A single GalNAc residue on Threonine-106 modifies the dynamics and the structure of Interferon alpha-2a around the glycosylation site.

Abstract

Abstract Enzymatic addition of a N-acetyl-galactosamine (GalNAc) to isotopically labeled interferon alpha-2a (IFNα2a) produced in Escherichia coli yielded the O-linked glycoprotein GalNAcα-13C,15N-IFNα2a. The three dimensional structure of the resulting 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 on Thr-106 significantly slows motions of the glycosylation loop in the nanosecond time scale. Subsequent addition of a galactose (Gal) unit produced Gal(β1,3)GalNAcα-13C,15N-IFNα2a. This extension resulted in a further decrease of 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.