Synthetic Mucins for Structural and Compositional Studies of Mucus Gels

摘要 粘液是生物体中调节与外界相互作用的主要物质，从人类到 水母。粘液凝胶在胚胎植入、吸收等过程中起到水合屏障的作用 营养物质、药物和病原体，同时也容纳了大部分微生物群。尽管有这些必要的 角色、粘液成分、物理特性和生物学仍未明确。这是因为少校 粘蛋白糖蛋白是一种天然的异质性物质，不能通过任何 当前的方法。这一障碍阻碍了我们对不同领域的上皮生物学的理解。 这项提议的总体目标是产生合成粘液作为转化材料，以探索 天然粘液的结构和功能，以及在治疗受损组织方面的生物医学应用。 我们假设，合成的多嵌段糖多肽可以模拟自然的多域凝胶形成 粘蛋白，但具有精确定义和可调节的组合物，能够选择性地调节凝胶特性 和生物活性。天然粘蛋白是一个由20+个糖蛋白组成的家族，具有巨大的杆状结构域 富含糖基化的丝氨酸/苏氨酸，以及在形成交联体中起作用的短末端结构域 粘蛋白通过半胱氨酸二硫化物和疏水相互作用而成束。粘蛋白表达与剪接变异 对每个组织和疾病都是独一无二的，蛋白质的糖基化模式是复合体的产物 代谢途径受&gt；1000基因控制。人们对这些途径知之甚少，也不可能 被任何目前的遗传或生化方法操纵的。总体而言，生物粘蛋白也是 异质，以探索许多具体的假设。糖共聚物已被探索为粘液模拟物，但 以前的例子未能概括天然粘蛋白的化学结构和生物物理。 在项目期间，我们将1)开发可调、可重复的合成粘蛋白，基于多个 封闭糖多肽，忠实地模拟天然粘蛋白的化学和生物物理特性， 2)揭示粘液成分(pH、离子、脂质、DNA、蛋白质)如何影响凝胶的物理性质 以及依赖于多糖的生物活性。我们将通过化学合成和化学合成来精确调整多糖模式 酶促糖基化：制备与糖结合蛋白相互作用的结合配体或控制配体。这些 属性不能由任何其他当前方法控制。我们将把糖多肽组装成 不同成分的凝胶，灵感来自对天然粘液的分析，我们将对我们的材料进行基准测试 对抗市面上可买到的粘蛋白。我们希望为我们的实验室和其他人提供新的研究工具 关于粘膜运输和与健康和疾病相关的生物学之前无法验证的假说。 拟议研究的成功预计将在不同的领域产生变革性的影响 材料科学和糖生物学涉及药剂学、免疫学、传染病、胃肠病学、 和妇科。

Abstract Mucus is the primary material that mediates interactions with the outside world in organisms from humans to jellyfish. Mucus gels function as a hydrating barrier involved in events such as embryo implantation, absorption of nutrients, drugs, and pathogens, while also housing the majority of the microbiome. Despite these essential roles, mucus composition, physical properties, and biology remain poorly defined. This is because the major component, mucin glycoproteins, is innately heterogeneous and cannot be reproducibly obtained by any current methodology. This roadblock has hindered our understanding of epithelial biology across diverse fields. The overall objective of this proposal is to generate synthetic mucus as transformative materials to probe the structure and function of native mucus, and with biomedical applications treating compromised tissues. We hypothesize that synthetic multi-block glycopolypeptides can emulate natural multi-domain gel-forming mucins, but with precisely defined and tunable compositions capable of selective modulation of gel properties and bioactivity. Native mucins are a family of 20+ glycoproteins characterized by massive rod-like domain rich in glycosylated-Ser/Thr, and short terminal domains that play a role in formation of cross-linked mucins bundles via Cys disulfides and hydrophobic interactions. Mucin expression and splice variation are unique to each tissue and disease, and the proteins' glycosylation patterns are the product of complex metabolic pathways controlled by >1000 genes. These pathways are poorly understood and cannot be manipulated by any current genetic or biochemical methods. Overall, biological mucins are too heterogeneous to probe many specific hypotheses. Glycopolymers have been explored as mucus-mimics, but prior examples have failed to recapitulate the chemical structures and biophysics of native mucins. During the project period, we will 1) develop tunable and reproducible synthetic mucins based on multi- block glycopolypeptides that faithfully emulate the chemical and biophysical properties of natural mucins, and 2) unravel how mucus composition (pH, ions, lipids, DNA, proteins) affects both gel physical properties and glycan-dependent bioactivity. We will precisely tune the glycan patterns by chemical synthesis and enzymatic glycosylation to prepare binding or control ligands to interact with glycan-binding proteins. These properties cannot be controlled by any other current methods. We will assemble the glycopolypeptides into gels with varied compositions inspired by analysis of native mucus, and we will benchmark our materials against commercially available mucins. We expect to provide new tools for our lab and others to study previously untestable hypotheses regarding mucosal transport and biology relevant to health and disease. Success of the proposed research is anticipated to make a transformative impact across diverse fields from materials science and glycobiology to pharmaceutics, immunology, infectious diseases, gasteroenterology, and gynecology.