Hyaluronan (HA) is a ubiquitous ECM component in vertebrates. HA is a powerful modulator of cell behavior and plays key roles in cell migration, development, cancer, wound healing and angiogenesis. The long-term hypothesis guiding this project is that HA synthesis and degradation must be tightly controlled for normal homeostasis and health. Birth defects, ulcerating wounds, arthritis, and cancer may be promoted by alterations in HA synthesis or degradation. The project contains two subprojects that are investigating HA synthesis and HA turnover. The first subproject is studying the streptococcal enzyme HA synthase (HAS), which we have no purified. This HAS makes the HA capsule of Group A and Group C cells, which is a significant virulence factor. Our hypothesis in this subproject is that structure-function analyses of the streptococcal HASs will allow us to understand how HAS works and how HA synthesis is regulated. The second subproject is investigating the final stage in mammalian HA turnover, which is the endocytosis of HA by liver endothelial cells (LECs). Monoclonal antibodies (MoAbs) to the rat LEC HA receptor (HAR) have enable us to block HA uptake by LECs and to affinity purify two HARs of 175kD and ~300kD. We have isolated a 3.9kb partial cDNA for the 17kD HAR, encoding a unique protein recognized by several of our MoAbs. Our hypothesis in this subproject is that LECs contain two highly similar isoreceptors for HA and that each HAR may be specialized to interact with either smaller or larger HA. We will employ techniques in biochemistry molecular biology and cell biology to address these hypotheses in the following aims. 1) To characterize streptococcal HAS mechanisms and structure-function relationships using biophysical techniques. 2) To create streptococcal HAS mutants with specific functional defects for structure-function studies. 3) To clone and express the mouse and human cDNAs for the 175kD HAR of LECs. 4) To clone and express the cDNAs for subunits of the 300kD HAR complex of LECs. 5) To elucidate structure-function relationships of the 17kD and 300kD HARs. The results from this project will allow us to investigate, for the first time, the role of the LEC HAR in various pathologies and diseases and could lead to new drugs for streptococcal diseases.
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