Biology subjects

Boons, G.-J.

Publications and source records attributed to Boons, G.-J..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Chemical Synthesis of Oligosaccharides Derived from Streptococcus pneumoniae Serotype 35B and D to Investigate Binding of Complement and Serum Factors

The wide use of Streptococcus pneumoniae capsular polysaccharide (CPS) conjugate vaccines is causing serotype replacement and the emergence of serotype 35B is concerning because of its multidrug resistance. CPS of 35B is composed of pentasaccharide repeating units that are linked through phosphodiester linkages. One of the galactofuranose residues of the pentasaccharide is acetylated, which distinguishes it from the invasive serotype 35D lacking the acetyl ester. Here, we describe a synthetic approach that can provide oligosaccharides derived of CPS 35B and 35D composed of up to 15 monosaccharides using a pentasaccharide building block equipped with four orthogonal protecting groups. The synthetic compounds were used to examine binding properties of ficolin-2, which is a protein that can activate the lectin pathway of the complement system. It was found that O-acetylation is essential for recognition by ficolin-2 requiring at least two repeating units. The data provides a rationale why 35D may escape immune detection and be more invasive. The synthetic oligosaccharides were also investigated for binding to pneumococcal serum factor 35a and 29b, which are employed for serotype identification. It indicates that immunization with 35B CPS will not provide protection against 35D and thus inclusion of 35B in vaccines may result in serotype replacement by 35D. On the other hand, antibodies that can bind 35D can recognize 35B and thus 35D CPS may provide cross protection. Our findings have direct implementation for the development of the next generation pneumococcal vaccine and provide an understanding of disease severity by the emerging serotype 35B and 35D.

biochemistry↗

bioRxiv2025

Sialic acid-containing glycolipids extend the receptor repertoire of Enterovirus-D68

Enterovirus D68 (EV-D68) emerged as a pathogen of increasing health concern globally, particularly due to its association with outbreaks of severe respiratory diseases and acute flaccid myelitis (AFM) in children. Knowledge regarding the tissue tropism and pathogenesis of EV-D68 within the respiratory tract and central nervous system remains limited, primarily due to an incomplete understanding of the host factors that facilitate EV-D68 entry into host cells. Several cellular receptors involved in EV-D68 infections have been identified, including ICAM-5, sialylated glycoproteins, and heparan sulfate (HS). Here, we investigate the receptor requirement of a panel of EV-D68 strains covering all clades focusing on HS and sialosides utilizing glycan arrays. We found that all EV-D68 strains binding to HS harbour a cell culture adaptative substitution in the structural protein VP1 at position 271 which changes the amino acid into a positive charged one. Glycan array analyses revealed that EV-D68 strains either prefer 2,6-linked sialic acids presented on N-glycans, 2,8 linked sialic acids on gangliosides, or both. Inhibition of glycolipid biosynthesis or multivalent glycolipid mimics confirmed that ganglioside structures serve as entry receptors for certain EV-D68 strains. Lastly, we examined whether EV-D68 strains that bind to HS or glycolipids require different uncoating mechanisms. Bafilomycin A1 minimally affected cell entry of HS-binding EV-D68 strains B2/039 and B2/947 and the ganglioside preferring B1/2013 other viruses were strongly inhibited. Together, we identified that EV-D68 strains can use disialoglycolipids as novel receptors and that different EV-D68 strains show a promiscuous sialic acid binding repertoire.

microbiology↗

bioRxiv2025

The Q226L mutation can convert a highly pathogenic H5 2.3.4.4e virus to bind human-type receptors

AbstractH5Nx viruses continue to wreak havoc in avian and mammalian species worldwide. The virus distinguishes itself by the ability to replicate to high titers and transmit efficiently in a wide variety of hosts in diverse climatic environments. Fortunately, transmission to and between humans is scarce. Yet, if such an event were to occur, it could spark a pandemic as humans are immunologically naive to H5 viruses. A significant determinant of transmission to and between humans is the ability of the influenza A virus hemagglutinin (HA) protein to shift from an avian-type to a human-type receptor specificity. Here, we demonstrate that a 2016 2.3.4.4e virus HA can convert to human-type receptor binding via a single Q226L mutation, in contrast to a cleavage-modified 2016 2.3.4.4b virus HA. Using glycan arrays, x-ray structural analyses, tissue- and direct glycan binding, we show that L133a{Delta} and 227Q are vital for this phenotype. Thus, whereas the 2.3.4.4e virus HA only needs a single amino acid mutation, the modified 2.3.4.4b HA was not easily converted to human-type receptor specificity.

microbiology↗

bioRxiv2024

Epistasis in the receptor binding domain of contemporary H3N2 viruses that reverted to bind sialylated diLacNAc repeats

Since the introduction of H3N2 influenza A viruses in the human population, these viruses have continuously evolved to escape human immunity, with mutations occurring in and around the receptor binding site. This process, called antigenic drift, recently resulted in viruses that recognize elongated glycans that are not abundantly displayed in the human respiratory tract. Such receptor specificities hampered our ability to pick and propagate vaccine strains. Using ELISA, glycan array, tissue staining, flow cytometry, and hemagglutinin assays, this study revealed that the most recent H3N2 viruses have expanded receptor specificity by regaining effective recognition to shorter glycans. In recent H3 strains, Y159 and T160 are responsible for restricted binding to elongated glycans; in contemporary strains, however, Y159N and T160I dominate with a consequent loss of strength in receptor binding. Yet, effective receptor interaction is rescued by a remote mutation in the 190-helix, Y195F. The results demonstrate epistasis of critical residues in three of the four structural elements composing the HA receptor-binding site (the 130-loop, 150-loop, and 190-helix), which synergistically contribute to shape receptor binding specificity. Interestingly, a positive correlation exists between binding to an asymmetrical N-glycan containing an 2,6 sialylated tri-LacNAc arm and binding to human and ferret respiratory tract tissues. Together, these results elucidate the epistatic nature of receptor binding specificity during influenza A virus H3N2 evolution.

microbiology↗

bioRxiv2024

A Light Sensitive Hyaluronic Acid-based Hydrogel for 3D Culturing, Differentiation, and Harvesting of Mesenchymal Stromal Cells

Hydrogels derived from natural sources are commonly used for 3D cell culturing due to their favourable interactions with cells. However, these biomaterials (e.g. Matrigel) suffer from variations in composition, limited mechanical tunability, the presence of xenogenic components and difficulties of cell retrieval. Semi-synthetic hydrogels are emerging to address these limitations, making these attractive for drug delivery and tissue engineering. Here, we describe a hydrogel platform based on hyaluronic acid (HA) modified by (1R,8S,9S)-bicycle[6.1.0]non-4-yn-9-ylmethanol (BCN) and a cross-linker composed of light sensitive o-nitrobenzyl moieties and polyethylene glycol (PEG) chains terminating in azides. The two components can undergo strain-promoted azide-alkyne cycloaddition (SPAAC) resulting in rapid gel formation. The stiffness of the hydrogel can be modulated by varying the crosslinker ratio and multi-functionalizing is possible by the incorporation of chemical cues modified by an azide. The fast hydrogelation enables easy encapsulation of cells and harvesting with preservation of viability is possible be short exposure to UV light. Moreover, it is demonstrated that visible light (405 nm) can soften the hydrogel resulting in phenotypical changes of human Mesenchymal Stromal Cells (hMSCs) co-cultured with endothelial colony-forming cells (ECFCs) with high viability. Our findings highlight that the photosensitive HA-based hydrogel provides a versatile and biocompatible platform for cell culture and tissue engineering applications, offering advantages over traditional 3D cell culturing platforms.

biochemistry↗

bioRxiv2024

A Chemoenzymatic Strategy for Site-Specific Glyco-Tagging of Native Proteins for the Development of Biologicals

Glycosylation is an attractive approach to enhance biological properties of pharmaceutical proteins, however, precise installation of glycans for structure-function studies remains challenging. Here, we describe a chemoenzymatic methodology for glyco-tagging of proteins by peptidoligase catalyzed modification of the N-terminus of a protein with a synthetic glycopeptide ester having an N-acetyl-glucosamine (GlcNAc) moiety to generate a N-GlcNAc modified protein. The GlcNAc moiety can be elaborated into complex glycans by trans-glycosylation using a well-defined sugar oxazolines and mutant forms of endo {beta}-N-acetylglucosaminidases (ENGases). The glyco-tagging methodology makes it possible to modify on-demand therapeutic proteins, including proteins heterologously expressed in E. coli, with diverse glycan structures. As a proof of principle, the N-terminus of interleukin (IL)-18 and interferon (IFN)-2a was modified by a glycopeptide harboring a complex N-glycan without compromising biological potencies. The glyco-tagging methodology was also used to prepare several glycosylated insulin variants that exhibit reduced oligomerization, aggregation and fibrillization yet maintained cell signaling properties, which is attractive for the development of insulins with improved shelf-lives. It was found that by employing different peptidoligases, it is possible to modify either the A or both chains of human insulin.

biochemistry↗

bioRxiv2024

A biofilm-tropic Pseudomonas aeruginosa bacteriophage uses the exopolysaccharide Psl as receptor

Bacteria in nature can exist in multicellular communities called biofilms. Biofilms also form in the course of many infections. Pseudomonas aeruginosa infections frequently involve biofilms, which contribute materially to the difficulty to treat these infections with antibiotic therapy. Many biofilm-related characteristics are controlled by the second messenger, cyclic-di-GMP, which is upregulated on surface contact. Among these factors is the exopolysaccharide Psl, which is a critically important component of the biofilm matrix. Here we describe the discovery of a P. aeruginosa bacteriophage, which we have called Clew-1, that directly binds to and uses Psl as a receptor. While this phage does not efficiently infect planktonically growing bacteria, it can disrupt P. aeruginosa biofilms and replicate in biofilm bacteria. We further demonstrate that the Clew-1 can reduce the bacterial burden in a mouse model of P. aeruginosa keratitis, which is characterized by the formation of a biofilm on the cornea. Due to its reliance on Psl for infection, Clew-1 does not actually form plaques on wild-type bacteria under standard in vitro conditions. This argues that our standard isolation procedures likely exclude bacteriophage that are adapted to using biofilm markers for infection. Importantly, the manner in which we isolated Clew-1 can be easily extended to other strains of P. aeruginosa and indeed other bacterial species, which will fuel the discovery of other biofilm-tropic bacteriophage and expand their therapeutic use.

microbiology↗

bioRxiv2024

Receptor Binding Specificity of a Bovine A(H5N1) Influenza Virus

Outbreaks in the US of highly pathogenic avian influenza virus (H5N1) in dairy cows have been occurring for months creating new possibilities for direct contact between the virus and humans. Eisfeld et al. examined the pathogenicity and transmissibility of a bovine HPAI H5N1 virus isolated from New Mexico in a series of in vitro and in vivo assays. They found the virus has a dual human- and avian virus-like receptor-binding specificity as measured in a solid phase glycan binding assay. Here, we examined the receptor specificity of a bovine HPAI H5N1 virus (A/bovine/OH/B24OSU-432/2024, H5N1, clade 2.3.4.4b) employing four different assays including glycan array technology, bio-layer interferometry (BLI), a solid phase capture assay and hemagglutination of glycan remodeled erythrocytes. As controls, well characterized avian (A/Vietnam/1203/2004, H5N1, clade 1) and human (A/CA/04/2009, H1N1) IAVs were included that bind 2,3- and 2,6-sialosides, respectively. We found that A/bovine/OH/B24OSU-432/2024 preferentially binds to "avian type" receptors (2,3-sialosides). Furthermore, sequence alignments showed that A/bovine has maintained amino acids in its HA associated with 2,3-sialoside (avian) receptor specificity. We conclude that while we find no evidence that A/bovine has acquired human virus receptor binding specificity, ongoing efforts must be placed on monitoring for this trait.

biochemistry↗

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↗

bioRxiv2024

Cell-Surface RNA Associates with Heparan Sulfate and RNA-Binding Proteins to Modulate Receptor-Ligand Interactions

Recent discoveries have shown the presence of RNA molecules on the cell surface, defying the traditional view that RNA only functions intracellularly. However, it is not well understood how cell-surface RNA (csRNA) is stably present on the plasma membrane and what functions it performs. We answer the pressing questions in the emerging field by taking integrated omic-wide approaches and multiple orthogonal validatory methods. Firstly, we exploited the RNA-sensing ability of TLR7 as a specific recombinant probe to detect csRNA. Coupling it with a genome-wide CRISPR-Cas9-knockout screening, we identified heparan sulfate (HS) as a crucial factor for RNA presentation on cells. Using the TLR7 probe, cell surface proximity labelling revealed that these HS-associated csRNAs (hepRNAs) are in vicinity with a plethora of RNA-binding proteins. The compelling observation led us to a molecular model where HS, RNA and RBP form ternary complexes at cell surface. A photochemical RNA-protein crosslinking technology termed SCOOPS were then established to validate the termolecular model in a TLR7-orthogonal manner. Moreover, enabled by SCOOPS, we unveiled identities of hepRNA using next-generation sequencing, and identified traits in RNA primary structures that facilitate HS association. We further show that hepRNA binds to killer cell immunoglobulin-like receptor 2DL5 (KIR2DL5), recruiting the protein to cell surface and potentially enhancing receptor-ligand interactions. Our findings provide a foundation for exploring how cell-surface ribonucleoproteins contribute to immune modulation.

molecular biology↗

bioRxiv2024

The HCoV-HKU1 N-terminal domain binds a wide range of 9-O-acetylated sialic acids presented on different glycan cores

Coronaviruses recognize a wide array of protein and glycan receptors using the S1 subunit of the spike (S) glycoprotein. The S1 subunit contains two functional domains: the N-terminal (S1-NTD) and C-terminal (S1-CTD). The S1-NTD of SARS-CoV-2, MERS-CoV, and HCoV-HKU1 possess an evolutionarily conserved glycan binding cleft that facilitates weak interactions with sialic acids on cell surfaces. HCoV-HKU1 employs 9-O-acetylated 2-8-linked disialylated structures for initial binding, followed by TMPRSS2 receptor binding and virus-cell fusion. Here, we demonstrate that HCoV-HKU1 NTD has a broader receptor binding repertoire than previously recognized. We presented HCoV-HKU1 NTD Fc chimeras on a nanoparticle system to mimic the densely decorated surface of HCoV-HKU1. These proteins were expressed by HEK293S GNTI- cells, generating species carrying Man-5 structures, often observed near the receptor binding site of CoVs. This multivalent presentation of high-mannose-containing NTD proteins revealed a much broader receptor binding profile compared to its fully glycosylated counterpart. Using glycan microarrays, we observed that 9-O-acetylated 2-3 linked sialylated LacNAc structures are also bound, comparable to OC43 NTD, suggesting an evolutionarily conserved glycan-binding modality. Further characterization of receptor specificity indicated promiscuous binding towards 9-O-acetylated sialoglycans, independent of the glycan core (glycolipids, N- or O-glycans). We demonstrate that HCoV-HKU1 may employ additional sialoglycan receptors to trigger conformational changes in the spike glycoprotein to expose the S1-CTD for proteinaceous receptor binding. (218)

microbiology↗

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↗

bioRxiv2023

Asymmetrical Bi-antennary Glycans Prepared by a Stop-and-Go Strategy Reveal Receptor Binding Evolution of Human Influenza A Viruses

Glycan binding properties of respiratory viruses have been difficult to probe due to a lack of biological relevant glycans for binding studies. Here, a stop-and-go chemoenzymatic methodology is presented that gave access to a panel of 32 asymmetrical bi-antennary N-glycans having various numbers of N-acetyl lactosamine (LacNAc) repeating units capped by 2,3- or 2,6-sialosides resembling structures found in airway tissues. It exploits that the branching enzymes MGAT1 and MGAT2 can utilize unnatural UDP-2-deoxy-2-trifluoro-N-acetamido-glucose (UDP-GlcNTFA) as donor. The TFA moiety of the resulting glycans can be hydrolyzed to give GlcNH2 at one of the antennae that temporarily blocks extension by glycosyl transferases. The N-glycans were printed as a microarray that was probed for receptor binding specificities of evolutionary distinct human A(H3N2) and A(H1N1)pdm09 viruses. It was found that not only the sialoside type but also the length of the LacNAc chain and presentation at the 1,3-antenna of N-glycans is critical for binding. Early A(H3N2) viruses bound to 2,6-sialosides at a single LacNAc moiety at the 1,3-antenna whereas later viruses required the sialoside to be presented at a tri-LacNAc moiety. Surprisingly, most of the A(H3N2) viruses that appeared after 2021 regained binding capacity to sialosides presented at a di-LacNAc moiety. As a result, these viruses agglutinate erythrocytes again, commonly employed for antigenic characterization of influenza viruses. Human A(H1N1)pdm09 viruses have similar receptor binding properties as recent A(H3N2) viruses. The data indicates that an asymmetric N-glycan having 2,6-sialoside at a di-LacNAc moiety is a commonly employed receptor by human influenza A viruses.

biochemistry↗

bioRxiv2023

Full-Length Single-Molecule Protein Fingerprinting

Proteins are the primary functional actors of the cell. Hence, their identification is pivotal to advance our understanding of cell biology and disease. Current protein analysis methods are of limited use for distinguishing proteoforms. In particular, mass spectrometric methods often provide only ambiguous information on post-translational modification sites, and sequences of co-existing modifications may not be resolved. Here we demonstrate FRET-based single-molecule protein fingerprinting to map the location of individual amino acids and a post-translational modification within single full-length protein molecules. Using an approach that relies on transient binding of fluorescently labeled DNA strands to probe the amino acids on a protein one by one we show that we can fingerprint intrinsically disordered proteins as well as folded globular proteins with sub-nanometer resolution. We anticipate that this technology will be used for proteoform identification in biological and translational research with ultimate sensitivity.

biophysics↗

bioRxiv2023

Exploiting Substrate Specificities of 6-O-Sulfotransferases to Enzymatically Synthesize Keratan Sulfate Oligosaccharides

Keratan sulfate (KS) is a glycosaminoglycan that is widely expressed in the extracellular matrix of various tissue types where it is involved in many biological processes. Herein, we describe a chemo-enzymatic approach to prepare well-defined KS oligosaccharides by exploiting known and newly discovered substrate specificities of relevant sulfotransferases. The premise of the approach is that recombinant GlcNAc-6-O-sulfotransferases (CHST2) only sulfates terminal GlcNAc moieties to give GlcNAc6S that can be galactosylated by B4GalT4. Furthermore, CHST1 can modify internal galactosides of a poly-LacNAc chain, however, it was found that a GlcNAc6S residue greatly increases the reactivity of CHST1 of a neighboring and internal galactoside. The presence of a 2,3-linked sialoside further modulates the site of modification by CHST1, and a galactoside flanked by 2,3-Neu5Ac and GlcNAc6S is preferentially sulfated over other Gal residues. The substrate specificities of CHST1 and 2 were exploited to prepare a panel of KS oligosaccharides including selectively sulfated N-glycans. The compounds and several other reference derivatives were used to construct a microarray that was probed for binding by several plant lectins, Siglec proteins and hemagglutinins of influenza viruses. It was found that not only the sulfation pattern but also presentation of epitopes as part of an O- or N-glycan determines binding properties.

biochemistry↗

bioRxiv2023

Sialoglycan binding triggers spike opening in a human coronavirus

Coronavirus (CoV) spikes mediate receptor binding and membrane fusion, making them prime targets for neutralising antibodies. In the cases of SARS-CoV, SARS-CoV-2, and MERS-CoV, spikes transition freely between open and closed conformations to balance host cell attachment and immune evasion. The open conformation exposes domain S1B, allowing it to bind to proteinaceous cell surface receptors. It also facilitates protein refolding during spike-mediated membrane fusion. However, with a single exception, the pre-fusion spikes of all other CoVs studied so far have been observed exclusively in the closed state. This raises the possibility of regulation, where spikes more commonly transition to open states in response to specific cues, rather than spontaneously. In our study, using cryo-EM and molecular dynamics simulations, we show that the spike protein of the common cold human coronavirus HKU1 undergoes local and long-range conformational changes upon binding a sialoglycan-based primary receptor to domain S1A. This binding triggers the transition of S1B domains to the open state via allosteric inter-domain cross-talk. Our findings paint a more elaborate picture of CoV attachment, with possibilities of dual receptor usage and priming of entry as a means of immune escape.

microbiology↗

bioRxiv2023

Probing Altered Receptor Specificities of Antigenically Drifting Human H3N2 Viruses by Chemoenzymatic Synthesis, NMR and Modeling

Prototypic receptors for human influenza viruses are cell surface N-glycans carrying 2,6-linked sialosides. Under immune pressure, A/H3N2 influenza viruses have emerged with altered receptor specificities that appear to recognize 2,6-linked sialosides presented on extended N-acetyl-lactosamine (LacNAc) moieties. Here, molecular recognition features of such drifted hemagglutinins (HAs) are examined by chemoenzymatic synthesis of complex N-glycans having 13C-labeled monosaccharides at strategic positions. The labeled glycans were employed in 2D STD-1H and 13C-HSQC NMR experiments to pinpoint which monosaccharides of the extended LacNAc chain engage with evolutionarily distinct HAs. The NMR data in combination with computational and mutagenesis studies demonstrate that mutations distal to the receptor binding domain of recent HAs have created an extended binding site that can directly interact with the extended LacNAc chain. A fluorine containing sialyl-LacNAc derivative is used as NMR probe to derive relative binding affinities and confirmed the contribution of the extended LacNAc chain for binding.

biochemistry↗

bioRxiv2023

Genipin Crosslinks the Extracellular Matrix to Rescue Developmental and Degenerative Defects, and Accelerates Regeneration of Peripheral Neurons

The peripheral nervous system (PNS) is essential for proper body function. A high percentage of the population suffer nerve degeneration or peripheral damage. For example, over 40% of patients with diabetes or undergoing chemotherapy develop peripheral neuropathies. Despite this, there are major gaps in the knowledge of human PNS development and therefore, there are no available treatments. Familial Dysautonomia (FD) is a devastating disorder that specifically affects the PNS making it an ideal model to study PNS dysfunction. FD is caused by a homozygous point mutation in ELP1 leading to developmental and degenerative defects in the sensory and autonomic lineages. We previously employed human pluripotent stem cells (hPSCs) to show that peripheral sensory neurons (SNs) are not generated efficiently and degenerate over time in FD. Here, we conducted a chemical screen to identify compounds able to rescue this SN differentiation inefficiency. We identified that genipin, a compound prescribed in Traditional Chinese Medicine for neurodegenerative disorders, restores neural crest and SN development in FD, both in the hPSC model and in a FD mouse model. Additionally, genipin prevented FD neuronal degeneration, suggesting that it could be offered to patients suffering from PNS neurodegenerative disorders. We found that genipin crosslinks the extracellular matrix, increases the stiffness of the ECM, reorganizes the actin cytoskeleton, and promotes transcription of YAP-dependent genes. Finally, we show that genipin enhances axon regeneration in an in vitro axotomy model in healthy sensory and sympathetic neurons (part of the PNS) and in prefrontal cortical neurons (part of the central nervous system, CNS). Our results suggest genipin can be used as a promising drug candidate for treatment of neurodevelopmental and neurodegenerative diseases, and as a enhancer of neuronal regeneration. One sentence summaryGenipin rescues the developmental and degenerative phenotypes of the peripheral neuropathy familial dysautonomia and enhances neuron regeneration after injury.

neuroscience↗