Intramolecular N-Glycan/Polypeptide Interactions Observed at Multiple N-Glycan Remodeling Steps through [¹³C,¹⁵N]-N-Acetylglucosamine Labeling of Immunoglobulin G1

Abstract

Asparagine-linked (N) glycosylation is a common eukaryotic protein modification that affects protein folding, function, and stability through intramolecular interactions between N-glycan and polypeptide residues. Attempts to characterize the structure–activity relationship of each N-glycan are hindered by inherent properties of the glycoprotein, including glycan conformational and compositional heterogeneity. These limitations can be addressed by using a combination of nuclear magnetic resonance techniques following enzymatic glycan remodeling to simultaneously generate homogeneous glycoforms. However, widely applicable methods do not yet exist. To address this technological gap, immature glycoforms of the immunoglobulin G1 fragment crystallizable (Fc) were isolated in a homogeneous state and enzymatically remodeled with [13C,15N]-N-acetylglucosamine (GlcNAc). UDP-[13C,15N]GlcNAc was synthesized enzymatically in a one-pot reaction from [13C]glucose and [15N-amido]glutamine. Modifying Fc with recombinantly expressed glycosyltransferases (Gnt1 and Gnt2) and UDP-[13C,15N]GlcNAc resulted in complete glycoform conversion as judged by mass spectrometry. Two-dimensional heteronuclear single-quantum coherence spectra of the Gnt1 product, containing a single [13C,15N]GlcNAc residue on each N-glycan, showed that the N-glycan is stabilized through interactions with polypeptide residues. Similar spectra of homogeneous glycoforms, halted at different points along the N-glycan remodeling pathway, revealed the presence of an increased level of interaction between the N-glycan and polypeptide at each step, including mannose trimming, as the N-glycan was converted to a complex-type, biantennary form. Thus, conformational restriction increases as Fc N-glycan maturation proceeds. Gnt1 and Gnt2 catalyze fundamental reactions in the synthesis of every glycoprotein with a complex-type N-glycan; thus, the strategies presented herein can be applied to a broad range of glycoprotein studies.

The Structural Role of Antibody N-Glycosylation in Receptor Interactions

“…This seems unlikely because the peak intensity does not change upon altering the glycan composition, and it is known that glycan extension stabilizes the N-glycan (Barb, 2015; Barb et al, 2012). An alternative and potentially more likely possibility is that the two (1)GlcNAc Fc peaks result from exchange of the nearby Y296 sidechain between two conformations as was observed by Kato and coworkers(Matsumiya et al, 2007).…”

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

“…This approach provides two advantages: glycan homogeneity as well as a mechanism to introduce selective carbohydrate labels with labeled sugar nucleotides. Multiple methods to prepare labeled sugar nucleotides are published, including UDP-[ 13 C]-galactose, [ 13 C, 15 N]-CMP-N-acetylneuraminic acid and [ 13 C, 15 N]-UDP-N-acetylglucosamine (Azurmendi, Vionnet, Wrightson, Trinh, Shiloach, & Freedberg, 2007; Barb, 2015; Barb & Prestegard, 2011; Macnaughtan et al, 2008; Yamaguchi et al, 1998). Many of these methods were developed to remodel glycoproteins with predominantly complex-type biantennary glycans with a varying level of galactose and N-acetylneuraminic acid residues following purification from human serum or HEK293F expression (Figure 2).…”

“…These glycans can be extended by one N-acetylglucosamine residue attached to the Manα1-3Manβ residue using Gnt1, expressed from HEK293F cells (Barb, 2015). Though the Man5 oligomannose N-glycan is the native substrate for this reaction, Gnt1 efficiently modifies the paucimannose N-glycan (containing two N-acetylglucosamine and three mannose residues).…”

“…The Man5 N-glycans from HEK293S expression (Figure 1) are effective acceptor substrates for Gnt1 to create a Man5 N-glycan with an N-acetylglucosamine residue on the Manα1-3Manβ residue (Barb, 2015). We have not identified a suitable mannosidase that expresses in sufficient quantities to convert the Man5-GlcNAc N-glycan in vitro into the cognate Gnt2 substrate, a Man3-GlcNAc N-glycan.…”

“…Our laboratory has used this approach extensively to study the IgG1 Fc N-glycans (Barb, 2015; Barb, Ho, Flanagan-Steet, & Prestegard, 2012; Barb, Meng, et al, 2012; Barb et al, 2011; Subedi et al, 2014). A wealth of labeling strategies and nuclei are accessible though labeled sugar nucleotides are time consuming or expensive to obtain.…”