Protein Secondary Structure Affects Glycan Clustering in Native Mass Spectrometry.

Hao Yan,Charlotte Uetrecht,Stefan Taube,Alvaro Mallagaray,Robert Creutznacher,Kevin Pagel,Gergo Peter Szekeres,Thomas Peters,Julia Lockhauserbäumer

doi:10.3390/life11060554

Abstract

Infection by the human noroviruses (hNoV), for the vast majority of strains, requires attachment of the viral capsid to histo blood group antigens (HBGAs). The HBGA-binding pocket is formed by dimers of the protruding domain (P dimers) of the capsid protein VP1. Several studies have focused on HBGA binding to P dimers, reporting binding affinities and stoichiometries. However, nuclear magnetic resonance spectroscopy (NMR) and native mass spectrometry (MS) analyses yielded incongruent dissociation constants (KD) for the binding of HBGAs to P dimers and, in some cases, disagreed on whether glycans bind at all. We hypothesized that glycan clustering during electrospray ionization in native MS critically depends on the physicochemical properties of the protein studied. It follows that the choice of a reference protein is crucial. We analysed carbohydrate clustering using various P dimers and eight non-glycan binding proteins serving as possible references. Data from native and ion mobility MS indicate that the mass fraction of β-sheets has a strong influence on the degree of glycan clustering. Therefore, the determination of specific glycan binding affinities from native MS must be interpreted cautiously.

Highlights

Human norovirus infection is the most common cause of acute gastroenteritis, leading to an estimated 685 million cases annually worldwide
Our results indicate that reference proteins need to be chosen carefully to match the structural properties of the target protein for glycan binding studies, and, crucially, they suggest the additional influence of structural dynamics that preclude glycan-binding studies in native mass spectrometry (MS) for Human norovirus (hNoV)
The tetrasaccharide of Histo blood group antigens (HBGAs) B type 1 is employed as a glycan known to bind to the wildtype and Gb4 as an all galactose glycan non-binder [9,10]

Summary

Introduction

Human norovirus (hNoV) infection is the most common cause of acute gastroenteritis, leading to an estimated 685 million cases annually worldwide. The elderly, immunocompromised patients and children under 5 years are the most severely affected. HNoV belongs to the family of Caliciviridae, non-enveloped viruses of icosahedral shape with the viral genome consisting of positive-sense single-stranded RNA. Histo blood group antigens (HBGAs) serve as attachment factors in viral infection [1,2]. Previous nuclear magnetic resonance spectroscopy (NMR), X-ray crystal structure and mass spectrometry (MS) investigations identified the L-fucose moieties within HBGAs as a minimal binding motif for hNoV attachment. L-galactose derived from L-fucose by substituting one hydrogen atom for a hydroxyl group at C6 is known not to bind [3,4]

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Discussion

Conclusion