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

描述(申请人提供)：糖基化是真核蛋白质最常见的翻译后修饰之一。多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，蛋白糖基化异常与包括癌症在内的许多疾病有关。此外，许多证据表明，N-葡聚糖(附着在ASN侧链的酰胺氮上)通过直接增加蛋白质的折叠速度和稳定性，在调节蛋白质折叠和分泌方面发挥着至关重要的作用。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储存疾病有关。这项拟议研究的长期目标是在原子细节上了解N-连接的糖基化如何影响蛋白质折叠能量，并最终阐明N-糖基化与分泌效率之间的关系。具体地说，这项拟议的工作旨在深入了解单个N-连接的N-乙酰-D-氨基葡萄糖(GlcNAc)在体外如何影响(3-折叠糖蛋白CD2ad)的折叠能量格局。为了实现这一目标，需要改变GlcNAc官能团的特性和立体化学构型以及CD2ad的氨基酸序列，并观察由此对CD2ad折叠能量格局的影响。这样的实验将需要完全均一的CD2ad糖形的化学合成，这些糖形包含任何所需的糖，而不仅仅是糖基化位点的GlcNAc。为此，拟议的工作涉及以下具体目标：(1)合成一系列在功能基团的同一性和/或立体化学构型上与GlcNAc不同的硫代功能化Asn连接糖；(2)分析这些硫醇功能化Asn连接糖促进连接连接处的短糖肽与多肽硫酯的化学连接的能力，这将有助于全长CD2ad的合成；(3)利用(1)-(2)中确定的糖辅助和表达的蛋白质连接的组合，制备单糖化的野生型和突变型CD2ad糖型；以及CD2ad类似物折叠能量图景的表征和比较，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，这与人类溶酶体储存疾病有关。

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.