Chemically Tunable Mucins to Probe Pathogenic Function in the Epithelial Milieu

摘要 人体上皮组织是分泌一种被称为粘液的独特水凝胶的基本生物屏障。 组织产生不同类型的粘液，这些粘液提供特定的生物功能，如水合、病原体 防御，并调节物质向细胞表面移动。粘液的主要成分， 粘蛋白对凝胶的结构和功能至关重要。粘蛋白是一个由20种蛋白质组成的多样化家族，其特征是 由一个大的杆状结构域组成，富含丝氨酸/苏氨酸和附加的糖或多糖。分子级粘液 由于组织和物种特有的不同的糖链模式，研究一直具有挑战性，例如 以及不同的蛋白质表达水平和导致具有不同长度和序列的结构的剪接。 粘蛋白表达、剪接和糖基化的错误调节会导致结构改变，从而可能影响 生物学功能与感染、炎症和癌症相关的结果。研究人员通常利用 从农场动物来源分离的粘蛋白，用于此类研究，但该来源存在批次之间的差异， 没有化学定义的结构，并且具有不能系统地 被更改了。目前，对可以在分子水平上调节的化学定义的粘蛋白的需求尚未得到满足 并具有人类糖基化模式。这些材料对于探索这些至关重要的角色是必不可少的 健康与疾病中的生物材料。拟议的研究将通过开发一种方法来解决这一关键需求 制备人工合成的粘蛋白，并将其应用于探测糖与病原体的相互作用。技术 从碳水化合物化学、氨基酸N-羧酸酐(NCA)聚合和酶 糖基化，将结合在一起，产生具有完全可调性能的材料。我将生成一个面板，其中 化学定义的具有不同长度、氨基酸组成和糖基化的粘蛋白糖多肽 密度和糖链结构。结构设计将以已发表的对原住民的血糖分析为指导 粘蛋白与呼吸道感染有关。将对所有糖多肽进行充分的物理化学表征 使用各种光谱、显微镜和生物化学方法来确定其性质。粘蛋白及其葡聚糖 以前已被证明影响致病微生物的活动，如粘连、生物膜的形成和 毒力特征。合成粘蛋白将被应用于揭示多糖提呈如何影响这些致病因子 功能。对于天然粘蛋白，这种研究是不可能的，因为糖基化不能控制，而且 通常甚至不是特色化的。总而言之，我的目标是阐明分子结构与功能的关系 在粘蛋白和微生物之间。这种跨学科的方法将结合多个领域的技术来 回答有关感染的重要问题，这些问题不能仅通过生物方法来解决。这个 建议的材料可能作为抗菌剂或在粘膜替代疗法中具有治疗应用。

Abstract Human epithelial tissues are essential biological barriers that secrete a unique hydrogel known as mucus. Tissues generate distinct types of mucus that provide specific biological functions like hydration, pathogen defense, and mediating the movement substances toward the cell surface. The major component of mucus, mucin proteins, is critical for gel structure and function. Mucins are a diverse family of 20+ proteins characterized by a large, rod-like domain rich serine/threonine with attached saccharides, or glycans. Molecular-level mucus studies have been challenging due to heterogeneous glycan patterns that are tissue and species specific, as well as varied protein expression levels and splicing that result in structures with varied lengths and sequences. Misregulation of mucin expression, splicing, and glycosylation results in altered structures that may affect biological function with outcomes relevant to infection, inflammation, and cancer. Researchers typically utilize mucins isolated from farm animal sources for such studies, but this source suffers from batch-to-batch variation, structures that are not chemically defined and have non-human glycan patterns that cannot be systematically altered. Currently, there is an unmet need for chemically-defined mucins that can be tuned at the molecular level and possess human glycosylation patterns. Such materials are essential to probe the role of these vital biomaterials in health and disease. The proposed research will address this critical need by developing a method to prepare synthetic human mucins, which will be applied to probe glycan-pathogen interactions. Techniques from carbohydrate chemistry, amino acid N-carboxyanhydride (NCA) polymerization, and enzymatic glycosylation, will be combined to generate materials with fully tunable properties. I will generate a panel of chemically-defined mucin glycopolypeptides with varied lengths, amino acid compositions, glycosylation densities, and glycan structures. Structure design will be guided by published glycomic analysis of native human mucins implicated in airway infections. All glycopolypeptides will be fully characterized for physicochemical properties using a variety of spectroscopic, microscopic, and biochemical methods. Mucins and their glycans have previously been shown to affect activity of pathogenic microbes such as adhesion, biofilm formation, and virulence traits. Synthetic mucins will be applied to reveal how glycan presentation affects these pathogenic functions. Such studies are not possible with native mucins since glycosylation cannot be controlled and is typically not even characterized. Overall, I aim to shed light on the molecular structure-function relationship between mucins and microbes. This interdisciplinary approach will combine techniques from multiple fields to answer important questions about infection that cannot be undertaken by biological methods alone. The proposed materials could have therapeutic applications as antimicrobials or in muco-replacement therapies.