Abstract
In human tissues, cells are surrounded by a heterogenous microenvironment of proteins, proteoglycans and polysaccharides. This so-called extracellular matrix (ECM) bears an essential role in tissue organization, specialization and repair processes. One of the major players in mammalian ECMs is hyaluronan (HA), a mixed linkage heteropolysaccharide of regularly alternating N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) sugars. HA is best recognized for its size-dependent influence on cellular physiology, acting as an acidic, viscoelastic scaffold that directs cell growth, prevents cellular desiccation and modulates inflammatory signaling. Mis-regulation of HA metabolism is a driver of arthritis in cartilaginous tissue, as well as tumor metastasis in breast and prostate cancers.
HA is synthesized by a single, membrane-embedded glycosyltransferase, HA synthase (HAS). HAS performs two functions – (1) it builds a nascent HA polymer from UDP-GlcNAc and UDP-GlcA substrates and (2) it secretes HA across the plasma membrane through a channel formed by its own transmembrane domain. Underlying both of these functions are a set of coordinated activities that confer HA sequence specificity and prevent premature polysaccharide release.
Previous work on a viral HAS demonstrated that HA biosynthesis is initiated through a self-priming event, where HAS binds UDP-GlcNAc to catalyze a hydrolysis reaction. The de novo GlcNAc product is prompted to diffuse to an acceptor site formed at HAS’ channel entrance, generating a reducing end cap for HA polymers to be built upon. In the subsequent reaction step, HAS must recruit its other substrate, UDP-GlcA, for a β-(1,3) transfer reaction with the priming GlcNAc residue. How HAS binds two chemically distinct UDP-sugars using a single active site, catalyzes temporally selective sugar transfer in the absence of a template, and positions the donor and acceptor molecules for glycosyl transfer remain important, unresolved questions.
To this end, I’ve utilized a combination of cryo-electron microscopy, enzyme kinetics, biophysical measurements and glycobiology to better understand the mechanism of alternating glycosyl transfer chemistry encoded by HAS. Firstly, I determined high resolution cryoEM structures of the viral HAS bound to its substrate UDP-GlcA, both in the presence and absence of a GlcNAc primer. Combining this work with quantification of substrate affinity and turnover efficiency, I discerned that UDP-GlcA stabilization for glycosyl transfer is driven by primer dependent conformational rearrangements. Secondly, through in vitro reconstitution of HA synthesis I captured cryoEM snapshots of an HA disaccharide bound at HAS’ channel entrance. Observations of disaccharide placement both before and after nucleotide release led me to conclude that HAS requires an additional driving force beyond the free-energy of glycosyl transfer to catalyze HA translocation. Lastly, observation of a dodecyl maltoside (DDM) bound HAS provided an unexpected blueprint for exploring patterns of acceptor promiscuity. To this end, I showed that HAS can semi-selectively transfer GlcA to β-linked disaccharides. This observation suggested that acceptor site features are preserved across closely related processive glycosyltransferases. Given the apparent stability of DDM at HAS’ acceptor site, I’ve postulated that this new cryoEM structure may serve as a productive starting point for the rational design of first-generation HAS inhibitors.
