Molecular Biology of Hyaluronan Biosynthesis

描述（由申请人提供）：脊椎动物中的细胞外基质为细胞提供结构支持，有助于细胞增殖调节，并且在介导 软结缔组织中的细胞-细胞相互作用，如软骨和皮肤。的主要组成部分 细胞外基质是透明质酸（HA），其是含有交替的N-乙酰葡糖胺（NAG）和葡糖醛酸（GA）残基的细胞外线性多糖。HA影响许多生理过程，从细胞粘附和迁移到细胞分化和胚胎发育。由于其对人体生理的广泛影响，大量病理状况，包括许多形式的癌症、自身免疫性疾病、炎症过程和类风湿性关节炎，与HA表达水平的改变相关。在分子水平上，HA由膜包埋的透明质酸合成酶（HAS）在细胞内产生。HAS是一种非凡的酶。它不仅催化从UDP活化的底物合成HA，而且还将生长的聚合物转运穿过细胞膜以将其存款在细胞外基质内。为了完成这项任务，HAS必须满足 几个功能。该酶结合底物UDP-NAG和-GA，它催化糖基转移反应以形成HA，并且它使生长的聚合物通过由其自身的跨膜区形成的孔易位穿过细胞膜。为了了解HA如何发挥其生理功能，并产生具有生物医学应用的定义属性的HA聚合物，我们必须首先解开HAS如何合成HA以及它如何将聚合物沉积在细胞外基质中。为此，我们提出了三个目标，这将揭示天然脂质膜中生物活性HAS亚基的组装，将确定HAS和易位HA聚合物之间的相互作用，并将使我们能够通过X射线晶体学确定HAS的结构。首先，我们将结合联合收割机免疫共沉淀研究与化学交联和光漂白技术，以可视化天然膜中的HAS寡聚体。然后将低分辨率结构数据与单体HAS的高分辨率结构整合，以重建天然的膜嵌入的HAS寡聚体。第二，通过将UV诱导的交联剂引入HAS的TM区，将绘制HAS与易位HA聚合物的相互作用。HA易位过程中的交联将识别与多糖紧密接近的位置，从而描绘HA跨膜通道的物理化学性质。第三，生物化学和低分辨率结构数据将与通过X射线晶体学获得的HAS的高分辨率结构相结合。确定HAS在洗涤剂溶解和膜包埋状态下的结构将揭示合酶的结构和低聚形式，使我们能够描绘这种奇妙的酶合成人体中最丰富的胞外多糖之一的机制。

DESCRIPTION (provided by applicant): The extracellular matrix in vertebrates provides structural support to the cell, aids in osmo-regulation, and is particularly important in mediating cell-cell interactions in soft connective tissues, such as cartilage and skin. A major component of the extracellular matrix is hyaluronan (HA), which is an extracellular linear polysaccharide containing alternating N-acetylglucosamine (NAG) and glucuronic acid (GA) residues. HA affects many physiological processes, from cell adhesion and migration to cell differentiation and embryological development. Because of its broad impact on human physiology, a large number of pathological conditions, including many forms of cancer, autoimmune diseases, inflammatory processes, and rheumatoid arthritis, correlate with altered expression levels of HA. On a molecular level, HA is produced inside the cell by the membrane-embedded hyaluronan synthase (HAS). HAS is a remarkable enzyme. It not only catalyzes the synthesis of HA from UDP-activated substrates, but it also transports the growing polymer across the cell membrane to deposit it within the extracellular matrix. In order to accomplish this task, HAS has to fulfill several functions. The enzyme binds the substrates UDP-NAG and -GA, it catalyzes the glycosyl transfer reaction to form HA, and it translocates the growing polymer across the cell membrane through a pore formed by its own transmembrane region. To understand how HA exerts its physiological function and to produce HA polymers with defined properties for biomedical applications, we must first unravel how HAS synthesizes HA and how it deposits the polymer in the extracellular matrix. To this end, we propose three aims that will reveal the assembly of biologically active HAS subunits in native lipid membranes, will identify the interactions between HAS and the translocating HA polymer, and will allow us to determine the structure of HAS by X-ray crystallography. First, we will combine co-immunoprecipitation studies with chemical cross-linking and photobleaching techniques to visualize HAS oligomers in native membranes. The low-resolution structural data will then be integrated with high-resolution structures of monomeric HAS to reconstruct the native, membrane-embedded HAS oligomer. Second, the interactions of HAS with the translocating HA polymer will be mapped by introducing UV-inducible cross-linkers into the TM-region of HAS. Cross-linking during HA translocation will identify positions that are in close proximity to the polysaccharide, thus delineating the physico-chemical properties of the HA transmembrane channel. Third, biochemical and low resolution structural data will be integrated with a high-resolution structure of HAS obtained by X-ray crystallography. Determining the structure of HAS both in a detergent-solubilized but also in a membrane-embedded state will reveal the architecture and oligomeric form of the synthase, allowing us to delineate the mechanism by which this marvelous enzyme synthesizes one of the most abundant extracellular polysaccharides in the human body.