Enzymatic Transglycosylation for N-Glycopeptide Synthesis

N-糖肽合成的酶促糖基转移

• 批准号: 7282571
• 负责人: LAI-XI WANG
• 金额: $ 21.85万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 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 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7282571
• 关键词: 4-methylumbelliferyl glycosides; Address; Antigens; Beta-N-Acetylglucosaminidase; Biological; Biological Assay; Cell Adhesion; Chemicals; Class; Complex; Coupled; DNA Shuffling; Development; Disaccharides; Doctor of Philosophy; Endoglycosidases; Energy Transfer; Engineering; Enzymes; Eukaryota; Eukaryotic Cell; Event; Facility Construction Funding Category; Fluorescence Resonance Energy Transfer; Genes; Glycopeptides; Glycoproteins; Glycosides; Goals; HIV Envelope Protein gp120; HIV-1; Heterogeneity; Hydrolysis; Immune response; Infection; Lead; Libraries; Link; Measures; Methods; Monosaccharides; Mutagenesis; Mutation; N acetylglucosaminidase; Neoplasm Metastasis; Oligosaccharides; Peptides; Pharmaceutical Preparations; Play; Polysaccharides; Post-Translational Protein Processing; Problem Solving; Proteins; Range; Recombinants; Research; Research Personnel; Role; Route; Screening procedure; Site-Directed Mutagenesis; Structure; Structure-Activity Relationship; Time; Trisaccharides; base; design; glycoprotein structure; glycosylation; glycosyltransferase; interest; mutant; novel; pathogen; programs; ribonuclease B; sugar; tumor

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）：拟研究的目标是开发高效和广泛应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 -链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、病原体感染和免疫应答等许多生物学事件中发挥重要作用。然而，对糖蛋白结构-功能关系的清晰理解往往受到其结构异质性的阻碍。天然糖蛋白通常是作为糖型的混合物产生的，这些糖型很难以纯形式分离出来。为了获得结构和生物学研究的均匀材料，我们建议系统地探索一类特殊的内糖苷酶，内切- β - n -乙酰氨基葡萄糖酶（ENGases）的反式糖基化活性，用于n -糖肽和n -糖蛋白的合成。一些engase具有独特的能力，可以将完整的低聚糖转移到含有glcnac的肽中，以区域和立体特异性的方式一步形成新的糖肽，从而为糖肽合成提供了高度收敛的途径。但该方法存在转糖基化产率低、供体底物受限、产物水解等严重问题。这些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。在特定目标1中，将合成并评估三种主要类型n -聚糖的一系列低聚糖供体底物，包括糖恶唑啉（假定的过渡态模拟物）和对硝基苯基苷（基态但动力学有利的底物）。具体目标2是通过筛选突变体文库，使用偶联酶实验和基于荧光共振能量转移（FRET）的实验，发现具有新的和/或增强的转糖基化活性的ENGase突变体。具体目标3侧重于几个大而复杂的HIV-1包膜糖蛋白片段的总合成，以及选定糖蛋白的半合成。从长远来看，所提出的研究将有助于糖蛋白类药物的开发。