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(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，帮助渗透调节，在调节细胞功能中尤为重要。 软结缔组织中的细胞间相互作用，如软骨和皮肤。的一个主要组成部分 细胞外基质为透明质酸(HA)，是一种含有N-乙酰氨基葡萄糖(NAG)和葡萄糖醛酸(GA)残基的胞外线性多糖。HA影响许多生理过程，从细胞黏附和迁移到细胞分化和胚胎发育。由于HA对人体生理的广泛影响，许多病理情况，包括多种形式的癌症、自身免疫性疾病、炎症过程和类风湿性关节炎，都与HA表达水平的变化有关。在分子水平上，透明质酸在细胞内由膜包埋的透明质酸合成酶(HAS)产生。HAS是一种了不起的酶。它不仅催化从UDP激活的底物合成HA，而且还将生长的聚合物穿过细胞膜，将其沉积在细胞外基质中。为了完成这项任务，必须完成 有几个功能。该酶与底物UDP-NAG和-GA结合，催化糖基转移反应形成HA，并通过自身跨膜区形成的孔将生长的聚合物转移到细胞膜上。为了了解HA如何发挥其生理功能，并生产出具有特定性质的HA聚合物用于生物医学，我们必须首先了解HA是如何合成的，以及它是如何将聚合物沉积在细胞外基质中的。为此，我们提出了三个目标，将揭示生物活性HAS亚单位在天然脂膜中的组装，将识别HAS与转位的HA聚合物之间的相互作用，并将使我们能够通过X射线结晶学来确定HAS的结构。首先，我们将结合免疫共沉淀研究与化学交联和光漂白技术来可视化天然膜中的HAS低聚物。然后将低分辨率的结构数据与高分辨率的单体HAS结构进行整合，重构天然的、膜包埋的HAS齐聚物。其次，通过将紫外光诱导的交联剂引入HAS的TM区，HAS与移位的HA聚合物的相互作用将被映射。在HA转运过程中的交联会识别靠近多糖的位置，从而描绘HA跨膜通道的物理化学性质。第三，生化和低分辨率结构数据将与通过X射线结晶学获得的HAS的高分辨率结构相结合。测定HAS在洗涤剂溶解和膜包埋状态下的结构将揭示合成酶的结构和低聚形式，使我们能够描绘这种神奇的酶合成人体中最丰富的胞外多糖之一的机制。