Mechanism of the Protein Specific Polysialylation of NCAM

描述(申请人提供)：ST8Sia IV(PST)是一种12，8-唾液酸基转移酶，催化有限数量的底物糖蛋白的多唾液酸化，以神经细胞黏附分子(NCAM)为主要底物。添加到NCAM中的聚唾液酸作为一种抗黏附作用来调节细胞黏附，阻止NCAM和其他邻近黏附分子介导的细胞相互作用。先前的研究表明，聚唾液酸化的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，多唾液酸化的NCAM已被发现在几种人类肿瘤中异常表达，促进其生长和侵袭性。尽管我们知道NCAM多唾液酸化的重要的有益和有害的影响，但PST识别和多唾液酸化NCAM的机制还不是很清楚。几个实验室的结果导致了这样的假设，即PST对NCAM的多唾液酸化是一个高度蛋白质特异性的过程，需要通过PST-NCAM蛋白质-蛋白质相互作用介导的初始识别步骤。这项研究的总体目标是进一步验证这一假说，并通过鉴定PST中负责识别和结合NCAM的氨基酸序列和残基来阐明NCAM蛋白特异性多唾液酸化的机制。在具体目标I中，将使用竞争、突变/重组和结合研究来鉴定识别NCAM所需的PST中的序列。初步结果表明，由1-140个氨基酸组成的截短的、催化失活的PST蛋白是SW2细胞NCAM聚唾液酸化的有力竞争者。进一步截短的一系列催化失活的PST蛋白在SW2小细胞肺癌细胞中与内源性PST竞争并阻断NCAM多唾液酸化的能力将通过抗多唾液酸抗体、OL.28、免疫印迹和FACS分析来评估。作为有效竞争者的PST序列将使用共免疫沉淀和等温滴定热量分析进一步测试其与NCAM结合的能力。这些与NCAM结合的PST序列将被替换在PST中，并插入到相关的12，8-唾液酸基转移酶ST8Sia III中，以确定它们是否对导致多唾液酸化的NCAM识别是必要的和充分的。在特定目标II中，将进行结构分析，以确定PST中的氨基酸残基和NCAM中介导PST-NCAM相互作用的氨基酸残基。其目的是获得PST多肽与最近结晶的NCAM Ig5-FN1片段(PST聚唾液酸化所需的最小结构域)的共晶结构，并启动针对获得可溶性PST晶体结构的实验。这些目标的实现将使人们对PST聚合NCAM的机制有更深入的了解，并可能成为开发针对PST-NCAM相互作用的治疗剂的基础。 与公共卫生相关：已发现神经细胞黏附分子(NCAM)用聚唾液酸(PSA)修饰发生在几种人类肿瘤的晚期，在那里它促进了这些肿瘤扩散到身体其他部位的能力。研究表明，将聚唾液酸添加到NCAM中需要NCAM与添加聚唾液酸的酶之间的相互作用。这项研究的目的是确定这种相互作用是如何发生的，以便为开发治疗方法来阻断癌细胞中的NCAM多唾液酸化奠定基础。

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. PUBLIC HEALTH RELEVANCE: The modification of the neural cell adhesion molecule (NCAM) with polysialic acid (PSA) has been found to occur in the advanced stages of several human tumors, where it promotes the ability of these tumors to spread to other parts of the body. Research has shown that the addition of polysialic acid to NCAM requires an interaction between NCAM and the enzymes that add polysialic acid. The goal of this research is to determine how this interaction takes place in order to lay the foundation for the development of therapeutics to block NCAM polysialylation in cancer cells.