Cells from all domains of life express glycan structures attached to lipids and proteins on their surface, called glycoconjugates. Cell-tocell contact mediated by glycan:glycan interactions have been considered to be low-affinity interactions that precede highaffinity protein-glycan or protein-protein interactions. In several pathogenic bacteria, truncation of surface glycans, lipooligosaccharide (LOS), or lipopolysaccharide (LPS) have been reported to significantly reduce bacterial adherence to host cells. Here, we show that the saccharide component of LOS/LPS have direct, high-affinity interactions with host glycans. Glycan microarrays reveal that LOS/LPS of four distinct bacterial pathogens bind to numerous host glycan structures. Surface plasmon resonance was used to determine the affinity of these interactions and revealed 66 high-affinity host-glycan:bacterial-glycan pairs with equilibrium dissociation constants (K D ) ranging between 100 nM and 50 μM. These glycan:glycan affinity values are similar to those reported for lectins or antibodies with glycans. Cell assays demonstrated that glycan:glycan interaction-mediated bacterial adherence could be competitively inhibited by either host cell or bacterial glycans. This is the first report to our knowledge of high affinity glycan:glycan interactions between bacterial pathogens and the host. The discovery of large numbers of glycan:glycan interactions between a diverse range of structures suggests that these interactions may be important in all biological systems.H ost surface glycosylation is ubiquitous and is targeted by pathogenic bacteria, viruses, fungi and parasites for adherence and toxin binding and by glycosidases (1). Escherichia coli type 1 fimbriae, FimH, is one of the most widely studied glycanrecognizing protein adhesins, with specificity for monomannose to oligomannose structures with the variability of the mannose structure bound leading to different tissue tropism (2). Other glycan-recognizing adhesins expressed by bacteria include the following: Pseudomonas aeruginosa lectins 1 and 2 (PA-IL and PA-IIL) that have specificity for galactose and fucose, respectively (3); Helicobacter pylori SabA, specific for sialic acid containing glycoconjugates including sialyLewis X; and BabAspecific for fucosylated glycoconjugates including Lewis B (4, 5). Although there are numerous known glycan binding adhesins, the adhesins of some bacteria that interact with host surface glycans remain unknown.Direct interactions between surface glycans (glycan:glycan interactions) have been reported in sea sponges as heterogenous glycan interactions, and in mouse embryo development and cancer where homodimers of Lewis X (LeX) or ganglioside structures play a role in cell adhesion and growth factor receptor interactions (6, 7). Outside of these reports, glycan:glycan interactions, when noted, have generally been considered to be low-affinity, weak interactions (8) that precede high-affinity protein:glycan or protein:protein interactions (1, 2, 5, 9).Interestingly, there...
We report here the exploitation of the 150-cavity in the active sites of group-1 neuraminidases for the design of new triazole-containing carbocycles related to oseltamivir. Inhibition studies with virus-like particles (VLPs) containing the influenza virus neuraminidase-1 (N1) activity indicate that several candidates are inhibitors, with K(i) values in the 10(-5)-10(-8) M range. In contrast, a known candidate that preserves the free amino group and a new candidate containing a guanidine function are better inhibitors, with K(i) values of 1.5 × 10(-9) and 4.6 × 10(-10) M, respectively. The most active inhibitor of the N1 enzyme in the triazole series was selective for the N1 class and showed significantly less inhibition (K(i) = 2.6 μM vs 0.07 μM) of the free influenza virus neuraminidase-2 (N2). In addition, saturation transfer difference (STD) NMR spectroscopic studies with this compound and the VLPs show that the entire molecule forms contacts with residues in the active site. These data taken together support our proposed binding mode in which the active site and the adjoining 150-cavity are both occupied.
The initial stage of host cell infection by influenza A viruses (IAV) is mediated through interaction of the viral haemagglutinin (HA) with cell surface glycans. The binding requirement of IAVs for Galβ(1,4)Glc/ GlcNAc (lactose/lactosamine) glycans with a terminal α(2,6)-linked (human receptors) or α(2,3)-linked (avian receptors) N-acetylneuraminic residue commonly found on N-glycans, is well-established. However the role and significance of sialylated Galβ(1,3)GalNAc (core 1) epitopes that are typical O-glycoforms in influenza virus pathogenesis remains poorly detailed. Here we report a multidisciplinary study using NMR spectroscopy, virus neutralization assays and molecular modelling, into the potential for IAV to engage sialyl-Galβ(1,3)GalNAc O-glycoforms for cell attachment. H5 containing virus like particles (VLPs) derived from an H5N1 avian IAV strain show a significant involvement of the O-glycan-specific GalNAc residue, coordinated by a EQTKLY motif conserved in highly pathogenic avian influenza (HPAI) strains. Notably, human pandemic H1N1 influenza viruses shift the preference from ‘human-like’ α(2,6)-linkages in sialylated Galβ(1,4)Glc/GlcNAc fragments to ‘avian-like’ α(2,3)-linkages in sialylated Galβ(1,3)GalNAc without involvement of the GalNAc residue. Overall, our study suggests that sialylated Galβ(1,3)GalNAc as O-glycan core 1 glycoforms are involved in the influenza A virus life cycle and play a particularly crucial role during infection of HPAI strains.
Saturation transfer difference (STD) (1)H NMR experiments were used to probe the epitope binding characteristics of the sialidase [EC 3.2.1.18] from the bacterium Vibrio cholerae, the causative agent of cholera. Binding preferences were investigated for N-acetylneuraminic acid (Neu5Ac, 1), the product of the sialidase catalytic reaction, for the known sialidase inhibitor 5-acetamido-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-non-2-enoic acid (Neu5Ac2en, 2), and for the uronic acid-based Neu5Ac2en mimetic iso-propyl 2-acetamido-2,4-dideoxy-alpha-L-threo-hex-4-enopyranosiduronic acid (3), in which the native glycerol side-chain of Neu5Ac2en is replaced with an O-iso-propyl ether. The STD experiments provided evidence, supporting previous studies, that Neu5Ac (1) binds to the sialidase as the alpha-anomer. Docking experiments using DOCK (version 4.0.1) revealed further information regarding the binding characteristics of the enzyme active site in complex with Neu5Ac2en (2) and the Neu5Ac2en mimetic (3), indicating an expected dominant interaction of the acetamide moiety with the protein.
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
We thank Shah et al 1 for their interest in the Portsmouth Glaucoma Refinement Scheme. 2 The scheme also uses Van Herick grading for anterior chamber depth-all patients with a Van Herick peripheral limbal anterior chamber depth of less than 25% of corneal thickness were referred to the virtual clinic for assessment by an ophthalmologist. Approximately 10% of all of those accepted from the Refinement Scheme virtual clinic to HES (from a total of 11 out of 100 referred to the virtual clinic, from our audit) were due to narrow angles suspected through Van Herick grading. Of these, 25% subsequently required laser peripheral iridotomy, slightly higher than the 17% positive predictive value, for the suggestion of occludable angles by an initial Van Herick test, outlined by Foster. 3 Conflict of interestThe authors declare no conflict of interest. References1 Shah B, Campbell P, Ford C, Goyal S, Lim KS. Re:The Portsmouth-based refinement scheme: a role for virtual clinics in the future? Eye 2013; 27(7): 892-893. 2 Trikha S, Macgregor C, Jeffery M, Kirwan J. ThePortsmouth-based refinement scheme: a role for virtual clinics in the future? Eye 2012; 26: 1288-1294. 3 Foster P. Advances in the understanding of primary angleclosure as a cause of glaucomatous optic neuropathy.
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