糖蛋白合成的研究进展

doi:10.12211/2096-8280.2024-018

摘要/Abstract

摘要：

糖基化修饰在各类生理活动中起着非常关键的作用。然而，天然糖蛋白中糖型结构的微观异质性，严重阻碍了糖型结构与糖蛋白功能之间构效关系的研究。因此，从分子层面精确控制糖蛋白结构的化学合成方法是解决这一难题的重要手段。随着蛋白质合成和糖工程技术的不断进步，研究者们发展多种蛋白质连接策略和糖苷键构建策略来精确合成均质糖蛋白。本综述将从化学、酶法构建糖苷键的角度梳理现有的糖蛋白全合成以及结合生物表达方法的半合成策略，分别介绍这些策略在合成带有N-糖基化、O-糖基化修饰等不同糖基化类型的复杂均质糖蛋白时所取得的成果，例如带有多个复杂N-糖基化修饰的HSV gD和含有长疏水片段的IL-2；以及运用这些合成的复杂糖蛋白了解糖基化修饰在各项生理过程中的构效关系所作出的突破，例如抗原糖蛋白中糖链长短与免疫原性的关系、O-GlcNAc调节神经元突触功能的机制等。最后总结了现有合成策略在糖苷键构建、纯化策略、蛋白溶解度等方面取得的进展，并指出选择性和合成产率的进一步优化仍是糖蛋白合成领域亟待解决的问题，而糖蛋白合成技术在开发免疫疗法以及理解各种疾病分子机理等方面的广泛应用，从理解原理到开发产品方面拓展了合成科学在生命健康领域的发展方向。

关键词: 糖蛋白, 糖肽, 构效关系, 化学合成, 化学酶法合成

Abstract:

Glycosylation modifications, extensively present on the surfaces of eukaryotic proteins as a type of post-translational modification, hold significant physiological and pathological implications. The microscopic heterogeneity of natural glycoproteins has led to the emergence of the chemical synthesis of homogeneous glycoproteins with defined structures as a crucial frontier in exploring the structure-function relationships of glycosylation modifications. With the flourishing development of protein synthesis and glycoengineering technologies, various protein ligation and polysaccharide synthesis strategies have been developed, enabling the preparation of glycoproteins containing hundreds of amino acid residues. The development of glycoprotein synthesis strategies primarily revolves around chemical and enzymatic approaches for glycosidic bond formation, leading to effective synthesis schemes such as Lansbury’s aspartic acid acylation, chemical strategies based on glycosyl amino acid building blocks, and glycan remodeling strategies using endoglycosidases and glycosyltransferases. This review will discuss the chemical and enzymatic construction of glycosidic bonds, examining existing strategies for the total synthesis of glycoproteins and semi-synthetic approaches that combine with biological expression methods. It will introduce these strategies’ achievements in synthesizing complex homogeneous glycoproteins with different types of glycosylation modifications, such as those with multiple complex N-glycosylation modifications like HSV gD and those containing long hydrophobic segments like IL-2. Additionally, this review will highlight breakthroughs in understanding the structure-function relationships of glycosylation modifications in various physiological processes through these synthetic complex glycoproteins, including the relationship between glycan chain length and immunogenicity in antigenic glycoproteins, and the mechanisms by which O-GlcNAc regulates synaptic function in neurons. Finally, it will summarize the progress made in glycosidic bond construction, purification strategies, and protein solubility, and point out that further optimization of selectivity and synthetic yield remains a pressing issue in the field of glycoprotein synthesis. The wide application of glycoprotein synthesis technology in developing immunotherapies and understanding the molecular mechanisms of various diseases expands the development directions of synthetic science in the field of life and health, from understanding principles to developing products.

Key words: glycoprotein, glycopeptide, structure-activity relationship, chemical synthesis, chemoenzymatic synthesis

中图分类号:

Q816

杨皓然, 叶发荣, 黄平, 王平. 糖蛋白合成的研究进展[J]. 合成生物学, DOI: 10.12211/2096-8280.2024-018.

Haoran YANG, Farong YE, Ping HUANG, Ping WANG. Recent advances in glycoprotein synthesis[J]. Synthetic Biology Journal, DOI: 10.12211/2096-8280.2024-018.

图/表 20

图1 N-连接糖基化修饰的特点与N-连接糖肽主要的化学合成方法

Fig.1 Characteristics of N-linked glycosylation modifications and the main chemical synthesis methods of N-linked glycopeptides

图2 假脯氨酸二肽策略解决汇聚法的天冬酰胺环化问题

Fig.2 Pseudoproline dipeptide strategy for resolving the aspartimide formation in convergent approach

图3 硒酯天冬氨酸活性酯改良方案

Fig.3 Improved scheme for selenoesterification in activating aspartate

图4 硫代酰胺定向激活天冬氨酸策略

Fig.4 Thioamide-directed activation strategy for aspartate

图5 基于表达蛋白连接（EPL）的半合成策略在复杂糖蛋白合成中的应用

Fig. 5 Application of semisynthetic strategy based on expressed protein ligation (EPL) in the synthesis of complex glycoproteins

图6 双功能硫代酸介导策略在白细胞介素-3（IL-3）全合成中的应用

Fig.6 Application of bifunctional thioacid mediated strategy in the total synthesis of interleukin-3 (IL-3)

图7 O-糖基化的存在形式和化学合成策略

Fig.7 Forms of O-glycosylation and chemical synthesis strategies

图8 白细胞介素-2（IL-2）的全合成与半合成策略

Fig.8 Total and semisynthetic strategies for interleukin-2 (IL-2)

图9 钯催化交叉偶联的SN2-糖基化策略

Fig. 9 Palladium catalysis cross-coupling-like SN2-glycosylation strategy

图10 O-糖基化的存在形式和化学合成策略

Fig.10 Forms of O-glycosylation and chemical synthesis strategies

图11 各种合成策略在O-GlcNAc修饰糖蛋白构效关系研究中的应用

Fig.11 Application of various synthesis strategies in study of structure-activity relationships of O-GlcNAc modified glycoproteins

图12 使用丝氨酸/苏氨酸连接策略全合成糖基化脂肪连蛋白胶原结构域（glyACD）（表格及糖基化羟基赖氨酸结构摘自文献［83］）

Fig.12 Total synthesis of glycosylated adiponectin collagen domain (glyACD) using serine/threonine ligation strategy(Table and structure of glycosylated hydroxylysine excerpted from literature [83])

图13 酶法合成糖蛋白概览

Fig.13 Overview of enzymatic synthesis of glycoproteins

图14 ENGase及其突变体催化糖苷键水解和转糖基化的作用机理

Fig.14 Catalytic mechanism of hydrolysis and transglycosylation via ENGase and its mutants

图15 内切糖苷酶在抗体修饰和功能化中的应用

Fig.15 Application of endoglycosidases in antibodies modification and functionalization

图16 内切糖苷酶快速构建含有复杂糖型的糖蛋白［35］

Fig.16 Endoglycosidase-mediated strategy for rapid construction of glycoproteins with complex glycan structures[35]

图17 糖基转移酶构建带有硫酸化修饰的复杂N-糖基化抗体

Fig.17 Glycosyltransferase-mediated construction of complex N-glycosylated antibodies with sulfation modifications

图18 一锅两酶法（OP2E）快速构建二糖肽

Fig.18 One-pot two-enzyme method (OP2E) for rapid construction of disaccharide peptides

图19 带有Tn、T、Sn糖型的MUC1糖蛋白的化学酶法合成

Fig.19 Chemoenzymatic synthesis of MUC1 glycoproteins with Tn, T, and STn glycoform

图20 O-GlcNAcylation辅助白介素-5（IL-5）折叠复性策略

Fig.20 O-GlcNAcylation-assisted folding and refolding strategy for interleukin-5 (IL-5)

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