A Single N-Acetylgalactosamine Residue at Threonine 106 Modifies the Dynamics and Structure of Interferon α2a around the Glycosylation Site

Houman Ghasriani,Pascal J.F Belcourt,Simon Sauvé,Derek J Hodgson,Denis Brochu,Michel Gilbert,Yves Aubin

doi:10.1074/jbc.m112.413252

Abstract

Enzymatic addition of GalNAc to isotopically labeled IFNα2a produced in Escherichia coli yielded the O-linked glycoprotein GalNAcα-[(13)C,(15)N]IFNα2a. The three-dimensional structure of GalNAcα-IFNα2a has been determined in solution by NMR spectroscopy at high resolution. Proton-nitrogen heteronuclear Overhauser enhancement measurements revealed that the addition of a single monosaccharide unit at Thr-106 significantly slowed motions of the glycosylation loop on the nanosecond time scale. Subsequent addition of a Gal unit produced Gal(β1,3)GalNAcα-[(13)C,(15)N]IFNα2a. This extension resulted in a further decrease in the dynamics of this loop. The methodology used here allowed the first such description of the structure and dynamics of an O-glycoprotein and opens the way to the study of this class of proteins.

Highlights

O-Glycans are known to modulate the biological activity and physicochemical properties of type I interferon
In this study, we propose an explanation of the effects of glycosylation on biological activity
Proton-nitrogen heteronuclear Overhauser enhancement measurements revealed that the addition of a single monosaccharide unit at Thr-106 significantly slowed motions of the glycosylation loop on the nanosecond time scale

Summary

Introduction

O-Glycans are known to modulate the biological activity and physicochemical properties of type I interferon. Results: A single sugar unit modulates the structure and dynamics of the protein. Conclusion: In this study, we propose an explanation of the effects of glycosylation on biological activity. Significance: The results will facilitate the study of any O-GalNAc glycoprotein. Enzymatic addition of GalNAc to isotopically labeled IFN␣2a produced in Escherichia coli yielded the O-linked glycoprotein GalNAc␣-[13C,15N]IFN␣2a. Proton-nitrogen heteronuclear Overhauser enhancement measurements revealed that the addition of a single monosaccharide unit at Thr-106 significantly slowed motions of the glycosylation loop on the nanosecond time scale. Subsequent addition of a Gal unit produced Gal(␤1,3)GalNAc␣-[13C,15N]IFN␣2a. This extension resulted in a further decrease in the dynamics of this loop. The methodology used here allowed the first such description of the structure and dynamics of an O-glycoprotein and opens the way to the study of this class of proteins

Objectives

Results

Conclusion