Giddens John P, Lomino Joseph V, Amin Mohammed N, Wang Lai-Xi
From the Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland 21201 and the Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742.
From the Institute of Human Virology, University of Maryland School of Medicine, Baltimore, Maryland 21201 and.
J Biol Chem. 2016 Apr 22;291(17):9356-70. doi: 10.1074/jbc.M116.721597. Epub 2016 Mar 10.
Chemoenzymatic synthesis is emerging as a promising approach to the synthesis of homogeneous glycopeptides and glycoproteins highly demanded for functional glycomics studies, but its generality relies on the availability of a range of enzymes with high catalytic efficiency and well defined substrate specificity. We describe in this paper the discovery of glycosynthase mutants derived from Elizabethkingia meningoseptica endoglycosidase F3 (Endo-F3) of the GH18 family, which are devoid of the inherent hydrolytic activity but are able to take glycan oxazolines for transglycosylation. Notably, the Endo-F3 D165A and D165Q mutants demonstrated high acceptorsubstrate specificity toward α1,6-fucosyl-GlcNAc-Asn or α1,6-fucosyl-GlcNAc-polypeptide in transglycosylation, enabling a highly convergent synthesis of core-fucosylated, complex CD52 glycopeptide antigen. The Endo-F3 mutants were able to use both bi- and triantennary glycan oxazolines as substrates for transglycosylation, in contrast to previously reported endoglycosidases derived from Endo-S, Endo-M, Endo-D, and Endo-A mutants that could not recognize triantennary N-glycans. Using rituximab as a model system, we have further demonstrated that the Endo-F3 mutants are highly efficient for glycosylation remodeling of monoclonal antibodies to produce homogeneous intact antibody glycoforms. Interestingly, the new triantennary glycan glycoform of antibody showed much higher affinity for galectin-3 than that of the commercial antibody. The Endo-F3 mutants represent the first endoglycosidase-based glycosynthases capable of transferring triantennary complex N-glycans, which would be very useful for glycoprotein synthesis and glycosylation remodeling of antibodies.
化学酶法合成正成为一种有前景的方法，用于合成功能糖组学研究中对均一糖肽和糖蛋白的大量需求，但它的通用性依赖于一系列具有高催化效率和明确底物特异性的酶的可用性。我们在本文中描述了从GH18家族的脑膜伊丽莎白菌内切糖苷酶F3（Endo-F3）衍生而来的糖基合酶突变体的发现，这些突变体没有固有的水解活性，但能够利用聚糖恶唑啉进行转糖基化。值得注意的是，Endo-F3 D165A和D165Q突变体在转糖基化中对α1,6-岩藻糖基-GlcNAc-Asn或α1,6-岩藻糖基-GlcNAc-多肽表现出高受体底物特异性，能够高度收敛地合成核心岩藻糖基化的复杂CD52糖肽抗原。与先前报道的源自Endo-S、Endo-M、Endo-D和Endo-A突变体的内切糖苷酶不同，Endo-F3突变体能够使用双天线和三天线聚糖恶唑啉作为转糖基化的底物，而这些内切糖苷酶无法识别三天线N-聚糖。以利妥昔单抗为模型系统，我们进一步证明Endo-F3突变体在单克隆抗体的糖基化重塑以产生均一完整抗体糖型方面非常高效。有趣的是，抗体的新三天线聚糖糖型对半乳糖凝集素-3的亲和力比商业抗体高得多。Endo-F3突变体代表了第一种基于内切糖苷酶的糖基合酶，能够转移三天线复杂N-聚糖，这对于糖蛋白合成和抗体的糖基化重塑将非常有用。
