NMR characterization of immunoglobulin G Fc glycan motion on enzymatic sialylation

Abstract

The terminal carbohydrate residues of the N-glycan on the immunoglobulin G (IgG) fragment crystallizable (Fc) determine whether IgG activates pro- or anti-inflammatory receptors. The IgG Fc alone becomes potently anti-inflammatory upon addition of α2-6-linked N-acetylneuraminic acid residues to the N-glycan, stimulating interest in use of this entity in novel therapies for autoimmune disease [Kaneko et al. (2006) Science313, 670-3]. Complete Fc sialylation has, however, been deemed challenging due to a combination of branch specificity and perceived protection by glycan-protein interactions. Here we report the preparation of high levels of disialylated Fc by using sufficient amounts of a highly active α2-6 sialyltransferase (ST6Gal1) preparation expressed in a transiently transformed human cell culture. Surprisingly, ST6Gal1 sialylated the two termini of the complex-type binantennary glycan in a manner remarkably similar to that observed for the free N-glycan, suggesting the Fc polypeptide does not greatly influence ST6Gal1 specificity. In addition, sialylation of either branch terminus does not appear to dramatically alter the motional behavior of the N-glycan as judged by solution NMR spectroscopy. Together these, data suggest the N-glycan occupies two distinct states: one with both glycan termini sequestered from enzymatic modification by an α1-6Man-branch interaction with the polypeptide surface and the other with both glycan termini exposed to the bulk solvent and free from glycan-polypeptide interactions. The results suggest new modes by which disialylated Fc can act as an anti-inflammatory effector.

Figure 1

The IgG N-glycan, attached to Asn297 of the Fc heavy chain (A), is required for Fc-mediated signaling. (B) Though the composition of the Fc glycan in serum is heterogeneous, there is remarkably little variability in the types of inter-residue linkages observed. Note the mannose residues (green circles) are attached with either an α1–3 or α1–6 linkage which denotes the identity of each glycan branch built upon those residues. The symbols for the individual carbohydrate residues follow the Consortium for Functional Glycomics standard (39). (C) Each of these three Fc glycoforms was studied here. “α1–3” refers to the α1–3Man-branch of the biantennary glycan as defined in the text. (D) Chemical structure of the glycan branch termini showing (from left to right) an N-acetylneuraminic acid α2–6 linked to a Gal residue β1–4 linked to an N-acetylglucosamine residue. Carbon positions enriched with ¹³C nuclei are shown with red type. Glycans were enriched with either [¹³C_U]- or [¹³C₂]-Gal.

Figure 2

MALDI-MS spectrum of the liberated and permethylated N-glycan following Fc sialylation using GFP-ST6Gal1. Primarily monosialylated (A) or disialylated (B) material was prepared depending on the amount of enzyme used and the length of incubation.

Figure 3

Purification of GFP-ST6Gal1 from transiently transformed human HEK293F cells. (A) Gel filtration chromatograph of GFP-ST6Gal1 followed initial purification by immobilized metal ion (Ni²⁺) chromatography. (B) SDS-PAGE gel of the purification steps and final purified material.

Figure 4

Plots of the accumulation of monosialyl and disialyl Fc in the presence of GFP-ST6Gal1 and CMP-N-acetylneuraminic acid were fitted to reveal the relative rates of sialylation on each glycan branch terminus. The reaction with 0.05 mg / mL (A) or 0.5 mg / mL (B) GFP-ST6Gal1 was monitored by MALDI-MS. By fitting these data to an equation that describes the accumulation of product according to a sequential addition model, the rate of the addition of the first residue was found to be ~11-fold faster than the rate of the second residue. This result is similar to the 9-fold difference observed when reanalyzing similar data for the sialylation of free N-glycan using bovine ST6Gal1 (18) or 8-fold for human ST6Gal1 (16).

Figure 5

NMR spectroscopy of terminal Gal and N-acetylneuraminic acid residues of the Fc-conjugated N-glycan shows distinct ¹H-¹³C correlations. (A) [¹³C_U]-Gal resonances observed in a ¹³C-HSQC spectrum of Gal-terminated Fc. (B) A similar experiment using Fc with a [¹³C_1,2,3]-N-acetylneuraminic acid residue attached to the (α1–3Man-branch)-Gal residue. (C) ¹³C-HSQC spectrum of di[¹³C_U]-Gal di[¹³C_1,2,3]-N-acetylneuraminic acid Fc. (D) and (E) ¹³C-HSQC spectrum of the ¹³C₃ N-acetylneuraminic acid region for the material in (B) and (C), respectively. “α1–3” and “α1–6” refer to the Man residue at the base of the glycan branch to which the residue is attached.

Figure 6

The two glycan branch termini behave differently according to NMR relaxation measurements of the Fc-conjugated N-glycan. The decay of signal intensity in Carr-Purcell R₂ relaxation experiments specific for [¹³C₂]-Gal (A–B) and [¹³C₃]-N-acetylneuraminic acid (C–D) nuclei is shown. Measurements were performed with both monosialyl-Fc (A and C) and disialyl Fc (B and D). The effect of ¹³C₃ - ¹³C₂ coupling during the ¹³C₃ N-acetylneuraminic acid measurements was removed by inverting the ¹³C₂ spin with a selective pulse coincident with ¹H decoupling. “SA” is shorthand notation for N-acetylneuraminic acid.