makes up over 80% of the O-glycoform abundance for both the ancestral or wildtype variants, which both tend to have Core 1 O-glycan structures.

Omicron, conversely, shows a single Core 2 structure comprising almost one in seventh of the total O-glycoform profile. They also found another structure of Core 2 type in this variant, making up a tenth or more of overall O-glycoform structures.  Though these were present in both wildtype and Delta, their relative abundance was <6%.

These findings show,

the distinct advantages of this top-down MS approach over glycopeptide-based bottom-up MS approaches.”

Looking at glycosites, Omicron showed both the expected and a new and unique glycosite, Thr376, found only here. This was located adjacent to a proline residue, which promotes O-glycosylation. “Fascinatingly, all detected S-RBD O-glycans for the WT and Delta variants were confidently assigned solely to Thr323.” Since Delta has fewer mutations than Omicron, this glycosite was intact in both wildtype and Delta.

However, Omicron shows both this and the new Thr376 glycosite, the latter being due to the presence of Pro373, a site-specific mutation unique to this variant. However, Thr376 has only <30% occupancy, and only the Core 1 GalNAcGal(NeuAc)2, which is most abundant, could be assigned with confidence. Other Core 2 O-glycoforms are also likely to have this modification, though this remains to be demonstrated.

Implications

This study, the first to explore the range of O-glycoform structures in the Omicron and Delta variants of SARS-CoV-2, showed Core 2 O-glycoforms to be used at higher rates in Omicron, vs Delta or the ancestral variant. They also found a new Omicron-specific glycosite.

This method could complement X-ray crystallography, or cryo-electron microscopy (cryo-EM), since it allows for direct analysis of O-glycan structure, unlike the other methods. This is due to the flexible nature of oligosaccharides coupled with their range of variation.

Importantly, this top-down MS approach establishes a necessary molecular foundation to understand the structure-function significance of S mutations and PTM changes in emerging SARS-CoV-2 variants. Our findings may provide new insights into how Omicron escapes immunological protection and can inform strategies for developing new variant-directed vaccines and therapeutics.”

*Important notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
Dr. Liji Thomas

Written by

Dr. Liji Thomas

Dr. Liji Thomas is an OB-GYN, who graduated from the Government Medical College, University of Calicut, Kerala, in 2001. Liji practiced as a full-time consultant in obstetrics/gynecology in a private hospital for a few years following her graduation. She has counseled hundreds of patients facing issues from pregnancy-related problems and infertility, and has been in charge of over 2,000 deliveries, striving always to achieve a normal delivery rather than operative.

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