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

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。聚糖在细胞内和细胞表面的分子识别事件中发挥着不同的作用，异常的蛋白质糖基化与许多疾病相关，包括癌症。此外，许多证据表明，N-聚糖（连接到Asn侧链的酰胺氮上）通过直接增加蛋白质折叠速率和稳定性在介导蛋白质折叠和分泌中发挥关键作用。这些作用是重要的，因为缓慢折叠或不正确折叠的蛋白质的过早降解与人类溶酶体贮积病有关。拟议研究的长期目标是在原子细节上了解N-连接糖基化如何影响蛋白质折叠能量学，并最终阐明N-糖基化与分泌效率之间的关系。具体而言，所提出的工作旨在提供对单个N-连接的N-乙酰基-D-葡糖胺（GlcNAc）如何影响体外β-折叠糖蛋白CD 2ad的折叠能量景观的深入了解。追求这一目标将涉及改变GlcNAc官能团的身份和立体化学构型以及CD 2ad的氨基酸序列，并观察对CD 2ad的折叠能量景观的影响。此类实验将需要化学合成完全均质的CD 2ad糖型，其含有任何所需的糖，而不仅仅是糖基化位点处的GlcNAc。（1）合成一系列与GlcNAc不同的巯基化的天冬酰胺连接糖，这些糖的官能团的特性和/或立体化学构型与GlcNAc不同;（2）分析了巯基化的Asn-连接的糖，以促进短糖肽与肽硫酯在连接点处的化学连接，这将对全-长度的CD 2ad合成;（3）通过糖辅助的和表达的蛋白质连接的组合，使用（1）-（2）中鉴定的Asn连接的糖和连接点制备单糖基化的野生型和突变的CD 2ad糖型;以及表征和比较CD 2ad类似物的折叠能量景观，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深入了解蛋白质糖基化如何影响蛋白质折叠和稳定性。这项工作可能最终阐明蛋白质糖基化和蛋白质分泌效率之间的关系，这与人类溶酶体贮积病有关。

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.