Molecular Biology of Hyaluronan Biosynthesis

透明质酸生物合成的分子生物学

• 批准号: 8816855
• 负责人: Jochen Zimmer
• 金额: $ 27.55万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-01 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8816855
• 关键词: Acetylglucosamine; Affect; Alginates; Amber; Anabolism; Architecture; Autoimmune Diseases; Binding; Biochemical; Biological Assay; Biological Models; Biological Process; Biotechnology; Carcinoma; Cartilage; Catalysis; Cell Adhesion; Cell Communication; Cell Differentiation process; Cell Surface Receptors; Cell membrane; Cell-Cell Adhesion; Cells; Cellulose; Charge; Chemicals; Chlorella; Co-Immunoprecipitations; Collaborations; Complex; Connective and Soft Tissue; Crystallization; Data; Deposition; Detergents; Development; Engineering; Enzymes; Escherichia coli; Event; Extracellular Matrix; Glucuronic Acids; Human; Human body; Hyaluronan; Imaging Techniques; Immunosuppressive Agents; In Vitro; Inflammatory; Laboratories; Lead; Length; Life; Link; Lipid Bilayers; Lipids; Malignant Neoplasms; Maps; Mediating; Medicine; Membrane; Membrane Biology; Membrane Lipids; Membrane Proteins; Methods; Microbial Biofilms; Microfilaments; Modeling; Molecular; Molecular Biology; Molecular Weight; Mono-S; Nature; Phase; Phenylalanine; Photobleaching; Physiological; Physiological Processes; Physiology; Polymers; Polysaccharides; Positioning Attribute; Process; Property; Proteins; Protocols documentation; Publishing; Reaction; Regulation; Research; Resistance; Resolution; Rheumatoid Arthritis; Robotics; Side; Signal Transduction; Site; Skin; Streptococcus; Structure; Techniques; Terminator Codon; Transferase; Transmembrane Domain; UV Radiation Exposure; Universities; Vertebrates; Viral; Virginia; Virus; X-Ray Crystallography; base; cell motility; chemical property; crosslink; dodecyldimethylamine oxide; extracellular; glycosyltransferase; hyaluronan synthase 1; member; membrane synthesis; mutant; physical property; proteoliposomes; public health relevance; reconstitution; research study; rho GTP-Binding Proteins; stoichiometry; sugar; three dimensional structure

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.

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OSMO调节，并且在介导 软性结缔组织中的细胞 - 细胞相互作用，例如软骨和皮肤。一个主要组成部分 细胞外基质是透明质酸（HA），它是含有交替的N-乙酰葡萄糖（NAG）和葡萄糖酸（GA）残基的细胞外线性多糖。 HA会影响许多生理过程，从细胞粘附和迁移到细胞分化和胚胎学发育。由于它对人类生理的广泛影响，包括多种形式的癌症，自身免疫性疾病，炎症过程和类风湿关节炎，与HA的表达水平改变相关。在分子水平上，HA是由膜膜的透明质酸合酶（HAS）在细胞内部产生的。有一个非凡的酶。它不仅催化了从UDP激活的底物中HA的合成，而且还可以将生长的聚合物跨过细胞膜传递，以将其沉积在细胞外基质中。为了完成这项任务，必须完成 几个功能。该酶结合底物UDP -NAG和-GA，它催化糖基转移反应形成HA，并通过其自身跨膜区域形成的孔迁移到整个细胞膜上的生长聚合物。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特性的HA聚合物，我们必须首先阐明如何合成HA及其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，这些目标将揭示生物学活性的组装在天然脂质膜中具有亚基，将确定HAS和转移HA聚合物之间的相互作用，并使我们能够通过X射线晶体学确定HAS的结构。首先，我们将共免疫沉淀研究与化学交联和光漂白技术相结合，以可视化天然膜的低聚物。然后，低分辨率的结构数据将与单体单体的高分辨率结构集成，以重建天然的，膜上包裹的膜具有低聚物。其次，将HAS与转运HA聚合物的相互作用将通过将UV诱导的交联链引入HAS的TM区域来映射。 HA易位期间的交联将识别与多糖近距离接近的位置，从而描绘出HA跨膜通道的物理化学特性。第三，生化和低分辨率结构数据将与通过X射线晶体学获得的高分辨率结构集成。确定在洗涤剂 - 溶解的和膜上包裹的状态中的结构，将揭示合酶的结构和低聚物形式，从而使我们能够描述这种奇妙的酶合成的机​​制，使这种机制是人类体内最丰富的细胞外多糖之一。