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

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 Man alpha 1,3Man arm of the biantennary N-glycan. The glycan chain was further elongated by bacterial beta 1,4 galactosyltransferase from Neiserria meningitidis and human beta 1,4 galactosyl-transferase 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 Fc gamma IIIa receptor.

Automated summary

This study describes the first in vitro enzymatic sulfation of biantennary complex type N-glycans using recombinant human CHST2. The sulfotransferase demonstrated high antennary preference and selectively sulfated the GlcNAc moiety on the Man alpha 1,3Man arm of the biantennary N-glycan. The sulfated N-glycans were further elongated and transferred to intact antibodies using a chemoenzymatic method, resulting in homogeneous sulfated glycoforms. Sulfation did not affect the binding affinity of the antibody for Fc gamma IIIa receptor.