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.
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