墨西哥海参(Holothuria mexicane)糖胺聚糖结构分析与抗P-选择素活性评价

	墨西哥海参(Holothuria mexicane)糖胺聚糖结构分析与抗P-选择素活性评价
	潘颖
导师	左之利
关键词	墨西哥海参，岩藻糖化硫酸软骨素，结构分析，二糖侧链，P-选择素 Holothuria mexicane, Fucosylated chondroitin sulfate, Structure analysis, Disaccharide branch, P-selectin
摘要	海参来源的岩藻糖化硫酸软骨素(Fucosylated chondroitin sulfate, FCS)是一类含侧链的糖胺聚糖衍生物。因其化学结构特殊，药理活性显著引起广泛关注。目前已知 FCS 主链类似硫酸软骨素(Chondroitin sulfate，CS)，硫酸化岩藻糖(L- Fucose，Fuc)以侧链形式连接于主链。但其确切结构存在多样性，且因物种来源不同而存在差异。在对 FCS 由简入繁地渐次分析中发现，天然 FCS 中侧链除存在硫酸化 Fuc 单糖外，还存在 Fuc 糖基化的二糖侧链；目前已确证二糖非还原端糖基包括乙酰氨基半乳糖(N-acetyl galactosamine, GalNAc)和半乳糖(D- Galactose, Gal)，尚未发现 Fuc 二糖侧链；多样的侧链硫酸化取代形式进一步丰富了 FCS 的结构多样性。天然 FCS 尤其是侧链类型复杂 FCS 的确切结构仍值得进一步研究。课题前期研究发现，墨西哥海参(Holothuria mexicane)来源的 FCS(Hm- FCS)NMR 谱图信号复杂，初步判断可能含二糖侧链，且侧链的确切结构类型较目前已知更加多样。已有多个研究组对 Hm-FCS 进行初步的结构分析，但对主/ 侧链硫酸化程度、侧链类型及侧链连接位点等结构信息报道不一致，其确切结构尚需确认。本论文以 Hm-FCS 为研究对象，系统分析了其理化性质；通过温和酸水解分析了侧链结构信息；经 β-消除解聚及寡糖纯化获得系列纯化寡糖；基于系列纯化寡糖及过氧化解聚产物，以“Bottom-up”策略推导原型 Hm-FCS 的确切结构。进一步分析了原型 Hm-FCS、低聚产物及纯化寡糖的 P-选择素抑制活性；探讨了 Hm-FCS 抑制 P-选择素的构效关系及其在炎症细胞黏附过程中的潜在应用价值。涉及方法及研究成果具体如下： Hm-FCS 的重均分子量为 5.5 kD，数均分子量为 4.3 kD；比旋光度为- 36.41°；单糖组成包括 Fuc、GalNAc 及葡萄糖醛酸(Glucuronic acid, GlcA)； SO 3 - 的含量为 31.2%。部分酸水解所得侧链组分分离得到 9种二糖及 1种单糖。初步显示 Hm-FCS 中侧链类型复杂多样。 Hm-FCS 的 β-消除解聚产物中，经凝胶柱层析及强阴离子交换-高效液相色谱制备法反复纯化，从中获得纯化寡糖化合物 1-15。对所得寡糖结构进行归类：排除非还原端Δ 4,5 不饱和己糖醛酸的形成因素，除化合物 3 外的所有寡糖主链均为 GalNAc 4S6S 和 GlcA 以 β1,3 和 β1,4 糖苷键交替连接形成，即硫酸软骨素主链；侧链均连接于主链 GlcA的 O-3位；侧链结构类型丰富，包括 Fuc单糖侧链及 D-GalNAc-α1,2-L-Fuc二糖侧链；侧链糖基的硫酸化程度及位点呈现多样性。侧链结构中除 GalNAc 的 O-3 位存在-OH 外，所有游离-OH 位置均可被硫酸化。由此进一步确认天然 Hm-FCS 中存在硫酸化形式多样的新型二糖侧链结构。化合物 3 为结构特殊的无侧链的线性六糖，非还原端二糖结构与上述寡糖主链结构一致；还原端四糖[D-GlcA-β(1,3)-D-Gal-β(1,3)-D-Gal 4S - β(1,4)-D-Xyl] 类似于 CS 与核心蛋白结合结构域[D-GlcA-β(1,3)-D-Gal-β(1,3) -D-Gal-β(1,4)-D- Xyl],区别在于 Gal 是否存在硫酸化。此前文献报道 FCS 在生物体内连接于核心蛋白从而以蛋白聚糖的形式存在，上述线性四糖应当是 FCS 与蛋白连接区域的具体结构。进一步通过过氧化解聚制备了 Hm-FCS 的系列分子量的低聚产物 dHm- FCS’1-6，结合寡糖结构特征回溯分析了分子量适宜的 dHm-FCS’5 的 2D NMR 谱图，通过“bottom up”策略确认天然 Hm-FCS 结构为：--{4-[R-α(1,2)-L-Fuc S - α(1,3)-]-D-GlcA-β(1,3)-D-GalNAc 4S6S -β(1,}--，R 为 H (53%)或 GalNAc S (47%)。单糖侧链和二糖侧链应为随机分布连接于主链。Hm-FCS 为迄今报道的侧链类型最复杂多样的 FCS。 P-选择素抑制活性分析结果表明，FCS 类具有显著的竞争性抑制 P-选择素的活性，活性受样品链长及侧链类型影响；进一步探究 FCS 对炎症细胞黏附的活性，结果表明分子量为 5.3 kD的解聚产物具有最佳抑制活性。; Glycosaminoglycan isolated from the body wall of sea cucumbers exclusively, is a branched glycosaminoglycan derivative. Due to its special chemical structure and remarkable pharmacological activity, holothurian glycosaminoglycan has attracted widespread attention. It is currently known that the backbone of holothurian glycosaminoglycan is similar to chondroitin sulfate (CS), and the sulfated fucose (Fuc) is attached to the backbone as branches. Thus, the holothurian glycosaminoglycan is also called fucosylated chondroitin sulfate (FCS). Numerous studies have shown that the exact structure of FCS varies with species of sea cucumber, so the structure were diverse. It was found that in addition to the sulfated Fuc monosaccharide branch, there were also exist fucosylated disaccharide branch. The non-reducing termini sugars of disaccharide branches include N-acetyl galactosamine (GalNAc) and Galactose (Gal), but the difucosyl branch has not been found. Diverse sulfated patterns further enrich the structural diversity of native FCS. The exact structure of native FCS, especially the FCS with complex branch types deserve further study. Our preliminary study found that the NMR spectrum signals of FCS from Holothuria mexicane (Hm-FCS) were complex. It is preliminarily judged that Hm-FCS may connect disaccharide branches, and the exact types of the branches are more diverse than those known so far. Several research groups have conducted preliminary structural analysis of Hm-FCS, but the reports on the structural information such as the sulfation pattern of backbone or branches, the type of branches and the attachment site of the branches were inconsistent. The exact structure needs to be confirmed further. In this thesis, Hm-FCS was taken as the research object, and its physicochemical properties were systematically analyzed. The structural information of the branch was analyzed by mild acid hydrolysis. A series of purified oligosaccharides were obtained by β-eliminative depolymerization and purification; The exact structure of the Hm-FCS was deduced based on a series of purified oligosaccharides. In addition, low molecular weight depolymerization products were prepared by peroxidative depolymerization. The P-selectin inhibitory activity of native Hm-FCS, oligomers and purified oligosaccharides were further analyzed. The structure-activity relationship of Hm-FCS and its potential application value in the process of blood cell adhesion were discussed.
语种	中文
	2022-05
学位授予单位	中国科学院大学
文献类型	学位论文
条目标识符	http://ir.kib.ac.cn/handle/151853/75173
专题	昆明植物所硕博研究生毕业学位论文
推荐引用方式 GB/T 7714	潘颖. 墨西哥海参(Holothuria mexicane)糖胺聚糖结构分析与抗P-选择素活性评价[D]. 中国科学院大学,2022.

条目包含的文件
文件名称/大小	文献类型	版本类型	开放类型	使用许可
潘颖-墨西哥海参(Holothuria （20066KB）	学位论文		限制开放	CC BY-NC-SA	请求全文