Sweet, I. | bioRxiv Science

bioRxiv2024

2,2-Di-Fluoro-Derivatives of Fucose Can Inhibit Cell Surface Fucosylation Without Causing Slow Transfer to Acceptors

Fucosyltransferases (FUTs) are enzymes that transfer fucose (Fuc) from GDP-Fuc to acceptor substrates resulting in fucosylated glycoconjugates that are involved in myriad of physiological and disease processes. Previously, it has been shown that per-O-acetylated 2-F-Fuc can be taken up by cells and converted into GDP-2-F-Fuc which is a competitive inhibitor of FUTs. Furthermore, it can act as a feedback inhibitor of de-novo biosynthesis of GDP-Fuc resulting in reduced glycoconjugate fucosylation. GDP-2-F-Fuc and several other reported analogs are slow substrates, which can result in unintended incorporation of unnatural fucosides. Here, we describe the design, synthesis, and biological evaluation of GDP-2,2-di-F-Fuc and corresponding prodrugs as inhibitor of FUTs. This compound lacks the slow transfer activity observed for the mono-fluorinated counterpart. Furthermore, it was found that GDP-2-F-Fuc and GDP-2,2-di-F-Fuc have similar Ki values for the various human fucosyl transferases while the corresponding phosphate prodrugs exhibit substantial differences in inhibition of cell surface fucosylation. Quantitative sugar nucleotides analysis by LC-MS indicates that the 2,2-di-F-Fuc prodrug has substantial greater feedback inhibitory activity. It was also found that by controlling the concentration of the inhibitor, varying degrees of inhibition of the biosynthesis of different types of fucosylated N-glycan structures can be achieved. These findings open new avenues for the modulation of fucosylation of cell surface glycoconjugates.

biochemistry↗

bioRxiv2023

H7 influenza A viruses bind sialyl-LewisX, a potential intermediate receptor between species

Influenza A viruses (IAVs) can overcome species barriers by adaptation of the receptor binding site of the hemagglutinin (HA). To initiate infection, HAs bind to glycan receptors with terminal sialic acids, which are either N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc), the latter is mainly found in horses and pigs but not in birds and humans. We investigated the influence of previously identified equine NeuGc-adapting mutations (S128T, I130V, A135E, T189A, and K193R) in avian H7 IAVs in vitro and in vivo. We observed that these mutations negatively affected viral replication in chicken cells, but not in duck cells, and positively affected replication in horse cells. In vivo, the mutations reduced virus virulence and mortality in chickens. Ducks excreted high viral loads for a longer time than chickens, although they appeared clinically healthy. To elucidate why chickens and ducks were infected by these viruses despite the absence of NeuGc, we re-evaluated the receptor binding of H7 HAs using glycan microarray and flow cytometry studies. This revealed that mutated avian H7 HAs also bound to 2,3-linked NeuAc and sialyl-LewisX, which have an additional fucose moiety in their terminal epitope, explaining why infection of ducks and chickens was possible. Interestingly, the 2,3-linked NeuAc and sialyl-LewisX epitopes were only bound when presented on tri-antennary N-glycans, emphasizing the importance of investigating the fine receptor specificities of IAVs. In conclusion, the binding of NeuGc-adapted H7 IAV to sialyl-LewisX enables viral replication and shedding by chickens and ducks, potentially facilitating interspecies transmission of equine-adapted H7 IAVs. (249 words) ImportanceInfluenza A viruses cause millions of deaths and illness in birds and mammals each year. The viral surface protein hemagglutinin initiates infection by binding to host cell terminal sialic acids. Hemagglutinin adaptations affect the binding affinity to these sialic acids and therefore the potential host species targeted. While avian and human IAVs tend to bind N-acetylneuraminic acid (a form of sialic acid), equine H7 viruses prefer binding to N-glycolylneuraminic acid (NeuGc). To better understand the function of NeuGc-specific adaptations in hemagglutinin and to elucidate interspecies transmission potential NeuGc-adapted viruses, we evaluated the effects of NeuGc-specific mutations in avian H7 viruses in chickens and ducks, important economic hosts and reservoir birds, respectively. We also examined the impact on viral replication and found a binding affinity to sialyl-LewisX, another terminal epitope. These findings are important as they contribute to the understanding of the role of sialyl-LewisX in avian influenza infection. (148 words)

molecular biology↗