STRUCTURE AND FUNCTION OF HA BINDING PROTEINS/RECEPTORS

透明质酸或透明质酸(HA)是一种重要的结构和 脊椎动物细胞外基质的调节成分。在人类身上 HA的体内总周转率和代谢率为4-10g/d。在我们四个人中 为了这个项目，我们将提纯和描述几个不同的 HA相关蛋白。我们的长期目标是了解这个结构 以及与HA相互作用的各种重要蛋白质的功能。 我们在下一个资助期的短期目标将是阐明 下列蛋白质的结构。(I)肝细胞HA/GAG结合 蛋白。大鼠肝细胞胞膜丰富 与透明质酸和其他糖胺多聚糖(GAG)结合的蛋白质复合体。这 复合体包含至少六个不同的亚基(M-r-S约34， 48、51、59、76和250 kD)通过二硫键结合在一起。医管局 结合亚基被鉴定为51kD亚基，并已纯化 大约35,000倍。其他亚基也将被提纯并 在分子和细胞水平上具有特征。序列信息 将在完整的亚基和/或纯化的蛋白水解物或 溴化碳碎片。针对纯化的亚基或合成的抗体 然后，基于氨基酸序列的寡核苷酸将用于 克隆Lambda gtlO中新发现的蛋白的cDNA 大鼠肝脏c DNA文库。细菌的亚细胞分布 HA/GAG结合复合体将通过荧光显微镜进行评估 和传输EM。(Ii)肝内皮细胞HA受体为 负责最终将HA从循环中移除，并负责 维持血液中低水平的透明质酸。然而，血清HA水平可以 某些癌症、类风湿性关节炎或 多种形式的肝硬变。我们已经鉴定出两种高相对分子质量蛋白质 在透过性LEC中用HA光亲和探针进行专门标记。 我们将提纯和鉴定这些HA受体亚基 分子和细胞水平，如上所述。(Iii)链球菌HA 合成酶。A组和C组链球菌，其中许多是人类 病原体表达一种HA合成酶，使它们能够合成 细胞外透明质酸胶囊，有助于其致病性和 帮助它们逃避宿主的免疫反应。我们将定义 链球菌RA合酶基因编码蛋白质的区域(S) 可以绑定HA的域。我们的目标是定义氨基酸序列 需要制作一个HA结合部位。然后我们将研究这一关系 通过改变DNA序列来改变蛋白质结构和功能 定点突变。

Hyaluronic acid or hyaluronan (HA) is an important structural and regulatory component of the vertebrate extracellular matrix. In humans the total body turnover and metabolism of HA is 4-10 g/day. In our four aims for this project, we will purify and characterize several different HA-related proteins. Our long term goal is to understand the structure and function of a variety of important proteins that interact with HA. Our short term goal during the next grant period will be to elucidate the structure of the following proteins. (i) Hepatocyte HA/GAG binding protein. Rat liver hepatocytes have an abundant intracellular membrane protein complex that binds HA and other glycosaminoglycans (GAGs). This complex contains at least six different subunits (M-r-s approximately 34, 48, 51, 59, 76 and >250 kD) held together by disulfide bonds. The HA binding subunit was identified as the 51 kD subunit and has been purified approximately 35,000-fold. The other subunits will also be purified and characterized at the molecular and cellular level. Sequence information will be obtained on the intact subunits and/or purified proteolytic or CNBr fragments. Antibodies to the purified subunits or synthetic oligonucleotides based on the amino acid sequence will then be used to clone the cDNA of novel, newly recognized proteins from lambda gtlO or lambda gtll rat liver cDNA libraries. The subcellular distribution of the HA/GAG binding complex will be assessed by fluorescence microscopy and transmission EM. (ii) The Liver endothelial cell (LEC) HA receptor is responsible for the final removal of HA from the circulation and for maintaining a low level of HA in the blood. However, serum HA levels can rise dramatically in patients with some cancers, rheumatoid arthritis or many forms of cirrhoses. We have identified two high MW proteins that are specifically labeled in permeable LEC with an HA photoaffinity probe. We will purify and characterize these HA receptor subunits at the molecular and cellular level as indicated above. (iii) Streptococcal HA synthase. Group A and C Streptococcal strains, many of which are human pathogens, express an HA synthase that allows them to synthesize an extracellular HA capsule that contributes to their pathogenicity and helps them escape the host immunological response. We will define the region(s) of the Streptococcal RA synthase gene that codes for protein domains that can bind HA. Our goal is to define the amino acid sequences needed to make an HA binding site. We will then examine the relationship between protein structure and function by altering the DNA sequence by site directed mutagenesis.