Enzymatic Transglycosylation for N-Glycopeptide Synthesis

N-糖肽合成的酶促糖基转移

• 批准号: 7146431
• 负责人: LAI-XI WANG
• 金额: $ 22.28万
• 依托单位: UNIVERSITY OF MD BIOTECHNOLOGY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7146431
• 关键词: HIV envelope protein; N glycosidase; bioengineering /biomedical engineering; chemical kinetics; enzyme activity; enzyme linked immunosorbent assay; enzyme substrate; fluorescence resonance energy transfer; genetic library; glycopeptides; glycosides; glycosylation; method development; mutant; oligosaccharides; peptide chemical synthesis; posttranslational modifications; protein engineering

DESCRIPTION (provided by applicant): The goal of the proposed research is to develop efficient and widely applicable chemoenzymatic methods for synthesizing N-glycopeptides and N-glycoproteins of biomedical significance. N-linked glycosylation is one of the most common posttranslational modifications of proteins in eukaryotes. Glycoproteins play important roles in many biological events such as cell adhesion, tumor metastasis, pathogen infection, and immune response. However, a clear understanding of the structure-function relationships of glycoproteins is often hampered by their structural heterogeneity. Natural glycoproteins are usually produced as a mixture of glycoforms that are extremely difficult to isolate in pure form. To obtain homogeneous materials for structural and biological studies, we propose to systematically explore the trans-glycosylation activity of a special class of endoglycosidases, the endo-beta-N-acetylglucosaminidases (ENGases), for N-glycopeptide and N-glycoprotein synthesis. Some ENGases possess unique ability to transfer an intact oligosaccharide to a GlcNAc-containing peptide in a single step to form a new glycopeptide in a regio- and stereo-specific manner, thus providing a highly convergent route to glycopeptide synthesis. But the method suffers with serious problems such as the low yield in transglycosylation, the limitation of donor substrates, and the product hydrolysis. The proposed studies intend to solve these problems by exploring new substrates and screening new enzyme mutants. Three specific aims are proposed to achieve the goal. In specific aim 1, a range of oligosaccharide donor substrates, including sugar oxazolines (presumed transition state mimics) and p-nitrophenyl glycosides (ground-state but kinetically favorable substrates) of three major types of N-glycans will be synthesized and evaluated. The specific aim 2 is to discover ENGase mutants with new and/or enhanced transglycosylation activity by screening mutant library, using the coupled enzyme assay and the fluorescent resonance energy transfer (FRET)-based assay. Specific aim 3 focuses on total synthesis of several large and complex HIV-1 envelope glycoprotein fragments, as well as semi- synthesis of selected glycoproteins. In the long term, the proposed studies will contribute to the development of glycoprotein-based drugs.

描述（由申请人提供）：本研究的目标是开发高效且广泛适用的化学酶方法来合成具有生物医学意义的N-糖肽和N-糖蛋白。 N-连接糖基化是真核生物中最常见的蛋白质翻译后修饰之一。糖蛋白在细胞粘附、肿瘤转移、病原体感染和免疫反应等许多生物事件中发挥着重要作用。然而，对糖蛋白结构与功能关系的清晰理解常常因其结构异质性而受到阻碍。天然糖蛋白通常以糖形式的混合物形式产生，极难以纯形式分离。为了获得用于结构和生物学研究的均质材料，我们建议系统地探索一类特殊的内切糖苷酶，内切-β-N-乙酰氨基葡萄糖苷酶（ENGases）的转糖基化活性，用于 N-糖肽和 N-糖蛋白的合成。一些 ENGase 具有独特的能力，可一步将完整寡糖转移至含 GlcNAc 的肽，以区域和立体特异性方式形成新的糖肽，从而为糖肽合成提供高度收敛的途径。但该方法存在糖基转移收率低、供体底物限制、产物水解等严重问题。拟议的研究旨在通过探索新的底物和筛选新的酶突变体来解决这些问题。为实现这一目标，提出了三个具体目标。在具体目标 1 中，将合成和评估一系列寡糖供体底物，包括糖恶唑啉（假定的过渡态模拟物）和三种主要类型 N-聚糖的对硝基苯基糖苷（基态但动力学有利的底物）。具体目标 2 是通过使用偶联酶测定和基于荧光共振能量转移 (FRET) 的测定筛选突变体库，发现具有新的和/或增强的转糖基活性的 ENGase 突变体。具体目标 3 侧重于几个大而复杂的 HIV-1 包膜糖蛋白片段的全合成，以及选定糖蛋白的半合成。从长远来看，拟议的研究将有助于糖蛋白药物的开发。