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. 2022 Nov:128:106070.
doi: 10.1016/j.bioorg.2022.106070. Epub 2022 Aug 1.

Site-selective sulfation of N-glycans by human GlcNAc-6-O-sulfotransferase 1 (CHST2) and chemoenzymatic synthesis of sulfated antibody glycoforms

Kun Huang  1 , Chao Li  1 , Guanghui Zong  1 , Sunaina Kiran Prabhu  1 , Digantkumar G Chapla  2 , Kelley W Moremen  2 , Lai-Xi Wang  3
Affiliations

Site-selective sulfation of N-glycans by human GlcNAc-6-O-sulfotransferase 1 (CHST2) and chemoenzymatic synthesis of sulfated antibody glycoforms

Kun Huang et al. Bioorg Chem. 2022 Nov.

Abstract

Sulfation is a common modification of glycans and glycoproteins. Sulfated N-glycans have been identified in various glycoproteins and implicated for biological functions, but in vitro synthesis of structurally well-defined full length sulfated N-glycans remains to be described. We report here the first in vitro enzymatic sulfation of biantennary complex type N-glycans by recombinant human CHST2 (GlcNAc-6-O-sulfotransferase 1, GlcNAc6ST-1). We found that the sulfotransferase showed high antennary preference and could selectively sulfate the GlcNAc moiety located on the Manα1,3Man arm of the biantennary N-glycan. The glycan chain was further elongated by bacterial β1,4 galactosyltransferase from Neiserria meningitidis and human β1,4 galactosyltransferase IV（B4GALT4）, which led to the formation of different sulfated N-glycans. Using rituximab as a model IgG antibody, we further demonstrated that the sulfated N-glycans could be efficiently transferred to an intact antibody by using a chemoenzymatic Fc glycan remodeling method, providing homogeneous sulfated glycoforms of antibodies. Preliminary binding analysis indicated that sulfation did not affect the apparent affinity of the antibody for FcγIIIa receptor.

Keywords: Chemoenzymatic synthesis; Glycoforms; Sulfated N-glycans; Sulfated antibody; Sulfation; Sulfotransferase.

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Conflict of interest statement

Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1.
Fig. 1.
LC-ESI-MS analysis of glycoengineered rituximab. (a) Intact antibody 9. (b) Intact antibody 10. (c) Intact antibody 11. (d) Intact antibody 12. The spectra are the deconvoluted spectra.
Fig. 2.
Fig. 2.
SPR sensorgrams of the binding of FcγRIIIa-V158 with different glycoforms of rituximab. A) G0F-rituximab (12); B) Sulfated G0F-rituximab (9); C) G2F-rituximab; D) Sulfated G2F-rituximab (11).
Scheme 1.
Scheme 1.
Chemoenzymatic synthesis of sulfated N-glycans.
Scheme 2.
Scheme 2.
Synthesis of sulfated N-glycan oxazolines.
Scheme 3.
Scheme 3.
Chemoenzymatic synthesis of sulfated glycoforms of rituximab.
See this image and copyright information in PMC

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