Enabling synthesis and biological studies of homogeneous heparan sulfate and chondroitin sulfate glyco-polypeptides and proteoglycans

蛋白多糖(PG)广泛存在于哺乳动物细胞表面和细胞外。 矩阵。PGS由硫酸乙酰肝素或硫酸软骨素糖胺多糖链组成 通过四糖连接物共价连接到核心蛋白上。PGS扮演着重要的角色 在许多生物过程中。然而，由于自然存在的PG的异质性，它是 提纯定义明确的结构以研究结构和活性具有极大的挑战性 两性关系。传统上，前列腺素的生物学功能被认为是由 附着的糖胺多糖链。越来越多的证据表明，核心蛋白可能显著 影响多聚糖的活性。在最后一次拨款期间，均相硫酸乙酰肝素和 硫酸软骨素糖肽已被合成，为这些复合体提供了途径 第一次发现了分子。然而，合成总共需要100多个化学步骤来 用硫酸乙酰肝素链来完成糖肽，这限制了合成产量。 此外，在目前的合成策略下，PG中的一些常见结构特征 是无法访问的。为了克服这些挑战，在这次续订申请中， 生物合成酶将被用来极大地扩展PG合成的能力。在AIM 1，将产生关键酶，以产生与核心的四糖连接区 多肽，并延长糖链。将开发自动合成策略，以进一步 加快合成速度。在目标2中，将建立连接策略来合成糖聚糖- 具有多个糖链的多肽。这将为扩展多肽提供一个强有力的工具 并探索核心蛋白可以调节糖链活性的假设。在……里面 目的3、完成硫酸软骨素蛋白多糖Bikunin样糖蛋白的合成。 比库宁是一种被批准用于治疗潜在威胁生命的脓毒症的药物，其 机制目前尚不清楚。在结构上定义明确的合成Bikunin的帮助下， 比库宁抗炎作用的分子机制将被建立， 展示合成在扩大对有趣的生物学的理解方面的力量 这些复杂分子的性质。

Proteoglycans (PGs) are ubiquitous on mammalian cell surfaces and in the extracellular matrix. PGs are made up of heparan sulfate or chondroitin sulfate glycosaminoglycan chains covalently attached to the core protein through tetrasaccharide linkers. PGs play important roles in many biological processes. However, due to the heterogeneity of naturally existing PGs, it is extremely challenging to purify well-defined structures to study the structure and activity relationship. Traditionally, the biological functions of PGs are believed to be dictated by the glycosaminoglycan chains attached. Evidence is emerging that the core protein may significantly impact the glycan activities. During the last grant period, homogeneous heparan sulfate and chondroitin sulfate glycopeptides have been synthesized, providing access to these complex molecules for the first time. However, the synthesis required total over 100 chemical steps to complete a glycopeptide with a heparan sulfate chain, which limited the synthetic output. Furthermore, with the current synthetic strategy, some of the common structural features in PGs are not accessible. To overcome these challenges, in this renewal application, the power of the biosynthetic enzymes will be harnessed to greatly expand the capability for PG synthesis. In aim 1, the key enzymes will be produced to generate the tetrasaccharide linkage region with the core peptide, and to extend the glycan chain. Automated synthesis strategy will be developed to further expedite the synthesis. In aim 2, ligation strategies will be established to synthesize glyco poly- peptides with multiple glycan chains. This will provide a powerful tool to extend the poly-peptide backbone, and to probe the hypothesis that the core protein can modulate glycan activities. In aim 3, synthesis of a chondroitin sulfate proteoglycan bikunin like glycoprotein will be completed. Bikunin is an approved drug to treat the potentially life-threatening sepsis conditions, and its mechanism is currently unclear. Aided by the structurally well-defined synthetic bikunin, the molecular mechanisms of the anti-inflammatory activities of bikunin will be established, demonstrating the power of synthesis in expanding the understanding of the interesting biological properties of these complex molecules.