Chemoenzymatic synthesis and Fcγ receptor binding of homogeneous glycoforms of antibody Fc domain. Presence of a bisecting sugar moiety enhances the affinity of Fc to FcγIIIa receptor

Abstract

Structurally well-defined IgG-Fc glycoforms are highly demanded for understanding the effects of glycosylation on an antibody's effector functions. We report in this paper chemoenzymatic synthesis and Fcγ receptor binding of an array of homogeneous IgG-Fc glycoforms. The chemoenzymatic approach consists of the chemical synthesis of defined N-glycan oxazolines as donor substrates, the expression of the Fc domain in a CHO cell line in the presence of an α-mannosidase inhibitor kifunensine, and an endoglycosidase-catalyzed glycosylation of the deglycosylated Fc domain (GlcNAc-Fc homodimer) with the synthetic glycan oxazolines. The enzyme from Arthrobacter protophormiae (Endo-A) was found to be remarkably efficient to take various modified N-glycan core oxazolines, including the bisecting sugar-containing derivatives, for Fc glycosylation remodeling, resulting in the formation of the corresponding homogeneous Fc glycoforms. Nevertheless, neither Endo-A nor the Mucor hiemalis endoglycosidase mutants (EndoM-N175A and EndoM-N175Q) were able to transfer full-length complex-type N-glycan to the Fc domain, implicating the limitations of these two enzymes in Fc glycosylation remodeling. Surface plasmon resonance (SPR) binding studies with the synthetic IgG-Fc glycoforms unambiguously proved that the presence of a bisecting GlcNAc moiety could significantly enhance the binding of Fc to FcγRIIIa, the activating Fcγ receptor, independent of Fc core-fucosylation. Interestingly, the Fc glycoforms carrying an unusual bisecting sugar moiety such as a mannose or a LacNAc moiety also demonstrated enhanced affinity to FcγRIIIa. On the orther hand, the presence of a bisecting GlcNAc or core-fucosylation had little effect on the affinity of Fc to the inhibitory Fcγ receptor, FcγRIIb. Our experimental data also showed that the α-linked mannose residues in the pentasaccharide Man3GlcNAc2 core was essential to maintain a high affinity of Fc to both FcγRIIIa and FcγRIIb. The synthetic homogeneous Fc glycoforms thus provide a useful tool for elucidating how a fine Fc N-glycan structure precisely affects the function of the Fc domain.

Figures

Figure 1

Schematic presentations of the natural and synthetic human IgG1-Fc glycoforms. A) natural heterogeneous Fc glycforms; B) synthetic homogeneous Fc glycoforms. The IgG1-Fc structure was modeled on the basis of the crystal structure of an anti-HIV antibody b12 (PDB code, 1hzh) ( E. O. Saphire et al, Science, 2001, 293, 1155). GlcNAc, N-acetylglucosamine; Man, mannose; Gal, galactose; Glc, glucose; Fuc, L-fucose; Sia, sialic acid. The dash lines represent variable decorations.

Figure 2

Structures of synthetic sugar oxazolines

Figure 3

SDS-PAGE and MALDI-TOF MS analysis of recombinant IgG1-Fc. A, SDS-PAGE: Lane M, protein marker; lane 1, HM-Fc (non-reducing condition); lane 2, HM-Fc, reducing condition; lane 3, PNGase F-treatment of HM-Fc; B, MALDI-TOF MS of HM-Fc; C, MALDI-TOF MS of N-glycans released from HM-Fc; D, MALDI-TOF MS of N-glycans released from CHO-expressed Fc (CT-Fc).

Figure 4

SDS-PAGE and MALDI-TOF MS analysis of GlcNAc-Fc and transglycosylation product Fc-1. A, SDS-PAGE: Lane 1, HM-Fc; lane 2, GlcNAc-Fc; lane 3, transglycosylation product Fc-1; lane 4, PNGase F-treatment of Fc-1; B, MALDI-TOF MS of GlcNAc-Fc; C, MALDI-TOF MS of product Fc-1; D, MALDI-TOF MS of N-glycans released from Fc-1.

Figure 5

SPR sensorgrams of the binding of various Fc glycoforms to FcγIIIa receptor from a representative experiment. Fc glycoforms were immobilized by Protein A capture and the binding was analyzed by injecting the respective Fcγ receptors at varied concentrations.

Scheme 1

Synthesis of bisecting GlcNAc- and LacNAc-containing N-glycan oxazolines a aReagents and conditions: (a) BSP, TTBP, Tf2O, CH2Cl2, 56%. (b) 80% aq. AcOH, 80%. (c) TBDMSCl, pyridine, 95%. (d) BF3·OEt2, CH2Cl2, 76%. (e) TFA, CH2Cl2, 88%. (f) TMSOTf, CH2Cl2, 54%. (g) 1) NH2NH2 monohydrate, EtOH, H2O; 2) Ac2O, pyridine, 77% (2 steps). (h) 1) Pd(OH)2-C, H2, CH2Cl2, MeOH; 2) Ac2O, pyridine, 89% (2 steps). (i) TMSBr, BF3·OEt2, 2,4,6-collidine, CH2Cl2, 48%. (j) MeONa, MeOH, quantitative. (k) MeONa, MeOH, quantitative. (l) DMC, Et3N, H2O, quantitative. (m) UDP-Gal, β-1,4-galactosyltransferase, buffer, quantitative. (n) DMC, Et3N, H2O, quantitative.

Scheme 2

Synthesis of a glucose-containing N-glycan oxazoline a a Reagents and conditions: (a) NIS, TfOH, dichloroethane, 63%. (b) DDQ, CH2Cl2, H2O, 87%. (c) Et3SiH, PhBCl2, CH2Cl2, 94%. (d) TMSOTf, CH2Cl2, 95%. (e) AcSH, pyridine, CHCl3, 78%. (f) 1) MeONa, MeOH, then Dowex (H+); 2) Pd(OH)2/C, H2, MeOH; 3) Ac2O, pyridine, 92% (three steps). (g) 1) MeONa, MeOH, then Dowex (H+); 2) DMC, Et3N, H2O, 95% (two steps).

Scheme 3

Chemoenzymatic synthesis of homogeneous glycoforms of human IgG1-Fc a a Reagents and conditions. The high-mannose IgG1-Fc glycoform was produced in CHO cells in the presence of kifunensine. Then the high-mannose N-glycan was removed by Endo-H to give the GlcNAc-containing IgG1-Fc, which served as an acceptor for the Endo-A catalyzed transglycosylation with respective synthetic sugar oxazolines (1 to 6) to afford the corresponding homogeneous IgG1-Fc glycoforms.