Intramolecular N-glycan/polypeptide interactions observed at multiple N-glycan remodeling steps through [(13)C,(15)N]-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 [(13)C,(15)N]-N-acetylglucosamine (GlcNAc). UDP-[(13)C,(15)N]GlcNAc was synthesized enzymatically in a one-pot reaction from [(13)C]glucose and [(15)N-amido]glutamine. Modifying Fc with recombinantly expressed glycosyltransferases (Gnt1 and Gnt2) and UDP-[(13)C,(15)N]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 [(13)C,(15)N]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.

Figure 1

Native IgG1 Fc N-glycan processing in the Golgi. Conversions catalyzed by the enzymes indicated above the solid arrows and labeled with black type largely proceed to completion. Reactions catalyzed by enzymes denoted with gray type a dashed arrow modify some but not all of the secreted IgG1. Glycoforms studied here by nuclear magnetic resonance are underlined. Carbohydrate residues are numbered according to ref (30) and represented using the CFG convention and shown in the inset (GlcNAc, N-acetylglucosamine). Glycosidic linkages of the human IgG1 Fc N-glycan are indicated.

Figure 2

Schemes for in vitro enzymatic conversions described in this study. (A) A one-pot synthesis of UDP-[¹³C,¹⁵N]GlcNAc utilizes enzymes from bacterial pathways and [¹³C]glucose. Carbohydrate remodeling started with Fc bearing either a mannose-type (B) or a complex-type (C) N-glycan. [¹³C,¹⁵N]GlcNAc is shown as a blue square with a white star in the cartoon figures and by “*N” in the glycan name; residue numbers corresponding to the convention introduced in Figure 1 are given in parentheses.

Figure 3

Gnt1-catalyzed remodeling of lec1^–/–-expressed IgG1 Fc with a Man5 N-glycan as monitored by MALDI-MS. Enzymatic remodeling occurs when the N-glycans are attached to the Fc polypeptide; however, the analysis shown here includes N-glycan removal followed by permethylation.

Figure 4

Generation of *N1F and *N2F Fc glycoforms from HEK293F-expressed Fc was confirmed using MALDI-MS analysis. These steps are shown in Figure 2C. Enzymatic remodeling occurs when the N-glycans are attached to the Fc polypeptide; however, the analysis shown here includes N-glycan removal followed by permethylation.

Figure 5

¹H–¹³C HSQC spectra of IgG1 Fc with a Man5 N-glycan following addition of [¹³C,¹⁵N]GlcNAc. (A) A 2D ¹H–¹³C HSQC spectrum of the *N-Man5 N-glycan following EndoF1-catalyzed hydrolysis is shown as gray contours. Blue contours show the positions of peaks from IgG1 Fc bearing a *N-Man5 N-glycan. Arrows indicate the direction of peak movement because of interactions with the Fc polypeptide. Peak labels that correspond to a figure of β-linked GlcNAc are shown (inset) and refer to the carbon position of the ¹H–¹³C peak. ¹J_C–C couplings are not resolved because of the limited resolution in the ¹³C dimension. (B) 1D ¹³C-observe NMR spectrum of *N-Man5 Fc. ¹J_C–C values are indicated. (C) 2D ¹H–¹⁵N HSQC spectra before and after N-glycan hydrolysis with the same colors used in panel A.

Figure 6

Overlay of ¹H–¹³C HSQC spectra collected with purified IgG1 Fc glycoforms that reveals shifts of peaks away from that of a hydrolyzed N-glycan. The positions of peaks from an Fc N-glycan following EndoF-catalyzed hydrolysis are indicated with X’s. Arrows show the direction of peak movement as the N-glycan matures. The C1 peak for the hydrolyzed glycan was obscured by residual water in the sample and was not observed.

Figure 7

Broad, low-intensity peaks appear in a ¹H–¹³C HSQC spectrum following Gnt2-catalyzed addition of a second [¹³C,¹⁵N]GlcNAc. The resonance assignments for the (5′)GlcNAc C3, C4, and C5 peaks were obtained by comparison to a report by Yamaguchi et al.

Figure 8

Structural model of the IgG1 Fc–N-glycan interface (based on Protein Data Bank entry 4ku1(71)). F243 directly contacts the (5′)GlcNAc residue. The (5)GlcNAc does not appear to make a direct contact with the polypeptide surface. Carbohydrate residues are shown in a stick model with the color of each residue corresponding to the key for each residue shown in Figure 1. Only ring atoms of the carbohydrates are shown for the sake of simplicity, and galactose residues present in the original Protein Data Bank model are not shown for the sake of clarity.