Biology subjects

Mycroft-West, C. J.

Publications and source records attributed to Mycroft-West, C. J..

7 recordsLinked to original sources

bioRxiv2026

Hexosyl transfer via a covalent intermediate within a dual retaining and inverting enzyme

Bifunctional glycosyltransferases underpin biosynthesis of essential glycans, including bacterial capsular antigens, and eukaryotic glycosaminoglycans. Typically, these enzymes transfer hexose sugars from activated nucleotide-phosphate donors, forming new glycosidic bonds with either retention or inversion of anomeric stereochemistry. Retaining hexosyl transfer typically proceeds via non-covalent SNi-type pathways, but recent studies of octulosyl Kdo-transferases have reignited debate over covalent mechanisms. Here we comprehensively dissected the activity of the archetypal bifunctional glycosyltransferase PmHS2, providing X-ray structures of ligand complexes alongside atomistic simulations to delineate full substrate-to-product reaction trajectories. Combined computational, biochemical and evolutionary evidence suggests that PmHS2 retaining GlcNAc-transfer likely proceeds via a previously undescribed covalent mechanism, involving considerable assistance from departing nucleotide-phosphate rotation. In mapping reaction pathways, we also report the first example of a glycosyl donor substrate coopted as an acceptor, which enables enzymatic self-priming for polymerization. These new covalent and self-priming functionalities highlight unexplored routes to modulate or inhibit polysaccharide biosynthesis.

biochemistry↗

bioRxiv2026

Molecular organization of the eukaryotic secretory pathway

The eukaryotic secretory pathway is an essential subcellular network comprised of the endoplasmic reticulum (ER) and Golgi organelles. It mediates the trafficking of protein, lipid and other biomolecular cargoes throughout the cell, and is also the site of enzymatic modifications such as glycosylation that are vital for biological function. Complex interactions between resident functional proteins and cargo biomolecules drive myriad activities within the secretory pathway, but how these components organize within native milieu is still poorly understood. Here, we used cryo-electron tomography to survey the HeLa secretory pathway, revealing detailed features of its organellar structure. By quantitatively mapping protein distributions, we find that interactions within the HeLa secretory pathway are dominated by small dimeric complexes, stochastically distributed across both ER and Golgi membranes. Our survey of secretory pathway biomolecules provides new insights into putative trafficking and modification mechanisms, and highlights links between molecular organization and function within intracellular compartments.

cell biology↗

bioRxiv2024

Enzymatic access to the rare ΔUA (1->4) Glc 3, 6, N-sulfated heparin disaccharide, implications for heparin quality control

The sulfated glycosaminoglycan heparin is the most commonly used pharmaceutical anticoagulant worldwide. Heparin, which is extracted primarily from porcine sources, has a complex heterogeneous structure, resulting in a highly variable pharmaceutical product susceptible to contamination. As a by-product of the food industry, heparin is also limited by production capacity, giving rise to concerns that demand will outstrip supply. The anticoagulant activity of heparin derives principally from the AGA*IA pentasaccharide sequence, containing a rare 3-O-sulfated glucosamine, which binds and activates antithrombin. Analytical heparin digestion by the widely used Pedobacter heparinus lyases has limited activity in regions of 3-O-sulfation, rendering these enzymes poorly suited to study anticoagulant sequences. Here, we provide structural and functional characterization of a Bacteroides eggerthii lyase that exhibits highly efficient heparin depolymerization, with specificity distinct to P. heparinus. Using a panel of biophysical and structural techniques, we demonstrate that B. eggerthii lyase effectively liberates the rare GA* disaccharide, a key indicator of anticoagulant potential, from the defined heparin pentasaccharide fondaparinux. We envision superior cleavage by B. eggerthii lyases will enable the future quantitative, direct detection of anticoagulant relevant 3-O-sulfated sequences, delivering complementary structural information to existing analytical methods, with clear utility for pharmaceutical quality control workflows.

biochemistry↗

bioRxiv2023

Structural and mechanistic characterization of bifunctional heparan sulfate N-deacetylase-N-sulfotransferase 1

Heparan sulfate (HS) polysaccharides are major constituents of the extracellular matrix, involved in myriad structural and signaling processes. Mature HS polysaccharides contain complex, non-templated patterns of sulfation and epimerization, which mediate interactions with diverse protein partners. Complex HS modifications form around initial clusters of glucosamine-N-sulfate (GlcNS) on nascent polysaccharide chains, but the mechanistic basis underpinning incorporation of the GlcNS modification itself into HS remains unclear. We have determined cryo-electron microscopy structures of human N-deacetylase-N-sulfotransferase (NDST)1, the bifunctional enzyme responsible for initial GlcNS modification of HS. Our structures reveal the architecture of both NDST1 deacetylase and sulfotransferase catalytic domains, alongside a previously unreported non-catalytic N-terminal domain. Surprisingly, the two catalytic domains of NDST1 adopt an unusual back-to-back topology that limits direct cooperativity. Binding analyses, aided by novel activity modulating nanobodies, suggest that sulfotransferase domain substrate anchoring initiates the NDST1 catalytic cycle, providing a plausible mechanism for cooperativity despite spatial domain separation. Our data shed light on key determinants of NDST1 activity, and describe tools to probe NDST1 function in vitro and in vivo.

biochemistry↗

bioRxiv2021

The hyperlipidaemic drug fenofibrate significantly reduces infection by SARS-CoV-2 in cell culture models

The SARS-CoV-2 pandemic has caused a significant number of fatalities and worldwide disruption. To identify drugs to repurpose to treat SARS-CoV-2 infections, we established a screen to measure dimerization of ACE2, the primary receptor for the virus. This screen identified fenofibric acid, the active metabolite of fenofibrate. Fenofibric acid also destabilized the receptor binding domain (RBD) of the viral spike protein and inhibited RBD binding to ACE2 in ELISA and whole cell binding assays. Fenofibrate and fenofibric acid were tested by two independent laboratories measuring infection of cultured Vero cells using two different SARS-CoV-2 isolates. In both settings at drug concentrations which are clinically achievable, fenofibrate and fenofibric acid reduced viral infection by up to 70%. Together with its extensive history of clinical use and its relatively good safety profile, these studies identify fenofibrate as a potential therapeutic agent requiring urgent clinical evaluation to treat SARS-CoV-2 infection. TeaserThe approved drug fenofibrate inhibits infection by SARS-COV-2

pharmacology and toxicology↗

bioRxiv2020

Pixatimod (PG545), a clinical-stage heparan sulfate mimetic, is a potent inhibitor of the SARS-CoV-2 virus

Heparan sulfate (HS) is a cell surface polysaccharide recently identified as a co-receptor with the ACE2 protein for recognition of the S1 spike protein on SARS-CoV-2 virus, providing a tractable new target for therapeutic intervention. Clinically-used heparins demonstrate inhibitory activity, but world supplies are limited, necessitating alternative solutions. Synthetic HS mimetic pixatimod is a drug candidate for cancer with immunomodulatory and heparanase-inhibiting properties. Here we show that pixatimod binds to and destabilizes the SARS-CoV-2 spike protein receptor binding domain (S1-RBD), and directly inhibits its binding to human ACE2, consistent with molecular modelling identification of multiple molecular contacts and overlapping pixatimod and ACE2 binding sites. Assays with multiple clinical isolates of live SARS-CoV-2 virus show that pixatimod potently inhibits infection of monkey Vero E6 and human bronchial epithelial cells at concentrations within its safe therapeutic dose range. Furthermore, in a K18-hACE2 mouse model pixatimod demonstrates that pixatimod markedly attenuates SARS-CoV-2 viral titer and COVID-19-like symptoms. This demonstration of potent anti-SARS-CoV-2 activity establishes proof-of-concept for targeting the HS-Spike protein-ACE2 axis with synthetic HS mimetics. Together with other known activities of pixatimod our data provides a strong rationale for its clinical investigation as a potential multimodal therapeutic to address the COVID-19 pandemic.

microbiology↗

bioRxiv2020

The 2019 coronavirus (SARS-CoV-2) surface protein (Spike) S1 Receptor Binding Domain undergoes conformational change upon heparin binding.

Many pathogens take advantage of the dependence of the host on the interaction of hundreds of extracellular proteins with the glycosaminoglycans heparan sulphate to regulate homeostasis and use heparan sulphate as a means to adhere and gain access to cells. Moreover, mucosal epithelia such as that of the respiratory tract are protected by a layer of mucin polysaccharides, which are usually sulphated. Consequently, the polydisperse, natural products of heparan sulphate and the allied polysaccharide, heparin have been found to be involved and prevent infection by a range of viruses including S-associated coronavirus strain HSR1. Here we use surface plasmon resonance and circular dichroism to measure the interaction between the SARS-CoV-2 Spike S1 protein receptor binding domain (SARS-CoV-2 S1 RBD) and heparin. The data demonstrate an interaction between the recombinant surface receptor binding domain and the polysaccharide. This has implications for the rapid development of a first-line therapeutic by repurposing heparin and for next-generation, tailor-made, GAG-based antivirals.

biochemistry↗