STRUCTURE AND FUNCTION OF HA BINDING PROTEINS/RECEPTORS

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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.