The Preparation and Solution NMR Spectroscopy of Human Glycoproteins Is Accessible and Rewarding

Abstract

The majority of proteins excreted by human cells and borne at the cell surface are modified with carbohydrates. Glycoproteins mediate a wide range of processes and adopt fundamental roles in many diseases. The carbohydrates covalently attached to proteins during maturation in the cell directly impact protein structure and function as integral and indispensable components. However, the ability to study the structure of glycoproteins to high resolution was historically limited by technical barriers including a limited availability of appropriate recombinant protein expression platforms, limited methods to generate compositional homogeneity and difficulties analyzing glycoprotein composition. Furthermore, glycoproteins and in particular the glycan moieties themselves often exhibit a high degree of conformational heterogeneity. Solution NMR spectroscopy is a powerful tool to study biological macromolecules that is capable of characterizing mobile elements of molecules with atomic-level resolution. Methods to express glycoproteins, incorporate stable isotope labels and analyze glycoproteins have recently opened new avenues to prepare and investigate glycoproteins. These methods are accessible to many laboratories with experience expressing and purifying proteins from prokaryotic expression hosts.

The weaker-binding Fc γ receptor IIIa F158 allotype retains sensitivity to N-glycan composition and exhibits a destabilized antibody-binding interface

“…Nglycans can be labeled by supplementing the expression medium with [ 13 C]-glucose, which is then incorporated into each N-glycan sugar and alanine methyls (33,34). One unique N-glycan proton-carbon correlation provides unique insight into each individual N-glycan at the point of attachment: the 1 H1-13 C1 correlation on the (1)GlcNAc residue that is covalently bonded to the N-glycosylated asparagine residue (35). NMR observation of this correlation provides a fingerprint of the glycoprotein N-glycans with one peak expected for each N-glycan.…”

Novel NMR Avenues to Explore the Conformation and Interactions of Glycans

“…31 Nevertheless, some of these strategies have been employed to study glycoproteins by NMR, suppressing the entire glycan ramifications that are not or barely important for structural integrity and activity. 32 Alternative studies on large glycancontaining proteins have made use of specific amino acid labeling strategies to simplify the 2D-HSQC spectra and to cope with line-broadening issues. These methodologies include [ 13 C]-labeling of methyl groups of methionines and/or the aliphatic amino acids (Ile, Leu, and Val), as well as labeling of the carbonyl groups.…”

Proceedings of workshop: “Neuroglycoproteins in health and disease”, INNOGLY cost action

“…Over the last few decades different efforts have been invested towards the development of new strategies for the use of Nuclear Magnetic Resonance (NMR) as a tool to report on key structural aspects on glycoprotein glycans. Most of these strategies are based on the incorporation of 13C-labels on the glycan moieties, for which different experimental protocols and schemes may be applied [ 59 – 62 ].…”