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功能性蛋白质， 脊椎动物细胞外基质的调节成分。 在人类中 HA的总身体周转和代谢为4-10 g/天。 在我们的四个 针对这个项目，我们将纯化和表征几种不同的 HA相关蛋白。 我们的长期目标是了解 与HA相互作用的多种重要蛋白质的功能。 我们在下一个资助期的短期目标是阐明 以下蛋白质的结构。(i)肝细胞HA/GAG结合 蛋白 大鼠肝细胞具有丰富的胞内膜 结合HA和其他糖胺聚糖（GAG）的蛋白质复合物。 这 复合物含有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 经鉴定，该结合亚基为51 kD亚基，并已纯化 大约35,000倍。 其他亚基也将被纯化， 在分子和细胞水平上表征。 序列信息 将在完整的亚基和/或纯化的蛋白水解或 溴化氰碎片。 针对纯化亚基或合成亚基的抗体 基于氨基酸序列的寡核苷酸然后将用于 从λ gt 10或λ gt 12中克隆新的、新识别的蛋白质的cDNA λ gtll大鼠肝cDNA文库。 的亚细胞分布 将通过荧光显微镜评估HA/GAG结合复合物 传输EM。(ii)肝内皮细胞（LEC）HA受体是 负责最终将HA从流通中清除， 保持血液中低水平的HA。 然而，血清HA水平可以 在某些癌症、类风湿性关节炎或 多种形式的装饰。 我们已经鉴定了两种高分子量蛋白质， 用HA光亲和探针在可渗透LEC中特异性标记。 我们将纯化和表征这些HA受体亚单位在 分子和细胞水平，如上所述。(iii)链球菌HA 合成酶 A组和C组链球菌菌株，其中许多是人类 病原体，表达HA合酶，使它们能够合成 有助于其致病性的细胞外HA包膜， 帮助它们逃避宿主的免疫反应 我们将定义 编码蛋白质的链球菌RA合酶基因的区域 可以结合HA的结构域。 我们的目标是确定 形成HA结合位点。 然后我们将研究 蛋白质结构和功能之间的联系， 定点诱变

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.