Department of Chemistry and Biochemistry, University of Maryland, 8051 Regents Drive, College Park, MD 20742, United States.
Complex Carbohydrate Research Center, University of Georgia, Athens 30602, Georgia.
Bioorg Chem. 2022 Nov;128:106070. doi: 10.1016/j.bioorg.2022.106070. Epub 2022 Aug 1.
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.
硫酸化是糖和糖蛋白的常见修饰方式。各种糖蛋白中都已鉴定出硫酸化 N-聚糖，并推测其具有生物学功能，但结构明确的全长硫酸化 N-聚糖的体外合成仍有待描述。我们在此报告首次通过重组人 CHST2（GlcNAc-6-O-磺基转移酶 1，GlcNAc6ST-1）对双天线复合型 N-聚糖进行体外酶促硫酸化。我们发现该磺基转移酶表现出高度的天线偏好性，可选择性地对双天线 N-聚糖的 Manα1,3Man 臂上的 GlcNAc 部分进行硫酸化。糖链进一步由脑膜炎奈瑟氏菌的细菌β1,4 半乳糖基转移酶和人β1,4 半乳糖基转移酶 IV（B4GALT4）延伸，导致形成不同的硫酸化 N-聚糖。使用利妥昔单抗作为模型 IgG 抗体，我们进一步证明了通过化学酶 Fc 聚糖重塑方法可以将硫酸化 N-聚糖有效地转移到完整抗体上，从而提供了抗体的同质硫酸化糖型。初步的结合分析表明，硫酸化不会影响抗体对 FcγIIIa 受体的表观亲和力。
