Karasinska, J. | Search bioRxiv

bioRxiv2025

Insights into substrate binding and utilization by hyaluronan synthase

Hyaluronan (HA), a heteropolysaccharide of alternating N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA), is an essential component of the vertebrate extracellular matrix. HA biosynthesis proceeds via three evolutionarily convergent reaction mechanisms, catalyzed by the membrane integrated class 1 enzymes that either elongate the non-reducing (NR) or reducing end of HA, as well as the class 2 hyaluronan synthase (HAS), a soluble non-processive enzyme. Class 1-NR HAS, found in both vertebrates and large double-stranded DNA viruses, is monomeric and couples HA polymerization via coordinated transfer of UDP-GlcNAc and UDP-GlcA substrates with the secretion of the nascent HA chain through its own transmembrane channel. How this HAS discriminates between two UDP-sugars using a single active site is a critical, yet unresolved question. Using single-particle cryo-EM, we reveal a two-step process by which the Chlorella virus HAS (CvHAS) recognizes and positions its substrate, UDP-GlcA, for glycosyl transfer. Further, we report greatly diminished turnover of UDP-GlcA in the absence of a primer, distinguishing acceptor free activity toward UDP-GlcNAc. Lastly, prompted by observation of a dodecyl maltoside bound HAS, we demonstrate the utility of non-canonical acceptors in priming of a UDP-GlcA transfer reaction. Altogether, this work clarifies the molecular basis for HAS dual substrate specificity and the role of UDP-GlcA recognition in integrity of HA synthesis.

biophysics↗

bioRxiv2020

Recombinant T cell receptors specific for HLA-A*02:01-restricted neoepitopes containing KRAS codon 12 hotspot mutations

KRAS codon 12 mutations are among the most common hotspot mutations in human cancer. Using a functional screening platform we set out to identify {beta} T-cell receptors (TCRs) as potential targeting reagents for KRASG12D and/or KRASG12V neoepitopes presented by the prevalent HLA-A*02:01 allele. Here we describe isolation and characterization of three distinct CD8+ T cell clones from a pre-treated 76 year old patient with pancreatic ductal adenocarcinoma (PDAC). One clone was KRASG12V reactive and two clones were KRASG12D reactive. Tetramer staining showed high specificity of each T cell clone for its cognate HLA-A*02:01 restricted KRASG12V or KRASG12D neoepitope (>98% tetramer positive) without appreciable cross-reactivity to wild-type KRAS (<2% tetramer positive). We amplified and sequenced the full-length TCR alpha and beta chains from each of the three T cell clones and determined that these three TCRs comprised distinct combinations of two different TCR alpha chains and two distinct TCR beta chains. We resynthesized these TCR alpha and beta chain nucleotide sequences and reconstituted the original pairs in healthy donor CD8+ T cells by lentiviral transduction, substituting the human {beta} TCR constant gene segments with murine {beta} TCR constant gene segments to prevent mispairing with endogenous TCR subunits. Tetramer analysis and IFN-{gamma} ELISpot analysis confirmed the specificity of each reconstituted TCR for its cognate HLA-A*02:01 restricted KRAS neoepitope. To test cytolytic activity TCR-transduced healthy donor CD8+ T cells were co-cultured with KRASG12V, KRASG12D or KRASwt peptide-pulsed K562-HLA-A*02:01 antigen presenting cells at an effector to target cell ratio of 4:1. Under these conditions we observed neoepitope-specific killing of 16.5% to 19.0% of target cell populations. To assess in vivo activity we developed a KRASG12V/A*02:01 patient-derived xenograft (PDX) mouse model. Over a 56-day period, PDX bearing mice infused with human TCR-transduced T cells had significantly reduced tumor growth and longer survival compared to mice infused with non-transduced control T cells. In conjunction with other therapeutic approaches, immune effector cell therapies expressing these TCRs may improve outcomes for HLA-A*02:01 patients with KRASG12V and/or KRASG12D positive tumors.

immunology↗