Structural Studies of Protein-Carbohydrate Interactions

本研究的重点是(1)探索抗体（Ab）对寡糖抗原的反应，(2)利用结构-功能方法（主要是x射线晶体学）合成和结合次级细胞壁聚合物（SCWPs）。首先，与蛋白质和多肽不同，碳水化合物免疫原通常不能诱导亲和成熟和类转换，这导致Ab种系经历了巨大的进化压力来直接识别碳水化合物。研究表明，抗原决定因素的多样性远远超过了Ab种系的组合潜力，这至少需要一些种系抗体显示多特异性。我们正在研究几组抗体。其中，在一个强大的多特异性演示中，我们正在探索模仿原始碳水化合物抗原的抗独特型抗体。另一方面，我们正在探索两种近种系抗体采用多种稳定构象的能力，通过这些构象选择，多特异性抗原识别可能会进行。在第三种情况下，我们正在研究糖肽特异性Ab的交叉反应电位。其次，自组装蛋白表面层（s层）是常见的原核细胞壁特征，参与关键的生理过程，包括营养物质分解、细胞壁组装，以及在感染的情况下，宿主细胞粘附。在革兰氏阳性细菌中，s层蛋白通过与特定肽聚糖连接的SCWPs连接的表面层同源（SLH）结构域固定在细胞壁上。在一些革兰氏阳性菌株中，这种相互作用依赖于丙酮化的SCWPs。值得注意的是，尽管丙酮酰化的SCWPs无处不在，但令人惊讶的是，在分子水平上对它们的生物合成、识别或由此带来的治疗抑制的见解知之甚少。来自模式生物肺泡芽孢杆菌CCM 2051T的SCWP由丙酮化的GlcNAc-ManNAc双糖重复序列组成。SCWP扩展涉及两个步骤：taga催化的n -乙酰甘露氨基基从UDP-ManNAc转移到末端的GlcNAc， csab催化的丙酮基从磷酸烯醇丙酮酸转移到末端的ManNAc-GlcNAc受体。我们在TagA（属于CAZy家族26，只有一个结构表征的例子）和CsaB（目前没有合适的模型）上取得了重大进展，并旨在确定它们的非配体结构和与合成供体和受体类似物的复合物。最后，我们扩展了SLH结构域SpaA的结构研究。我们最近在Nature common上发表了SpaA与末端SCWP单糖配合物的结构。揭示了蛋白质中意想不到的构象变化，我们证明了这一变化具有生物学意义。我们正朝着使用更长的、更具生物学相关性的合成SCWP片段来表征SpaA - SLH相互作用的方向发展。

This proposal is focused (1) on the exploring antibody (Ab) response to oligosaccharide antigens and (2) on the synthesis and binding of secondary cell wall polymers (SCWPs) using a structure-function approach, primarily X-ray crystallography.    First, unlike proteins and peptides, a carbohydrate immunogen cannot generally induce affinity maturation and class switching, which results in the Ab germline experiencing great evolutionary pressure to recognize carbohydrates directly. It has been shown that the diversity of antigenic determinants far exceeds the combinatorial potential of the Ab germline, which requires at least some germline Abs to display polyspecificity.    We are studying several groups of Abs. In one, in a powerful demonstration of polyspecificity, we are exploring anti-idiotypic Abs that mimic the original carbohydrate antigens. In another, we are exploring the ability of two near-germline Abs to adopt multiple stable conformations through which polyspecific antigen recognition may proceed via conformational selection. In a third, we are studying the interesting cross-reactive potential of an Ab specific for a glycopeptide.    Second, self-assembling protein surface layers (S-layers) are common prokaryotic cell wall features involved in key physiological processes including nutrient breakdown, cell wall assembly and, in the case of infection, host cell adhesion. In Gram-positive bacteria, S-layer proteins are anchored to the cell wall by surface layer homology (SLH) domains linked to specific peptidoglycan-linked SCWPs. In several Gram-positive bacterial strains this interaction relies on pyruvylated SCWPs. Remarkably, despite the ubiquity of pyruvylated SCWPs, surprisingly little is known at the molecular level about their biosynthesis, recognition, or the concomitant insights into therapeutic inhibition such understanding would bring.    The SCWP's from model organism Paenibacillus alvei CCM 2051T consists of pyruvylated GlcNAc-ManNAc disaccharide repeats.  Two steps involved in SCWP extension are TagA-catalyzed transfer of a N-acetylmannosaminyl group from UDP-ManNAc to the terminal GlcNAc, and CsaB-catalyzed transfer of a pyruvyl group from phosphoenolpyruvate to the terminal ManNAc-GlcNAc acceptor. We have made significant progress on TagA (which belongs to CAZy family 26 with only one structurally characterized example) and CsaB (for which there is currently no suitable model), and aim to determine their structures unliganded and in complex with  synthetic donor and acceptor analogues.    Finally, we are extending structural studies of the SLH domain SpaA. Our recent publication of the structure of SpaA in complex with the terminal SCWP monosaccharide reported in Nature Commun. revealed an unexpected conformational shift in the protein which we demonstrated had biological significance. We are moving toward characterizing SpaA SLH interactions using longer and more biologically relevant synthetic SCWP fragments.