Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Motooka 744, Nishi-ku, Fukuoka 819-0395, Japan.
J Biol Chem. 2019 Nov 8;294(45):17143-17154. doi: 10.1074/jbc.RA119.010842. Epub 2019 Sep 23.
-Linked glycans play important roles in various cellular and immunological events. Endo-β--acetylglucosaminidase (ENGase) can release or transglycosylate -glycans and is a promising tool for the chemoenzymatic synthesis of glycoproteins with homogeneously modified glycans. The ability of ENGases to act on core-fucosylated glycans is a key factor determining their therapeutic utility because mammalian -glycans are frequently α-1,6-fucosylated. Although the biochemistries and structures of various ENGases have been studied extensively, the structural basis for the recognition of the core fucose and the asparagine-linked GlcNAc is unclear. Herein, we determined the crystal structures of a core fucose-specific ENGase from the caterpillar fungus (Endo-CoM), which belongs to glycoside hydrolase family 18. Structures complexed with fucose-containing ligands were determined at 1.75-2.35 Å resolutions. The fucose moiety linked to GlcNAc is extensively recognized by protein residues in a round-shaped pocket, whereas the asparagine moiety linked to the GlcNAc is exposed to the solvent. The -glycan-binding cleft of Endo-CoM is Y-shaped, and several lysine and arginine residues are present at its terminal regions. These structural features were consistent with the activity of Endo-CoM on fucose-containing glycans on rituximab (IgG) and its preference for a sialobiantennary substrate. Comparisons with other ENGases provided structural insights into their core fucose tolerance and specificity. In particular, Endo-F3, a known core fucose-specific ENGase, has a similar fucose-binding pocket, but the surrounding residues are not shared with Endo-CoM. Our study provides a foothold for protein engineering to develop enzymatic tools for the preparation of more effective therapeutic antibodies.
连接聚糖在各种细胞和免疫学事件中发挥着重要作用。内-β-N-乙酰氨基葡萄糖苷酶（ENGase）可以释放或转移-β-聚糖，是化学酶法合成具有均匀修饰聚糖的糖蛋白的有前途的工具。ENGases 作用于核心岩藻糖基化聚糖的能力是决定其治疗效用的关键因素，因为哺乳动物的-β-聚糖经常是α-1,6-岩藻糖基化的。尽管已经广泛研究了各种 ENGases 的生物化学和结构，但识别核心岩藻糖和天冬酰胺连接的 GlcNAc 的结构基础尚不清楚。在此，我们确定了来自毛毛虫真菌（Endo-CoM）的核心岩藻糖特异性 ENGase 的晶体结构，它属于糖苷水解酶家族 18。用含岩藻糖的配体确定了结构复合物的分辨率为 1.75-2.35Å。与 GlcNAc 连接的岩藻糖部分被圆形口袋中的蛋白质残基广泛识别，而与 GlcNAc 连接的天冬酰胺部分暴露于溶剂中。Endo-CoM 的-β-聚糖结合裂缝呈 Y 形，并且在其末端区域存在几个赖氨酸和精氨酸残基。这些结构特征与 Endo-CoM 在利妥昔单抗（IgG）上含岩藻糖聚糖的活性及其对唾液酸双天线底物的偏好一致。与其他 ENGases 的比较为它们对核心岩藻糖的耐受性和特异性提供了结构上的见解。特别是，已知的核心岩藻糖特异性 ENGase Endo-F3 具有相似的岩藻糖结合口袋，但周围的残基与 Endo-CoM 不同。我们的研究为开发用于制备更有效治疗性抗体的酶工具的蛋白质工程提供了立足点。
