Revealing complexity of hyaluronan-protein interactions: novel tools and insights

透明质酸（HA）是一种由单糖重复单元组成的大型线性生物聚合物，自5亿年前进化以来，这种分子在脊椎动物中高度保守。透明质酸存在于所有哺乳动物组织的细胞外基质中，具有一系列惊人的生物学功能。透明质酸决定组织结构、弹性、水合作用和通透性，并通过与细胞表面受体（如CD44和LYVE-1）的结合来指导细胞行为。透明质酸涉及广泛的生理和病理过程，包括发育、炎症和组织修复、免疫反应、肿瘤发展和病毒/细菌感染。透明质酸通过与一系列透明质酸结合蛋白的相互作用，对多种生物功能有广泛的贡献。就其本身而言，HA不采用已定义的结构，但本质上仍然是无序的。在与不同的ha结合蛋白结合时，它以不同的方式组织、采用和呈现各种构象和相互作用模式，有点类似于符合其容器形状的水。透明质酸与蛋白质相互作用的复杂程度很高。我们可以用核酸做一个类比；然而，与DNA和RNA相比，我们对在蛋白质影响下支持HA功能的分子和物理机制的理解仍处于起步阶段。造成这种情况的主要原因是技术上的：我们缺乏生物物理工具来探测涉及HA的分子相互作用的复杂性，以及制造定义的“设计”HA分子的能力。这个项目将直接解决这些技术瓶颈。它的目的是产生一个新的工具包，以揭示支持HA和HA结合蛋白的复杂相互作用的生物物理现象。我们用工具包解决的具体问题是：HA细胞表面受体如何识别HA链的拓扑结构（例如，通过选择性地结合到自由链末端），以及蛋白质如何沿着HA链滑动（类似于沿着DNA滑动的聚合酶）？这些问题的答案将有助于确定ha受体相互作用关键支持免疫细胞运输的分子机制。细胞外基质蛋白聚糖是如何与透明质酸链组装成超螺旋结构的？这种透明质酸/蛋白质“超分子弹簧”的机械特性是什么？这与在组织发育和修复过程中形成的软的和动态的富含ha的细胞外基质的弹性有关，也与肿瘤发展和其他疾病的早期阶段形成的弹性有关，以促进细胞的迁移。ha蛋白相互作用控制细胞如何识别彼此，形成组织，并为免疫监视和组织修复迁移。它们也会被癌细胞利用，转移到远处的器官，被致病菌/病毒感染组织/细胞。揭示透明质酸及其与蛋白质相互作用的复杂性，而不仅仅是单纯的存在或缺失，将最终为更好地控制这些过程（例如，阻止组织移植后破坏性免疫反应的启动）开辟新的途径，并设计具有新功能的生物材料，用于再生医学和免疫调节。该项目还将为其他生物物理学家提供新的试剂和方案，以研究ha -蛋白质相互作用，以及更广泛的糖胺聚糖-蛋白质相互作用，并利用生物物理学方法揭示支撑其许多功能的分子机制。

Hyaluronan (HA) is a large linear biopolymer made of simple sugar repeat units, a molecule highly conserved in vertebrates since their evolution 500 million years ago. Present in the extracellular matrix of all mammalian tissues, HA exhibits an astonishing array of biological functions. HA dictates tissue architecture, elasticity, hydration and permeability, and also directs cell behaviour via engagement with cell surface receptors, such as CD44 and LYVE-1. HA is implicated in a wide range of physiological and pathological processes, including development, inflammation and tissue repair, immune response, tumour development and virus/bacteria infections.HA contributes widely to a diversity of biological functions through its interaction with a repertoire of HA-binding proteins. On its own, HA does not adopt defined structures but remains intrinsically disordered. It differentially organises, adopts and assumes various conformations and modes of interactions upon binding to different HA-binding proteins, somewhat similar to water conforming to the shape of its container. The level of complexity in how HA interacts with proteins is high. An analogy can be made to nucleic acids; however, compared to DNA and RNA, our understanding of the molecular and physical mechanisms underpinning HA functions under the influence of proteins is still in its infancy. The reasons for this are primarily technical: we lack the biophysical tools to probe the complexity of molecular interactions involving HA, and the ability to make defined 'designer' HA molecules.This project will directly address these technical bottlenecks. It aims to generate a new toolkit required to reveal the biophysical phenomena underpinning the complex interactions of HA and HA binding proteins. Specific questions that we address with the toolkit are:How do the HA cell surface receptors recognise the topology of HA chains (e.g., by selectively binding to free chain ends), and how can proteins slide along HA chains (similar to polymerase sliding along DNA)? Answers to these questions will help defining the molecular mechanisms by which HA-receptor interactions critically support immune cell trafficking.How do extracellular matrix proteoglycans assemble with an HA chain into a super-helix, and what are the mechanical characteristics of such HA/protein 'supramolecular springs'? This is relevant for the elasticity of soft and dynamic HA-rich extracellular matrices that are formed during tissue development and repair, but also during early stages of tumour development and other diseases, to promote the migration of cells.HA-protein interactions control how cells recognise each other, shape tissues, and migrate for immune surveillance and tissue repair. They are also opportunistically exploited by cancer cells for metastasis in distant organs, and by pathogenic bacteria/viruses to infect tissues/cells. Revealing the complexity, but not only the mere presence of absence, of HA and its interactions with proteins, will ultimately open up new avenues to better control such processes (e.g., to block the priming of damaging immune responses following tissue transplantation), and to design biomaterials with novel functions for applications in regenerative medicine and immune regulation. The project will also lead the way, by providing new reagents and protocols, for other biophysicists to study HA-protein interactions, and glycosaminoglycan-protein interactions more generally, with biophysical methods to reveal the molecular mechanisms underpinning their many functions.