Mechanism of the Protein Specific Polysialylation of NCAM

描述（由申请人提供）：ST8Sia IV （PST）是一种12,8-唾液基转移酶，可催化有限数量的底物糖蛋白的多唾液基化，其中神经细胞粘附分子（NCAM）是主要底物。添加到NCAM中的聚唾液酸通过充当抗粘合剂来调节细胞粘附，阻止由NCAM和其他附近粘附分子介导的细胞相互作用。先前的研究表明，聚唾液化的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，已发现多唾液化的NCAM在几种人类肿瘤中异常表达，促进肿瘤的生长和侵袭性。尽管我们知道NCAM聚唾液酰化的重要有益和有害影响，但PST识别和聚唾液酰化NCAM的机制尚不清楚。几个实验室的研究结果表明，PST对NCAM的多唾液化作用是一个高度蛋白质特异性的过程，需要通过PST-NCAM蛋白-蛋白相互作用介导的初始识别步骤。本研究的总体目标是进一步验证这一假设，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蛋白特异性多唾液化的机制。在Specific Aim I中，竞争、突变/重组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组成的截断的无催化活性PST蛋白是SW2细胞NCAM多唾液化的有力竞争者。一系列进一步截断的催化失活PST蛋白在SW2小细胞肺癌细胞中与内源性PST竞争并阻断NCAM多唾液化的能力将通过抗多唾液酸抗体OL.28、免疫印迹和FACS分析进行评估。作为有效竞争者的PST序列将进一步测试其与NCAM结合的能力，使用共免疫沉淀和等温滴定量热分析。这些结合NCAM的PST序列将在PST中被替换，并插入到相关的12,8-唾液基转移酶ST8Sia III中，以确定它们对于NCAM识别是否必要和充分，从而导致多唾液基化。在Specific Aim II中，将进行结构分析，以确定PST和NCAM中介导PST-NCAM相互作用的氨基酸残基。本研究的目的是获得PST肽与最近结晶的NCAM Ig5-FN1片段（PST多唾液化所需的最小结构域）的共晶结构，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多唾液化的机制提供更深入的了解，并可能为开发靶向PST-NCAM相互作用的治疗剂提供基础。

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.