Molecular basis of hyaluronan synthesis

Hyaluronan (HA) is an essential component of the vertebrate extracellular matrix (ECM). It is enriched in soft connective tissues, the vitreous of the eye, and cartilage. HA affects important physiological processes, including cell migration, proliferation, and adhesion. Accordingly, many pathological conditions from autoimmune diseases to infertility and cancer correlate with altered HA production levels. Yet the molecular basis of how HA is synthesized and exerts its physiological functions remains poorly understood.
HA’s physiological activities depend on its length. High molecular weight (HMW) HA contributes to wound healing and has anti-inflammatory properties, whereas low molecular weight (LMW) HA is involved in cell migration, and in high concentration, pathologies such as cancer metastasis. Large quantities of unusually HMW HA are believed to be the reason behind cancer resistance and longevity of some subterranean rodents.
HA is a heteropolysaccharide of alternating units of N-acetylglucosamine and glucuronic acid synthesized by HA-synthase (HAS). HAS is an unusual enzyme for three reasons. Firstly, it recognizes its two uridine-diphosphate (UDP)-activated substrates through a single binding pocket. Second, it catalyzes formation of HA by incorporating the substrates into the growing polysaccharide in a strictly alternating fashion. Third, HAS translocates HA during synthesis across the plasma membrane through a channel formed by its membrane-embedded region for integration into the ECM. Vertebrates express three HAS isoforms that produce HA of different sizes. HAS isoform 2 is essential.
Determining the molecular basis for HA synthesis and length control requires detailed structural and functional insights. In my graduate research, I characterized a heterologously expressed vertebrate HAS to determine factors controlling substrate polymerization and product length. I used cryogenic electron microscopy to determine the first structural snapshots of a vertebrate HAS homolog and provided insights into HA biosynthesis and translocation. I revealed the coordination of the UDP product by a conserved gating loop and captured the opening of the translocation channel to coordinate the translocating HA polymer. Using site-directed mutagenesis studies, I identified active site- and channel-lining residues that modulate HA product lengths. By integrating structural and biochemical analyses, my research provided unprecedented insights into the biosynthesis of one of the most abundant extracellular polysaccharides in the human body and established the molecular basis for understanding its function in health and disease.