Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602.
Department of Chemistry, University of Georgia, Athens, GA 30602.
Proc Natl Acad Sci U S A. 2021 Jan 19;118(3). doi: 10.1073/pnas.2012935118.
Humans express seven heparan sulfate (HS) 3--sulfotransferases that differ in substrate specificity and tissue expression. Although genetic studies have indicated that 3--sulfated HS modulates many biological processes, ligand requirements for proteins engaging with HS modified by 3--sulfate (3-OS) have been difficult to determine. In particular, the context in which the 3-OS group needs to be presented for binding is largely unknown. We describe herein a modular synthetic approach that can provide structurally diverse HS oligosaccharides with and without 3-OS. The methodology was employed to prepare 27 hexasaccharides that were printed as a glycan microarray to examine ligand requirements of a wide range of HS-binding proteins. The binding selectivity of antithrombin-III (AT-III) compared well with anti-Factor Xa activity supporting robustness of the array technology. Many of the other examined HS-binding proteins required an IdoA2S-GlcNS3S6S sequon for binding but exhibited variable dependence for the 2-OS and 6-OS moieties, and a GlcA or IdoA2S residue neighboring the central GlcNS3S. The HS oligosaccharides were also examined as inhibitors of cell entry by herpes simplex virus type 1, which, surprisingly, showed a lack of dependence of 3-OS, indicating that, instead of glycoprotein D (gD), they competitively bind to gB and gC. The compounds were also used to examine substrate specificities of heparin lyases, which are enzymes used for depolymerization of HS/heparin for sequence determination and production of therapeutic heparins. It was found that cleavage by lyase II is influenced by 3-OS, while digestion by lyase I is only affected by 2-OS. Lyase III exhibited sensitivity to both 3-OS and 2-OS.
人类表达七种不同的肝素硫酸（HS）3-硫酸转移酶，它们在底物特异性和组织表达上有所不同。尽管遗传研究表明 3-硫酸化 HS 调节许多生物学过程，但与 3-硫酸化（3-OS）修饰的 HS 结合的蛋白的配体要求一直难以确定。特别是，3-OS 基团需要呈现以结合的上下文在很大程度上是未知的。本文描述了一种模块化的合成方法，可以提供具有和不具有 3-OS 的结构多样的 HS 寡糖。该方法用于制备 27 种己糖，这些糖被打印成糖基微阵列，以检查广泛的 HS 结合蛋白的配体要求。抗凝血酶-III（AT-III）的结合选择性与抗因子 Xa 活性很好地匹配，支持了阵列技术的稳健性。许多其他检查的 HS 结合蛋白需要 IdoA2S-GlcNS3S6S 序列基序才能结合，但对 2-OS 和 6-OS 部分以及紧邻中心 GlcNS3S 的 GlcA 或 IdoA2S 残基表现出可变的依赖性。还检查了 HS 寡糖作为单纯疱疹病毒 1 细胞进入的抑制剂，令人惊讶的是，它们缺乏 3-OS 的依赖性，表明它们不是与糖蛋白 D（gD）竞争结合，而是与 gB 和 gC 竞争结合。这些化合物还用于检查肝素酶的底物特异性，肝素酶是用于 HS/肝素解聚以进行序列测定和生产治疗性肝素的酶。发现裂解酶 II 的切割受 3-OS 影响，而裂解酶 I 的消化仅受 2-OS 影响。裂解酶 III 对 3-OS 和 2-OS 均敏感。
