Mechanism of the Protein Specific Polysialylation of NCAM

NCAM 蛋白质特异性多唾液酸化机制

• 批准号: 8370587
• 负责人: Joseph Louis Zapater
• 金额: $ 4.72万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8370587
• 关键词: Adhesives; Affect; Amino Acid Sequence; Amino Acids; Antibodies; Binding; Biological Assay; Body part; Calorimetry; Cell Adhesion; Cell Adhesion Molecules; Cell Communication; Cells; Chimera organism; Co-Immunoprecipitations; Complex; Crystallization; Data; Densitometry; Development; Enzymes; Fibronectins; Fluorescence-Activated Cell Sorting; Foundations; Glycoproteins; Goals; Growth; Human; Immunoblotting; Immunofluorescence Microscopy; In Vitro; Knowledge; Laboratories; Lead; Length; Link; Literature; Lung; Malignant Epithelial Cell; Mediating; Membrane; Modification; Molecular; Mono-S; Mutagenesis; Nerve Regeneration; Nervous system structure; Neural Cell Adhesion Molecules; Neurons; Peptides; Polysaccharides; Polysialic Acid; Process; Proteins; Research; Role; Series; Sialic Acids; Sialyltransferases; Staging; Structure; Surface; Testing; Therapeutic Agents; Titrations; Western Blotting; Work; base; cancer cell; design; insight; polymerization; prevent; protein protein interaction; reconstitution; research study; therapeutic development; therapeutic target; tumor

DESCRIPTION (provided by applicant): ST8Sia IV (PST) is an 12,8-sialyltransferase that catalyzes the polysialylation of a limited number of substrate glycoproteins, with the neural cell adhesion molecule (NCAM) being the primary substrate. Polysialic acid addition to NCAM modulates cell adhesion by acting as an anti-adhesive that prevents cell interactions mediated by NCAM and other nearby adhesion molecules. Previous studies have demonstrated that polysialylated NCAM is absolutely necessary for proper development and plasticity of the mammalian nervous system and the regeneration of damaged neurons. Interestingly, polysialylated NCAM has been found to be aberrantly expressed in several human tumors, where it promotes their growth and invasiveness. Despite our knowledge of the important beneficial and detrimental effects of NCAM polysialylation, the mechanism by which PST recognizes and polysialylates NCAM is not well understood. Results from several laboratories lead to the hypothesis that polysialylation of NCAM by PST is a highly protein-specific process requiring an initial recognition step that is mediated through PST-NCAM protein-protein interactions. The overall goal of the proposed research is to further test this hypothesis and elucidate the mechanism of NCAM protein-specific polysialylation by identifying the amino acid sequences and residues within PST that are responsible for recognition and binding of NCAM. In Specific Aim I, competition, mutagenesis/reconstitution, and binding studies will be used to identify sequences in PST required for NCAM recognition. Preliminary results have demonstrated that a truncated, catalytically inactive PST protein consisting of amino acids 1-140 acts as a strong competitor of SW2 cell NCAM polysialylation. The ability of a series of further truncated catalytically inactive PST proteins to compete with endogenous PST in SW2 small cell lung carcinoma cells and block NCAM polysialylation will be evaluated using the anti-polysialic acid antibody, OL.28, immunoblotting and FACS analysis. PST sequences that function as effective competitors will further be tested for their ability to bind to NCAM using co-immunoprecipitation and isothermal titration calorimetry analyses. Those PST sequences that bind NCAM will be replaced in PST and inserted into a related 12,8-sialyltransferase, ST8Sia III, to determine whether they are necessary and sufficient for NCAM recognition leading to polysialylation. In Specific Aim II, structural analyses will be performed to identify amino acid residues within PST and those in NCAM that mediate the PST-NCAM interaction. The goal of this aim is to obtain a co-crystal structure of a PST peptide with the recently crystallized NCAM Ig5-FN1 fragment (minimal domain required for polysialylation by PST), and to initiate experiments directed at obtaining the crystal structure of soluble PST. Accomplishment of these aims will provide greater insight into the mechanism of NCAM polysialylation by PST and may serve as the basis for the development of therapeutic agents targeting the PST-NCAM interaction.

描述（由申请人提供）：ST 8 Sia IV（PST）是一种12，8-唾液酸转移酶，可催化有限数量的底物糖蛋白的聚唾液酸化，神经细胞粘附分子（NCAM）是主要底物。向NCAM中添加聚唾液酸通过充当抗粘附剂来调节细胞粘附，所述抗粘附剂防止由NCAM和其它附近粘附分子介导的细胞相互作用。先前的研究已经证明，多唾液酸化的NCAM对于哺乳动物神经系统的正常发育和可塑性以及受损神经元的再生是绝对必要的。有趣的是，已经发现多唾液酸化NCAM在几种人类肿瘤中异常表达，在那里它促进它们的生长和侵袭性。尽管我们知道NCAM多聚唾液酸化的重要有益和有害作用，但PST识别和多聚唾液酸化NCAM的机制还没有很好地理解。来自几个实验室的结果导致了这样的假设，即PST对NCAM的聚唾液酸化是一种高度蛋白特异性的过程，需要通过PST-NCAM蛋白-蛋白相互作用介导的初始识别步骤。拟议研究的总体目标是进一步验证这一假设，并通过鉴定PST内负责识别和结合NCAM的氨基酸序列和残基来阐明NCAM蛋白特异性聚唾液酸化的机制。在特定目标I中，竞争、诱变/重建和结合研究将用于鉴定NCAM识别所需的PST序列。初步结果表明，由氨基酸1-140组成的截短的无催化活性的PST蛋白充当SW 2细胞NCAM多聚唾液酸化的强竞争者。将使用抗聚唾液酸抗体OL. 28、免疫印迹和FACS分析评估一系列进一步截短的无催化活性的PST蛋白与SW 2小细胞肺癌细胞中的内源性PST竞争并阻断NCAM聚唾液酸化的能力。将使用免疫共沉淀和等温滴定量热法分析进一步测试作为有效竞争者的PST序列与NCAM结合的能力。结合NCAM的那些PST序列将在PST中被替换并插入相关的12，8-唾液酸转移酶ST 8 Sia III中，以确定它们是否是NCAM识别所必需和足够的，从而导致聚唾液酸化。在特定目标II中，将进行结构分析，以鉴定PST内的氨基酸残基和NCAM中介导PST-NCAM相互作用的氨基酸残基。该目的的目标是获得PST肽与最近结晶的NCAM Ig 5-FN 1片段（PST聚唾液酸化所需的最小结构域）的共晶体结构，并启动旨在获得可溶性PST晶体结构的实验。这些目标的实现将为PST对NCAM多聚唾液酸化的机制提供更深入的了解，并可作为开发靶向PST-NCAM相互作用的治疗剂的基础。