Glycosylation States on Intact Proteins Determined by NMR Spectroscopy.

Audra A Hargett,Aaron M Marcella,Lai-Xi Wang,Chao Li,Darón I Freedberg,Marcos D Battistel,Jared Orwenyo,Huifeng Yu

doi:10.3390/molecules26144308

Abstract

Protein glycosylation is important in many organisms for proper protein folding, signaling, cell adhesion, protein-protein interactions, and immune responses. Thus, effectively determining the extent of glycosylation in glycoprotein therapeutics is crucial. Up to now, characterizing protein glycosylation has been carried out mostly by liquid chromatography mass spectrometry (LC-MS), which requires careful sample processing, e.g., glycan removal or protein digestion and glycopeptide enrichment. Herein, we introduce an NMR-based method to better characterize intact glycoproteins in natural abundance. This non-destructive method relies on exploiting differences in nuclear relaxation to suppress the NMR signals of the protein while maintaining glycan signals. Using RNase B Man5 and RNase B Man9, we establish reference spectra that can be used to determine the different glycoforms present in heterogeneously glycosylated commercial RNase B.

Highlights

Glycosylation is one of the most common post-translational modifications (PTM).There are two main types of glycosylation: (i) O-linked glycosylation, in which glycans are covalently linked to the hydroxyl oxygen of serine (S) or threonine (T) residues [1,2], and (ii) N-linked glycosylation, where glycans are attached to asparagine (N) residues within the N-X-S/T sequon [3,4,5]
We show that the glycans in intact folded RNase B can be characterized by NMR spectroscopy
We show that using a T2 filter in small glycoproteins reduces the spectral complexity that arises from the protein peaks yet captures the glycosylation microheterogeneity by retaining glycan peaks

Summary

Introduction

Glycosylation is one of the most common post-translational modifications (PTM).There are two main types of glycosylation: (i) O-linked glycosylation, in which glycans are covalently linked to the hydroxyl oxygen of serine (S) or threonine (T) residues [1,2], and (ii) N-linked glycosylation, where glycans are attached to asparagine (N) residues within the N-X-S/T sequon [3,4,5]. Because protein glycosylation is not template driven, it is inherently heterogeneous, with several factors contributing to the final glycan structure, such as protein structure [6,7], enzyme protein levels [8], Golgi transport mechanism [9], and secretory protein load [10] Overall, this process yields heterogeneously glycosylated proteins, such as IgG, which has 32 possible glycans for its one N-linked glycosylation site at N297 [11]. In Hepatitis C virus envelope 2 protein, the loss of either N2 or N4 glycan results in total loss of HCV infectivity [23] These are just a few glycoproteins where the location and type of glycan are critical to protein function. Developing tools to characterize intact glycoproteins will aid in the understanding of optimal glycosylation for a given function, especially in protein therapeutics

Methods

Results

Conclusion