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Abstract：
Polysialic acid (polySia) is an unusual glycan that posttranslational modifies neural cell adhesion molecule (NCAM) proteins in mammalian cells. The up-regulated expression of polySia-NCAM is associated with tumor progression in many metastatic human cancers and in neurocognitive processes. Two members of the ST8Sia family of α2,8-polysialyltransferases (polySTs), ST8Sia II (STX) and ST8Sia IV (PST) both catalyze synthesis of polySia when activated cytidine monophosphate(CMP)-Sialic acid (CMP-Sia) is translocate into the lumen of the Golgi apparatus. Two key polybasic domains in the polySTs, the polybasic region (PBR) and the polysialyltransferase domain (PSTD) areessential forpolysialylation of the NCAM proteins. However, the precise molecular details to describe the interactions required for polysialylation remain unknown. In this study, we hypothesize that PSTD interacts with both CMP-Sia and polySia to catalyze polysialylation of the NCAM proteins. To test this hypothesis, we synthesized a 35-amino acid-PSTD peptide derived from the ST8Sia IV gene sequence and used it to study its interaction with CMP-Sia, and polySia. Our results showed for the PSTD-CMP-Sia interaction,the largest chemical-shift perturbations (CSP) were in amino acid residues V251 to A254 in the short H1 helix, located near the N-terminus of PSTD. However, larger CSP values for the PSTD-polySia interaction were observed in amino acid residues R259 to T270 in the long H2 helix. These differences suggest that CMP-Sia preferentially binds to the domain between the short H1 helix and the longer H2 helix. In contrast, polySia was principally bound to the long H2 helix of PSTD. For the PSTD-polySia interaction, a significant decrease in peak intensity was observed in the 20 amino acid residues located between the N-and C-termini of the long H2 helix in PSTD, suggesting a slower motion in these residues when polySia bound to PSTD. Specific features of the interactions between PSTD-CMP-Sia, and PSTD-polySia were further confirmed by comparing their 800 MHz-derived HSQC spectra with that of PSTD-Sia, PSTD-TriSia (DP 3) and PSTD-polySia. Based on the interactions between PSTD-CMP-Sia, PSTD-polySia, PBR-NCAM and PSTD-PBR, these findingsprovide a greater understanding of the molecular mechanisms underlying polySia-NCAM polysialylation, and thus provides a new perspective for translational pharmacological applications and development by targeting the two polysialyltransferases.
摘要：
聚唾液酸（polySia）是一种不寻常的聚糖，其翻译后修饰哺乳动物细胞中的神经细胞粘附分子（NCAM）蛋白。polySia-NCAM的上调表达与许多转移性人类癌症和神经认知过程中的肿瘤进展有关。α2,8-聚唾液酸转移酶（polySTs）的ST8Sia家族的两个成员，当活化的单磷酸胞苷(CMP)-唾液酸(CMP-Sia)易位到高尔基体的内腔中时,ST8SiiaII(STX)和ST8SiiaIV(PST)都催化polySia的合成。polyST中的两个关键多元结构域，多碱基区（PBR）和多唾液酸转移酶结构域（PSTD）是NCAM蛋白的多聚唾液酸化必需的。然而,描述聚唾液酸化所需的相互作用的精确分子细节仍然未知。在这项研究中,我们假设PSTD与CMP-Sia和polySia相互作用以催化NCAM蛋白的聚唾液酸化。为了检验这个假设，我们合成了来自ST8SiaIV基因序列的35个氨基酸的PSTD肽，并将其用于研究其与CMP-Sia的相互作用，和PolySia。我们的结果表明，对于PSTD-CMP-Sia相互作用，最大的化学位移扰动（CSP）是在短H1螺旋中的氨基酸残基V251至A254，位于PSTD的N端附近。然而,在长H2螺旋中的氨基酸残基R259至T270中观察到用于PSTD-polySia相互作用的更大的CSP值。这些差异表明CMP-Sia优先结合短H1螺旋和较长H2螺旋之间的结构域。相比之下,polysia主要与PSTD的长H2螺旋结合。对于PSTD-polysia相互作用，在PSTD中长H2螺旋的N和C末端之间的20个氨基酸残基中观察到峰强度的显着降低，表明当polySia与PSTD结合时，这些残基的运动较慢。PSTD-CMP-Sia之间相互作用的特定特征，通过将其800MHz衍生的HSQC光谱与PSTD-Sia的光谱进行比较，进一步证实了PSTD-polysia，PSTD-TriSia(DP3)和PSTD-polysia。基于PSTD-CMP-Sia之间的相互作用，PSTD-polysia,PBR-NCAM和PSTD-PBR,这些发现提供了对polySia-NCAM多聚唾液酸化的分子机制的更深入的理解，因此，通过靶向两种聚唾液酸转移酶，为翻译药理学应用和开发提供了新的视角。
