CHARACTERIZATION OF THE ENZYMES PmHAS AND MUTANTS OF PmHS2 IN THE SYNTHESES OF HYALURONIC ACID AND HEPARAN SULFATE, RESPECTFULLY

Glycosaminoglycans (GAGs) are essential components of the extracellular matrix (ECM) with diverse biological functions in development, homeostasis, and disease. Among them, hyaluronic acid (HA) and heparan sulfate (HS) stand out for their unique structural properties and distinct biological functions. HA, a non-sulfated, linear GAG, is primarily involved in maintaining tissue hydration, while HS, a highly sulfated GAG, interacts with a wide array of proteins to modulate growth factor signaling and cellular communication. Their respective biosynthetic enzymes, PmHAS and PmHS2, derived from Pasteurella multocida, have become valuable tools for studying GAG biosynthesis due to their ability to polymerize hyaluronic acid and heparosan, respectively, with these efforts still ongoing. HA is known to accumulate in the bloodstream during liver injury, making it a promising non-invasive biomarker for liver fibrosis and other chronic liver diseases. However, the precise chain length of the accumulated HA remains unclear. This thesis aims to deepen our understanding of the PmHAS enzyme, which is essential for synthesizing HA in a size-controlled manner for its potential use as a biomarker. In contrast, PmHS2 is a better-characterized enzyme, and the work presented here further explores how changes in specific amino acid residues affect donor substrate specificity and enhance the incorporation of pre-modified sulfated residues. These insights support the efficient synthesis of HS structures, with potential applications in the development of novel therapeutics.