The role of N-glycosylation on beta-sheet protein folding energetics

N-糖基化对β-折叠蛋白折叠能量学的作用

• 批准号: 7680779
• 负责人: Joshua L Price
• 金额: $ 4.72万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-18 至 2011-08-17
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7680779
• 关键词: Acetylglucosamine; Affect; Amides; Amino Acid Sequence; Cell surface; Cells; Chemicals; Disease; Event; Glycopeptides; Glycoproteins; Goals; Human; In Vitro; Kinetics; Length; Ligation; Light; Link; Lysosomal Storage Diseases; Malignant Neoplasms; Mannose; Measures; Mediating; Nitrogen; Peptides; Play; Polysaccharides; Post-Translational Protein Processing; Preparation; Protein Glycosylation; Protein Secretion; Proteins; Research; Role; Series; Side; Site; Sulfhydryl Compounds; Testing; Thermodynamics; Trisaccharides; Variant; Vertebral column; Work; analog; beta pleated sheet; chemical synthesis; functional group; glycosylation; insight; man; molecular recognition; mutant; premature; protein folding; public health relevance; research study; sugar; thioester

DESCRIPTION (provided by applicant): Glycosylation is one of the most common post-translational modifications of eukaryotic proteins. Glycans play diverse roles in molecular recognition events inside the cell and at the cell surface, and aberrant protein glycosylation is associated with a number of diseases, including cancer. In addition, much evidence suggests that N-glycans (attached to the amide nitrogen of an Asn side-chain) play crucial roles in mediating protein folding and secretion by directly increasing protein folding rates and stability. These roles are important because premature degradation of slowly folding or improperly folded proteins is related to the human lysosomal storage diseases. The long-term goal of the proposed research is to understand, in atomic detail, how N-linked glycosylation affects protein-folding energetics, and ultimately to shed light on the relationship between N-glycosylation and secretion efficiency. Specifically, the proposed work seeks to provide insight into how a single N-linked N-acetyl-D-glucosamine (GlcNAc) affects the folding energy landscape of the (3-sheet glycoprotein CD2ad in vitro. Pursuit of this goal will involve varying the identity and stereochemical configuration of the GlcNAc functional groups as well as the amino acid sequence of CD2ad and observing the resulting effect on the folding energy landscape of CD2ad. Such experiments will require the chemical synthesis of completely homogeneous CD2ad glycoforms which contain any desired sugar, not just GlcNAc at the glycosylation site. To that end, the proposed work involves the following specific aims: (1) synthesis of a series of thiolfunctionalized Asn-linked sugars which differ from GlcNAc in the identity and/or stereochemical configuration of their functional groups; (2) analysis of the ability of these thiol-functionalized Asn-linked sugars to facilitate the chemical ligation of short glycopeptides to peptide thioesters at ligation junctions that would be useful for full-length CD2ad synthesis; (3) preparation of monoglycosylated wild type and mutant CD2ad glycoforms by a combination of sugar-assisted and expressed protein ligation using the Asn-linked sugars and ligation junctions identified in (1 )-(2); and characterization and comparison of the folding energy landscape of CD2ad analogs, attributing changes in energetic parameters to specific protein/sugar contacts. PUBLIC HEALTH RELEVANCE The proposed research aims to provide insight into how protein glycosylation affects protein folding and stability. This work could ultimately shed light on the relationship between protein glycosylation and protein secretion efficiency, which is related to human lysosomal storage diseases.

描述（由申请人提供）：糖基化是真核蛋白最常见的翻译后修饰之一。聚糖在细胞内部和细胞表面的分子识别事件中起多种作用，并且异常的蛋白质糖基化与包括癌症在内的多种疾病有关。此外，许多证据表明，通过直接提高蛋白质折叠率和稳定性，N-聚糖（与ASN侧链的酰胺氮相连）在介导蛋白质折叠和分泌方面起着至关重要的作用。这些角色很重要，因为缓慢折叠或不当折叠蛋白的过早降解与人类溶酶体储存疾病有关。拟议的研究的长期目标是通过原子细节了解N连接的糖基化如何影响蛋白质折叠能量，并最终阐明N-糖基化和分泌效率之间的关系。具体而言，拟议的工作旨在提供有关单个N-连接的N-乙酰基-D-葡萄糖（GLCNAC）如何影响（体外3页糖蛋白CD2AD。追求该目标的折叠能量景观。对CD2AD的折叠能量景观的影响将需要化学合成完全均匀的CD2AD糖型，这些糖型包含任何所需的糖，而不仅仅是GlcNAC，而不仅仅是糖基化的糖基化。其功能组的立体化学构型； （3）通过使用ASN链接糖和（1） - （2）中鉴定的ASN链接糖和连接连接的组合，制备了单糖基化的野生型野生型和突变型CD2AD糖型，并通过糖辅助和表达的蛋白质连接组合制备；以及CD2AD类似物的折叠能景观的表征和比较，将能量参数的变化归因于特定的蛋白质/糖接触。公共卫生相关性拟议的研究旨在提供有关蛋白质糖基化如何影响蛋白质折叠和稳定性的见解。这项工作最终可以阐明蛋白质糖基化与蛋白质分泌效率之间的关系，这与人类溶酶体储存疾病有关。