Understanding hyaluronan crosslinking mechanisms in ovulation and inflammation: CryoEM structural and interaction analysis of HC-HA/PTX3 complexes

细胞外基质存在于我们体内几乎每个细胞的周围和之间，正是这种物质将细胞组织成器官，并为我们的组织提供了特殊的机械性能。例如，骨头是硬的，大脑是软的，皮肤是结实但有弹性的。一个重要的基质成分是透明质酸，简称HA。透明质酸是由数千个排列在长链上的糖分子组成的大型聚合物（“多糖”）。透明质酸存在于哺乳动物的每一个组织中，据信在5亿多年前就已经进化出来了。正是透明质酸给我们的组织补水，使它们抵抗压力。例如，透明质酸为软骨提供弹性，在我们走路和跑步时缓冲我们的关节。透明质酸在人类生殖中也起着至关重要的作用，在排卵时卵子从卵巢释放出来之前，在卵子周围形成一层非常柔软但有弹性的果冻状涂层；这种有弹性的果冻可以让卵子沿着输卵管向下移动，这是捕获健康精子所必需的。透明质酸也是胚胎发育所必需的，在新器官形成过程中指导细胞的运动。鉴于透明质酸在哺乳动物生物学中具有如此重要的作用，因此在疾病期间经常受到影响也就不足为奇了；例如，在癌症和炎症期间产生过多的透明质酸。透明质酸还会导致纤维化，这一过程导致了大约40%的死亡，因为组织变得僵硬，不再正常运作。虽然已知血凝素在健康和疾病中发挥着广泛的作用，但它是如何发挥作用的尚不清楚。尽管透明质酸很大，但它是一个非常简单的分子，这使得了解透明质酸如何有助于实现如此多样化的功能变得很有趣。证据有力地表明，解释在于透明质酸与蛋白质的关联。然而，目前尚不清楚这在分子层面上是如何发生的。我们已经提出，透明质酸与不同组织位置的不同类型的蛋白质结合，这允许形成具有不同机械和功能特性的不同范围的透明质酸/蛋白质“复合材料”。换句话说，透明质酸就像一种生物塑料，可以根据与之结合的蛋白质的不同，被塑造成不同的形状，具有不同程度的柔软度和柔韧性。我们还假设，这些不同的透明质酸/蛋白质复合物在细胞外基质中编码不同的分子信号，这些信号可以被细胞解码和解释，告诉它们该做什么。拟议研究的目的是在分子水平上确定HA/蛋白质复合材料如何组织成三维网络，以及这如何决定它们的机械性能。这将使用一种相对较新的方法（低温电子显微镜）来确定连接HA链的蛋白质的精确形状（在原子分辨率上），因为更传统的方法无法实现这一点。我们的研究将产生基本的新见解，将开始让我们了解透明质酸如何介导其多样化和重要的功能，提供新的分子概念，可以广泛应用于哺乳动物生物学。为了实现我们的目标，我们将重点关注HA/蛋白质复合物的特定亚型（称为“HC-HA/PTX3复合物”），其组成和功能已经部分建立。例如，这种HA/蛋白复合物亚型在排卵和受精中起着至关重要的作用，在关节炎期间在我们的关节腔中形成，并且根据其形成的环境可以促进或预防纤维化。因此，我们研究的详细结果将有可能促进许多生物医学应用。其中包括设计量身定制的HC-HA/PTX3复合物，用于改善生育治疗，以及针对多种炎症和纤维化疾病的新型再生医学策略。

The extracellular matrix is found around and between virtually every cell in our bodies and it is this substance that organises cells into organs and provides our tissues with their particular mechanical properties. For example, bone is hard, brain is soft, and skin is strong but elastic. One important matrix component is hyaluronic acid, or HA for short. HA is a large polymer comprised of thousands of sugar molecules arranged in a long chain (a 'polysaccharide'). HA is present in every tissue of mammals and is believed to have evolved more than 500 million years ago. It is HA that hydrates our tissues and makes them resistant to compressive forces. For example, HA provides cartilage with its resilience, cushioning our joints when we walk and run. HA also plays a critical role in human reproduction, forming a very soft, but elastic, jelly-like coating around the egg just prior to it being released from the ovary at ovulation; here this elastic jelly allows the egg to travel down the oviduct and is required for the capture of a healthy sperm. HA is also necessary for embryonic development, directing the movement of cells during the formation of new organs. Given that HA has such an important role in mammalian biology, it is not surprising that HA is often affected during disease; for example, too much HA is made during cancer and during inflammatory conditions. HA also contributes to fibrosis, a process that contributes to approximately 40% of all deaths, because tissues become stiffened and no longer function correctly.Although HA is known to play a wide range of roles in health and disease, how it does this is not well understood. Despite HA being large, it is a very simple molecule, making it intriguing to understand how HA can contribute to such diverse functions. Evidence strongly suggests that the explanation lies in the association of HA with proteins. However, it is not clear exactly how this occurs in molecular terms. We have suggested that HA associates with different types of protein in different tissue locations and that this allows the formation of a diverse range of HA/protein 'composites' with distinct mechanical and functional properties. In other words, HA is like a biological plastic that can be moulded into different shapes with different degrees of softness and flexibility depending on which proteins it is combined with. We have also hypothesised, that these various HA/protein composites encode distinct molecular signals within the extracellular matrix that can be decoded and interpreted by cells, telling them what to do.The aim of the proposed research is to determine at a molecular level how HA/protein composites are organised into a three-dimensional network and how this dictates their mechanical properties. This will use a relatively new method (cryo-electron microscopy) to determine the precise shape (at atomic resolution) of the proteins that link together the HA chains, since more traditional methods have failed to achieve this. Our studies will generate fundamental new insights that will begin to allow us to understand how HA mediates its diverse and important functions, providing novel molecular concepts that can be applied widely across mammalian biology. To achieve our goals we will focus on a particular subtype of HA/protein composite (known as "HC-HA/PTX3 complexes") with compositions and functions that are already partly established. For example, this subtype of HA/protein composite has an essential role in ovulation and fertilisation, is formed in our joint cavities during arthritis, and can either promote or prevent fibrosis depending on the context in which it is made. Thus, the detailed findings of our research will have the potential to facilitate numerous biomedical applications. These include the design of tailored HC-HA/PTX3 complexes for use in improved fertility treatments and novel regenerative medicine strategies for a wide range of inflammatory and fibrotic diseases.