Ferrets exclusively synthesize Neu5Ac and express naturally humanized influenza A virus receptors

Mammals express the sialic acids ​N-acetylneuraminic acid (​Neu5Ac) and ​N-glycolylneuraminic acid (​Neu5Gc) on cell surfaces, where they act as receptors for pathogens, including influenza A virus (IAV). ​Neu5Gc is synthesized from ​Neu5Ac by the enzyme cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH). In humans, this enzyme is inactive and only ​Neu5Ac is produced. Ferrets are susceptible to human-adapted IAV strains and have been the dominant animal model for IAV studies. Here we show that ferrets, like humans, do not synthesize ​Neu5Gc. Genomic analysis reveals an ancient, nine-exon deletion in the ferret CMAH gene that is shared by the Pinnipedia and Musteloidia members of the Carnivora. Interactions between two human strains of IAV with the sialyllactose receptor (sialic acid—α2,6Gal) confirm that the type of terminal sialic acid contributes significantly to IAV receptor specificity. Our results indicate that exclusive expression of ​Neu5Ac contributes to the susceptibility of ferrets to human-adapted IAV strains

The pneumococcal alpha-glycerophosphate oxidase enhances nasopharyngeal colonization through binding to host glycoconjugates

Streptococcus pneumoniae (the pneumococcus) is a major human pathogen, causing a broad spectrum of diseases including otitis media, pneumonia, bacteraemia and meningitis. Here we examined the role of a potential pneumococcal meningitis vaccine antigen, alpha-glycerophosphate oxidase (SpGlpO), in nasopharyngeal colonization. We found that serotype 4 and serotype 6A strains deficient in SpGlpO have significantly reduced capacity to colonize the nasopharynx of mice, and were significantly defective in adherence to human nasopharyngeal carcinoma cells in vitro. We also demonstrate that intranasal immunization with recombinant SpGlpO significantly protects mice against subsequent nasal colonization by wild type serotype 4 and serotype 6A strains. Furthermore, we show that SpGlpO binds strongly to lacto/neolacto/ganglio host glycan structures containing the GlcNAcβ1-3Galβ disaccharide, suggesting that SpGlpO enhances colonization of the nasopharynx through its binding to host glycoconjugates. We propose that SpGlpO is a promising vaccine candidate against pneumococcal carriage, and warrants inclusion in a multi-component protein vaccine formulation that can provide robust, serotype-independent protection against all forms of pneumococcal disease

Characterization of the N-glycans of female Angiostrongylus cantonensis worms

Glycoconjugates play a crucial role in the host-parasite relationships of helminthic infections, including angiostrongyliasis. It has previously been shown that the antigenicity of proteins from female Angiostrongylus cantonensis worms may depend on their associated glycan moieties. Here, an N-glycan profile of A. cantonensis is reported. A total soluble extract (TE) was prepared from female A. cantonensis worms and was tested by western blot before and after glycan oxidation or N- and O-glycosidase treatment. The importance of N-glycans for the immunogenicity of A. cantonensis was demonstrated when deglycosylation of the TE with PNGase F completely abrogated IgG recognition. The TE was also fractionated using various lectin columns [Ulex europaeus (UEA), concanavalin A (Con A), Arachis hypogaea (PNA), Triticum vulgaris (WGA) and Lycopersicon esculentum (LEA)], and then each fraction was digested with PNGase F. Released N-glycans were analyzed with matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) and MALDI-TOF/TOF-MS/MS. Complex-type, high mannose, and truncated glycan structures were identified in all five fractions. Sequential MALDI-TOF-TOF analysis of the major MS peaks identified complex-type structures, with a α1-6 fucosylated core and truncated antennas. Glycoproteins in the TE were labeled with BodipyAF558-SE dye for a lectin microarray analysis. Fluorescent images were analyzed with ProScanArray imaging software followed by statistical analysis. A total of 29 lectins showed positive binding to the TE. Of these, Bandeiraea simplicifolia (BS-I), PNA, and Wisteria floribunda (WFA), which recognize galactose (Gal) and N-acetylgalactosamine (GalNAc), exhibited high affinity binding. Taken together, our findings demonstrate that female A. cantonensis worms have characteristic helminth N-glycans.Office of the Snr Dep Vice Chancellor, Institute for GlycomicsNo Full Tex

Neurotoxin-mediated potent activation of the axon degeneration regulator SARM1

Axon loss underlies symptom onset and progression in many neurodegenerative disorders. Axon degeneration in injury and disease is promoted by activation of the nicotinamide adenine dinucleotide (NAD)-consuming enzyme SARM1. Here, we report a novel activator of SARM1, a metabolite of the pesticide and neurotoxin vacor. Removal of SARM1 completely rescues mouse neurons from vacor-induced neuron and axon death in vitro and in vivo. We present the crystal structure the Drosophila SARM1 regulatory domain complexed with this activator, the vacor metabolite VMN, which as the most potent activator yet know is likely to support drug development for human SARM1 and NMNAT2 disorders. This study indicates the mechanism of neurotoxicity and pesticide action by vacor, raises important questions about other pyridines in wider use today, provides important new tools for drug discovery, and demonstrates that removing SARM1 can robustly block programmed axon death induced by toxicity as well as genetic mutation

Lectin activity of the pneumococcal pilin proteins

Streptococcus pneumoniae is a leading cause of morbidity and mortality globally. The Pilus-1 proteins, RrgA, RrgB and RrgC of S. pneumoniae have been previously assessed for their role in infection, invasive disease and as possible vaccine candidates. In this study we have investigated the glycan binding repertoire of all three Pilus-1 proteins, identifying that the tip adhesin RrgA has the broadest glycan recognition of the three proteins, binding to maltose/cellobiose, α/β linked galactose and blood group A and H antigens. RrgB only bound mannose, while RrgC bound a subset of glycans also recognized by RrgA. Adherence of S. pneumoniae TIGR4 to epithelial cells was tested using four of the oligosaccharides identified through the glycan array analysis as competitive inhibitors. The blood group H trisaccharide provided the best blocking of S. pneumoniae TIGR4 adherence. Adherence is the first step in disease, and host glycoconjugates are a common target for many adhesins. This study has identified Pilus-1 proteins as new lectins involved in the targeting of host glycosylation by S. pneumoniae.Christopher J. Day, Adrienne W. Paton, Richard M. Harvey, Lauren E. Hartley-Tassell, Kate L. Seib, Joe Tiralongo, Nicolai Bovin, Silvana Savino, Vega Masignani, James C. Paton and Michael P. Jenning

Identification of NuoX and NuoY Ligand binding specificity in the Campylobacter Jejuni Complex I

The components of the proton pump NADH:ubiquinone (Complex I) of the respiration pathway have been identified in the C. jejuni genome. However, the paradigm genes nuoE and nuoF encoding subunits of the NADH dehydrogenase module of Complex I are absent. Instead the genes cj1575c and cj1574c encoding NuoX and NuoY are present in the loci corresponding to nuoE and nuoF, respectively. Bioinformatics analyses showed the presence of nuoX and nuoY homologues in all sequenced strains of C. jejuni and in other Campylobacter species, as well as the presence of orthologues in other ɛ-Proteobacteria. To understand the involvement of the NuoX and NuoY proteins in the respiration of C. jejuni and to characterize their ligand binding specificity and affinity, a tricarboxylic acid cycle array was developed as a tool to identify proteins that can bind to intermediates of this cycle as well as other metabolites. This array showed that NuoX bound FAD2+, and NuoY bound FAD2+ and the electron donors malate and lactate. Saturation Transfer Difference Nuclear Magnetic Resonance studies confirmed the NuoY binding ligands, and suggested that the flavin moiety of FAD2+ interacted more strongly with NuoY than the adenine moiety. Affinity data generated by Surface Plasmon Resonance indicated that NuoY bound to FAD2+ with a KD of 337 nM; NuoX and NuoY had an affinity for NADH of a KD of 403 nM and 478 nM, respectively, and a ten-fold lower affinity for both NAD+ and FAD2+. The data suggested that the flavin-adenine dinucletoide could be bound preferentially to the NAD in the Complex I of C. jejuni

Characterization of H type 1 and type 1 N-acetyllactosamine glycan epitopes on ovarian cancer specifically recognized by the anti-glycan monoclonal antibody mAb-A4.

Cancer-specific glycans of ovarian cancer are promising epitopes for targeting with monoclonal antibodies (mAb). Despite their potential, structural characterization of these glycan epitopes remains a significant challenge in mAb preclinical development. Our group generated the monoclonal antibody mAb-A4 against human embryonic stem cells (hESC), which also bound specifically to N-glycans present on 11 out of 19 ovarian cancer (OC) and 8 out of 14 breast cancer cell lines tested. Normal cell lines and tissue were unstained by mAb-A4. To characterize the N-linked glycan epitopes on OC cell lines targeted by mAb-A4, we used glycosidases, glycan microarray, siRNA and advanced high-sensitivity matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The mAb-A4 epitopes were found to be Fuc α1-2Galβ1-3GlcNAcβ (H Type 1) and Galβ1-3GlcNAcβ (Type 1 LacNAc). These structures were found to be present on multiple proteins from hESC and OC. Importantly, endo-β-galactosidase coupled with MALDI-MS allowed these two epitopes, for the first time, to be directly identified on the polylactosamines of N-glycans of SKOV3, IGROV1, OV90 and OVCA433. Furthermore, siRNA knockdown of B3GALT5 expression in SKOV3 demonstrated that mAb-A4 binding was dependent on B3GALT5, providing orthogonal evidence of the epitopes' structures. The recognition of oncofetal H Type 1 and Type 1 LacNAc on OC by mAb-A4 is a novel and promising way to target OC and supports the theory that cancer can acquire stem-like phenotypes. We propose that the orthogonal framework used in this work could be the basis for advancing anti-glycan mAb characterization

Lectin activity of the TcdA and TcdB toxins of Clostridium difficile

Clostridium difficile is a major cause of hospital-acquired antibioticassociated diarrhea. C. difficile produces two cytotoxins, TcdA and TcdB; both toxins are multidomain proteins that lead to cytotoxicity through the modification and inactivation of small GTPases of the Rho/Rac family. Previous studies have indicated that host glycans are targets for TcdA and TcdB, with interactions thought to be with both - and -linked galactose. In the current study, screening of glycan arrays with different domains of TcdA and TcdB revealed that the binding regions of both toxins interact with a wider range of host glycoconjugates than just terminal - and -linked galactose, including blood groups, Lewis antigens, N-acetylglucosamine, mannose, and glycosaminoglycans. The interactions of TcdA and TcdB with ABO blood group and Lewis antigens were assessed by surface plasmon resonance (SPR). The blood group A antigen was the highest-affinity ligand for both toxins. Free glycans alone or in combination were unable to abolish Vero cell cytotoxicity by TcdB. SPR competition assays indicate that there is more than one glycan binding site on TcdB. Host glycoconjugates are common targets of bacterial toxins, but typically this binding is to a specific structure or related structures. The binding of TcdA and TcdB is to a wide range of host glycans providing a wide range of target cells and tissues in vivo.Full Tex

The cholesterol-dependent cytolysins pneumolysin and streptolysin O require binding to red blood cell glycans for hemolytic activity

The cholesterol-dependent cytolysin (CDC) pneumolysin (Ply) is a key virulence factor of Streptococcus pneumoniae. Membrane cholesterol is required for the cytolytic activity of this toxin, but it is not clear whether cholesterol is the only cellular receptor. Analysis of Ply binding to a glycan microarray revealed that Ply has lectin activity and binds glycans, including the Lewis histo-blood group antigens. Surface plasmon resonance analysis showed that Ply has the highest affinity for the sialyl LewisX (sLeX) structure, with a K(d) of 1.88 × 10(-5) M. Ply hemolytic activity against human RBCs showed dose-dependent inhibition by sLeX. Flow cytometric analysis and Western blots showed that blocking binding of Ply to the sLeX glycolipid on RBCs prevents deposition of the toxin in the membrane. The lectin domain responsible for sLeX binding is in domain 4 of Ply, which contains candidate carbohydrate-binding sites. Mutagenesis of these predicted carbohydrate-binding residues of Ply resulted in a decrease in hemolytic activity and a reduced affinity for sLeX. This study reveals that this archetypal CDC requires interaction with the sLeX glycolipid cellular receptor as an essential step before membrane insertion. A similar analysis conducted on streptolysin O from Streptococcus pyogenes revealed that this CDC also has glycan-binding properties and that hemolytic activity against RBCs can be blocked with the glycan lacto-N-neotetraose by inhibiting binding to the cell surface. Together, these data support the emerging paradigm shift that pore-forming toxins, including CDCs, have cellular receptors other than cholesterol that define target cell tropism.Lucy K. Shewell, Richard M. Harvey, Melanie A. Higgins, Christopher J. Day, Lauren E. Hartley-Tassell, Austen Y. Chen, Christine M. Gillen, David B. A. James, Francis Alonzo, Victor J. Torres, Mark J. Walker, Adrienne W. Paton, James C. Paton, and Michael P. Jenning