STRUCTURE AND FUNCTION OF HA BINDING PROTEINS/RECEPTORS

人类每天要消耗几克透明质酸（hyaluronan，HA）， 脊椎动物细胞外基质的重要结构成分。HA 还具有多种生物学效应，并且越来越多地被 被认为是能够调节细胞内 反应和行为。HA参与转移和伤口愈合， 疾病，如类风湿性关节炎和骨关节炎，是一种毒力因子 化脓性链球菌（一种重要的人类病原体）。日益 HA在临床和外科应用以及药物递送中的应用 强调了理解HA合成和 在健康和疾病状态下控制降解。 我们的长期 目的是了解重要HA的结构-功能关系 受体，酶和结合蛋白，以及HA如何影响细胞行为。 我们在这个延续期的主要目标是描述两个关键的 HA代谢中的蛋白质，最近克隆的细菌HA合酶和 介导HA内吞和清除的特异性肝受体 从血液中。我们的具体目标是：1）表征纯化的 链球菌HA合酶的生化和酶。表达 含有C-末端6xHis簇的构建体（在pKK 223 -3中）将允许 用镍螯合物亲和纯化正常或变异的酶 层析;固定化的酶反过来允许亲和性 纯化酶的抗体。我们将确定二硫化物 键排列，关键半胱氨酸的位置，酶的拓扑结构，UDP- 糖结合位点、HA合成方向以及引物是否 必需的. 2)为了阐明结构-功能关系， 重组链球菌HA合酶。将针对7种药物开发的试验 酶的结合和酶活性将用于 筛选/选择由随机和定向产生的有缺陷的变体 诱变程序。这种方法将识别蛋白质结构域， 参与这7种活性的序列或残基。3)克隆并 鉴定大鼠肝脏内吞HA受体的cDNA 窦状隙内皮细胞将克隆175 kDa受体cDNA 使用基于肽序列数据的寡核苷酸，和/或新的 制备了多克隆抗体。4)建立真核细胞转染子 表达内吞大鼠HA受体。 表达系统将允许 我们进一步表征细胞功能和分布的 HA受体，以亲和纯化抗体并进行结构-功能 问题研究虽然不是目前的目标，但抗体和 这种受体的核酸探针将允许研究 首次发现其可能参与病理状态， 疾病这四个目标将采用化学， 生物化学和分子生物学方法。

Humans turn over grams/day of hyaluronic acid (hyaluronan, HA), which is an important structural component of vertebrate extracellular matrices. HA also elicits diverse biological effects, and is increasingly being recognized as a pharmacologic agent capable of modulating cellular responses and behavior. HA is involved in metastasis and wound healing, in diseases such as rheumatoid- and osteoarthritis, and is a virulence factor for Streptococcus pyogenes, an important human pathogen. The increasing use of HA in clinical and surgical applications and in drug delivery underscores the importance of understanding how HA synthesis and degradation are controlled in healthy and disease states. Our long-term goal is to understand the structure-function relationships of important HA receptors, enzymes and binding proteins, and how HA affects cell behavior. Our major goals in this continuation period are to characterize two key proteins in HA metabolism, the recently cloned bacterial HA synthase and the specific liver receptor that mediates endocytosis and clearance of HA from the blood. Our specific aims are: 1) To characterize the purified Streptococcus HA synthase biochemically and enzymatically. Expression constructs (in pKK223-3) containing a C-terminal 6xHis cluster will allow affinity-purification of normal or variant synthases by Ni-chelate chromatography; the immobilized enzyme in turn allows affinity purification of antibodies to the enzyme. We will determine the disulfide bond arrangement, position of critical cysteines, enzyme topology, UDP- sugar binding sites, direction of HA synthesis, and whether a primer is required. 2) To elucidate structure-function relationships within the recombinant Streptococcus HA synthase. Assays to be developed for the 7 binding and enzymatic activities of the enzyme will be used to screen/select defective variants generated by random and directed mutagenic procedures. This approach will identify protein domains, sequences or residues involved in these 7 activities. 3) To clone and characterize the cDNA for the endocytic HA receptor of rat liver sinusoidal endothelial cells. The 175 kDa receptor cDNA will be cloned using oligonucleotides based on peptide sequence data, and/or a newly developed polyclonal antibody. 4) To establish eukaryotic transfectants expressing the endocytic rat HA receptor. Expression systems will allow us to characterize further the cellular function and distribution of the HA receptor, to affinity-purify antibody and to do structure-function studies. Although not a present objective, the development of antibody and nucleic acid probes for this receptor will allow investigation for the first time of its potential involvement in pathological conditions and diseases. These four aims will employ a combination of chemical, biochemical and molecular biological approaches.