Akkermansia muciniphila exoglycosidases target extended blood group antigens to generate ABO-universal blood.

Matching donor and recipient blood groups based on red blood cell (RBC) surface ABO glycans and antibodies in plasma is crucial to avoid potentially fatal reactions during transfusions. Enzymatic conversion of RBC glycans to the universal group O is an attractive solution to simplify blood logistics and prevent ABO-mismatched transfusions. The gut symbiont Akkermansia muciniphila can degrade mucin O-glycans including ABO epitopes. Here we biochemically evaluated 23 Akkermansia glycosyl hydrolases and identified exoglycosidase combinations which efficiently transformed both A and B antigens and four of their carbohydrate extensions. Enzymatic removal of canonical and extended ABO antigens on RBCs significantly improved compatibility with group O plasmas, compared to conversion of A or B antigens alone. Finally, structural analyses of two B-converting enzymes identified a previously unknown putative carbohydrate-binding module. This study demonstrates the potential utility of mucin-degrading gut bacteria as valuable sources of enzymes for production of universal blood for transfusions.

N-glycoproteomic analyses of human intestinal enteroids, varying in histo-blood group geno- and phenotypes, reveal a wide repertoire of fucosylated glycoproteins.

Human noroviruses, globally the main cause of viral gastroenteritis, show strain specific affinity for histo-blood group antigens (HBGA) and can successfully be propagated ex vivo in human intestinal enteroids (HIEs). HIEs established from jejunal stem cells of individuals with different ABO, Lewis and secretor geno- and phenotypes, show varying susceptibility to such infections. Using bottom-up glycoproteomic approaches we have defined and compared the N-linked glycans of glycoproteins of seven jejunal HIEs. Membrane proteins were extracted, trypsin digested, and glycopeptides enriched by hydrophilic interaction liquid chromatography and analyzed by nanoLC-MS/MS. The Byonic software was used for glycopeptide identification followed by hands-on verifications and interpretations. Glycan structures and attachment sites were identified from MS2 spectra obtained by higher-energy collision dissociation through analysis of diagnostic saccharide oxonium ions (B-ions), stepwise glycosidic fragmentation of the glycans (Y-ions), and peptide sequence ions (b- and y-ions). Altogether 694 unique glycopeptides from 93 glycoproteins were identified. The N-glycans encompassed pauci- and oligomannose, hybrid- and complex-type structures. Notably, polyfucosylated HBGA-containing glycopeptides of the four glycoproteins tetraspanin-8, carcinoembryonic antigen-related cell adhesion molecule 5, sucrose-isomaltase and aminopeptidase N were especially prominent and were characterized in detail and related to donor ABO, Lewis and secretor types of each HIE. Virtually no sialylated N-glycans were identified for these glycoproteins suggesting that terminal sialylation was infrequent compared to fucosylation and HBGA biosynthesis. This approach gives unique site-specific information on the structural complexity of N-linked glycans of glycoproteins of human HIEs and provides a platform for future studies on the role of host glycoproteins in gastrointestinal infectious diseases.

Strain-specific requirements of susceptibility to rhesus enteric calicivirus infection.

Recently, we identified the coxsackie and adenovirus receptor (CAR) as the entry receptor for rhesus enteric calicivirus (ReCV) isolate FT285 and demonstrated that co-expression of the CAR and the type B histo-blood group antigen (HBGA) is required to convert the resistant CHO cell line susceptible to infection. To address whether the CAR is also the functional entry receptor for other ReCV isolates and the requirement for specific HBGAs or other glycans, here we used a panel of recombinant CHO cell lines expressing the CAR and the type A, B, or H HBGAs alone or in combination. Infection studies with three diverse ReCV strains, the prototype GI.1 Tulane virus (TV), GI.2 ReCV-FT285, and GI.3 ReCV-FT7, identified that cell surface expression of the CAR is an absolute requirement for all three strains to promote susceptibility to infection, while the requirement for HBGAs varies among the strains. In addition to the CAR, ReCV-FT285 and TV require type A or B HBGAs for infection. In the absence of HBGAs, TV, but not Re-CV FT285, can also utilize sialic acids, while ReCV-FT7 infection is HBGA-independent and relies on CAR and sialic acid expression. In summary, we demonstrated strain-specific diversity of susceptibility requirements for ReCV infections and that CAR, type A and B HBGA, and sialic acid expression control susceptibility to infection with the three ReCV isolates studied. Our study also indicates that the correlation between in vitro HBGA binding and HBGAs required for infection is relatively high, but not absolute. This has direct implications for human noroviruses.IMPORTANCEHuman noroviruses (HuNoVs) are important enteric pathogens. The lack of a robust HuNoV cell culture system is a bottleneck for HuNoV cell culture-based studies. Often, cell culture-adapted caliciviruses that rapidly replicate in conventional cell lines and recapitulate biological features of HuNoVs are utilized as surrogates. Particularly, rhesus enteric caliciviruses (ReCVs) display remarkable similarities, including the primate host, clinical manifestation of gastroenteritis, genetic/antigenic diversity, and reliance on histo-blood group antigens (HBGAs) for attachment. While the HuNoV entry receptor(s) is unknown, the coxsackie and adenovirus receptor (CAR) has recently been identified as the ReCV entry receptor. Here, we identified the CAR, the type A and B HBGAs, and sialic acids as critical cell surface molecules controlling susceptibility to ReCV infections. The CAR is required for all ReCV isolates studied. However, the requirement for the different carbohydrate molecules varies among different ReCV strains. Our findings have direct implications for HuNoVs.

The disordered protein SERF promotes α-Synuclein aggregation through liquid-liquid phase separation.

The aggregation of α-Synuclein (α-Syn) into amyloid fibrils is the hallmark of Parkinson's disease. Under stress or other pathological conditions, the accumulation of α-Syn oligomers is the main contributor to the cytotoxicity. A potential approach for treating Parkinson's disease involves preventing the accumulation of these α-Syn oligomers. In this study, we present a novel mechanism involving a conserved group of disorderly proteins known as small EDRK-rich factor (SERF), which promotes the aggregation of α-Syn through a cophase separation process. Using diverse methods like confocal microscopy, fluorescence recovery after photobleaching assays, solution-state NMR spectroscopy, and Western blot, we determined that the N-terminal domain of SERF1a plays a role in the interactions that occur during cophase separation. Within these droplets, α-Syn undergoes a gradual transformation from solid condensates to amyloid fibrils, while SERF1a is excluded from the condensates and dissolves into the solution. Notably, in vivo experiments show that SERF1a cophase separation with α-Syn significantly reduces the deposition of α-Syn oligomers and decreases its cellular toxicity under stress. These findings suggest that SERF1a accelerates the conversion of α-Syn from highly toxic oligomers to less toxic fibrils through cophase separation, thereby mitigating the biological damage of α-Syn aggregation.

Dantu Blood Group Erythrocytes Form Large Plasmodium falciparum Rosettes Less Commonly.

Dantu erythrocytes, which express a hybrid glycophorin B/A protein, are protective against severe malaria. Recent studies have shown that Dantu impairs Plasmodium falciparum invasion by increasing erythrocyte membrane tension, but its effects on pathological host-parasite adhesion interactions such as rosetting, the binding of uninfected erythrocytes to P. falciparum-infected erythrocytes, have not been investigated previously. The expression of several putative host rosetting receptors-including glycophorin A (GYPA), glycophorin C (GYPC), complement receptor 1 (CR1), and band 3, which complexes with GYPA to form the Wrightb blood group antigen-are altered on Dantu erythrocytes. Here, we compare receptor expression, and rosetting at both 1 hour and 48 hours after mixing with mature trophozoite-stage Kenyan laboratory-adapted P. falciparum strain 11019 parasites in Dantu and non-Dantu erythrocytes. Dantu erythrocytes showed lower staining for GYPA and CR1, and greater staining for band 3, as observed previously, whereas Wrightb and GYPC staining did not vary significantly. No significant between-genotype differences in rosetting were seen after 1 hour, but the percentage of large rosettes was significantly less in both Dantu heterozygous (mean, 16.4%; standard error of the mean [SEM], 3.2) and homozygous donors (mean, 15.4%; SEM, 1.4) compared with non-Dantu erythrocytes (mean, 32.9%; SEM, 7.1; one-way analysis of variance, P = 0.025) after 48 hours. We also found positive correlations between erythrocyte mean corpuscular volume (MCV), the percentage of large rosettes (Spearman's rs = 0.5970, P = 0.0043), and mean rosette size (rs = 0.5206, P = 0.0155). Impaired rosetting resulting from altered erythrocyte membrane receptor expression and reduced MCV might add to the protective effect of Dantu against severe malaria.

Culture of Human Rotaviruses in Relevant Models Shows Differences in Culture-Adapted and Nonculture-Adapted Strains.

Rotavirus (RV) is the leading cause of acute gastroenteritis (AGE) in children under 5 years old worldwide, and several studies have demonstrated that histo-blood group antigens (HBGAs) play a role in its infection process. In the present study, human stool filtrates from patients diagnosed with RV diarrhea (genotyped as P[8]) were used to infect differentiated Caco-2 cells (dCaco-2) to determine whether such viral strains of clinical origin had the ability to replicate in cell cultures displaying HBGAs. The cell culture-adapted human RV Wa model strain (P[8] genotype) was used as a control. A time-course analysis of infection was conducted in dCaco-2 at 1, 24, 48, 72, and 96 h. The replication of two selected clinical isolates and Wa was further assayed in MA104, undifferentiated Caco-2 (uCaco-2), HT29, and HT29-M6 cells, as well as in monolayers of differentiated human intestinal enteroids (HIEs). The results showed that the culture-adapted Wa strain replicated more efficiently in MA104 cells than other utilized cell types. In contrast, clinical virus isolates replicated more efficiently in dCaco-2 cells and HIEs. Furthermore, through surface plasmon resonance analysis of the interaction between the RV spike protein (VP8*) and its glycan receptor (the H antigen), the V7 RV clinical isolate showed 45 times better affinity compared to VP8* from the Wa strain. These findings support the hypothesis that the differences in virus tropism between clinical virus isolates and RV Wa could be a consequence of the different HBGA contents on the surface of the cell lines employed. dCaco-2, HT29, and HT29M6 cells and HIEs display HBGAs on their surfaces, whereas MA104 and uCaco-2 cells do not. These results indicate the relevance of using non-cell culture-adapted human RV to investigate the replication of rotavirus in relevant infection models.

A single nanobody neutralizes multiple epochally evolving human noroviruses by modulating capsid plasticity.

Acute gastroenteritis caused by human noroviruses (HuNoVs) is a significant global health and economic burden and is without licensed vaccines or antiviral drugs. The GII.4 HuNoV causes most epidemics worldwide. This virus undergoes epochal evolution with periodic emergence of variants with new antigenic profiles and altered specificity for histo-blood group antigens (HBGA), the determinants of cell attachment and susceptibility, hampering the development of immunotherapeutics. Here, we show that a llama-derived nanobody M4 neutralizes multiple GII.4 variants with high potency in human intestinal enteroids. The crystal structure of M4 complexed with the protruding domain of the GII.4 capsid protein VP1 revealed a conserved epitope, away from the HBGA binding site, fully accessible only when VP1 transitions to a "raised" conformation in the capsid. Together with dynamic light scattering and electron microscopy of the GII.4 VLPs, our studies suggest a mechanism in which M4 accesses the epitope by altering the conformational dynamics of the capsid and triggering its disassembly to neutralize GII.4 infection.

Intestinal colonization with Escherichia fergusonii enhances infectivity of GII.12 human norovirus in gnotobiotic pigs.

The role of gut microbiota [especially, histo-blood group antigen (HBGA)-expressing bacteria] in influencing human norovirus (HuNoV) infections is unclear. We investigated if infectivity of GII.12 HuNoV in gnotobiotic (Gn) pigs is altered by intestinal colonization with Escherichia fergusonii known to express HBGA A and H on their cell surface. Fifteen piglets were randomly grouped: (1) E. fergusonii + HuNoV (n = 6), (2) HuNoV alone (n = 6), and (3) Mock-inoculated (n = 3). Pigs (8-11-day-old) were inoculated orally with GII.12 HuNoV strain HS206 (9.5 log10 genomic equivalents/pig) or mock. For 2 days prior to viral inoculation, pigs were inoculated orally with E. fergusonii [8 log10 colony forming units/pig/day]. Daily fecal consistency, fecal viral RNA or E. fergusonii shedding, and histopathology (at euthanasia) were evaluated. Unlike the reduced infectivity of GII.4 HuNoV observed previously in Gn pigs colonized with Enterobacter cloacae known to express HBGA A, B, and H on the surface, E. fergusonii + HuNoV pigs exhibited significantly higher cumulative fecal HuNoV RNA shedding at PIDs 6-14 and 1-21 compared with HuNoV alone pigs. Mean days of fecal HuNoV RNA shedding were also significantly greater in E. fergusonii + HuNoV pigs (11.8 ± 1.6 days) compared with HuNoV alone pigs (7.0 ± 1.0 days). By immunofluorescent staining, HuNoV antigen-positive bacteria were detected on the surface of the intestinal epithelium, possibly enhancing attachment of HuNoV to enterocytes, suggesting a potential mechanism by which intestinal colonization with E. fergusonii promoted infectivity of GII.12 HuNoV in Gn pigs.

Species-Specific N-Glycomes and Methylation Patterns of Oysters Crassostrea gigas and Ostrea edulis and Their Possible Consequences for the Norovirus-HBGA Interaction.

Noroviruses, the major cause of acute viral gastroenteritis, are known to bind to histo-blood group antigens (HBGAs), including ABH groups and Lewis-type epitopes, which decorate the surface of erythrocytes and epithelial cells of their host tissues. The biosynthesis of these antigens is controlled by several glycosyltransferases, the distribution and expression of which varies between tissues and individuals. The use of HBGAs as ligands by viruses is not limited to humans, as many animal species, including oysters, which synthesize similar glycan epitopes that act as a gateway for viruses, become vectors for viral infection in humans. Here, we show that different oyster species synthesize a wide range of N-glycans that share histo-blood A-antigens but differ in the expression of other terminal antigens and in their modification by O-methyl groups. In particular, we show that the N-glycans isolated from Crassostrea gigas and Ostrea edulis exhibit exquisite methylation patterns in their terminal N-acetylgalactosamine and fucose residues in terms of position and number, adding another layer of complexity to the post-translational glycosylation modifications of glycoproteins. Furthermore, modeling of the interactions between norovirus capsid proteins and carbohydrate ligands strongly suggests that methylation has the potential to fine-tune the recognition events of oysters by virus particles.

Intestinal mucin-type O-glycans: the major players in the host-bacteria-rotavirus interactions.

Rotavirus (RV) causes severe diarrhea in young children and animals worldwide. Several glycans terminating in sialic acids (SAs) and histo-blood group antigens (HBGAs) on intestinal epithelial cell (IEC) surface have been recognized to act as attachment sites for RV. IECs are protected by the double layer of mucus of which O-glycans (including HBGAs and SAs) are a major organic component. Luminal mucins, as well as bacterial glycans, can act as decoy molecules removing RV particles from the gut. The composition of the intestinal mucus is regulated by complex O-glycan-specific interactions among the gut microbiota, RV and the host. In this review, we highlight O-glycan-mediated interactions within the intestinal lumen prior to RV attachment to IECs. A better understanding of the role of mucus is essential for the development of alternative therapeutic tools including the use of pre- and probiotics to control RV infection.

Direct Blockade of the Norovirus Histo-Blood Group Antigen Binding Pocket by Nanobodies.

Noroviruses are the leading cause of outbreaks of acute gastroenteritis. These viruses usually interact with histo-blood group antigens (HBGAs), which are considered essential cofactors for norovirus infection. This study structurally characterizes nanobodies developed against the clinically important GII.4 and GII.17 noroviruses with a focus on the identification of novel nanobodies that efficiently block the HBGA binding site. Using X-ray crystallography, we have characterized nine different nanobodies that bound to the top, side, or bottom of the P domain. The eight nanobodies that bound to the top or side of the P domain were mainly genotype specific, while one nanobody that bound to the bottom cross-reacted against several genotypes and showed HBGA blocking potential. The four nanobodies that bound to the top of the P domain also inhibited HBGA binding, and structural analysis revealed that these nanobodies interacted with several GII.4 and GII.17 P domain residues that commonly engaged HBGAs. Moreover, these nanobody complementarity-determining regions (CDRs) extended completely into the cofactor pockets and would likely impede HBGA engagement. The atomic level information for these nanobodies and their corresponding binding sites provide a valuable template for the discovery of additional "designer" nanobodies. These next-generation nanobodies would be designed to target other important genotypes and variants, while maintaining cofactor interference. Finally, our results clearly demonstrate for the first time that nanobodies directly targeting the HBGA binding site can function as potent norovirus inhibitors. IMPORTANCE Human noroviruses are highly contagious and a major problem in closed institutions, such as schools, hospitals, and cruise ships. Reducing norovirus infections is challenging on multiple levels and includes the frequent emergence of antigenic variants, which complicates designing effective, broadly reactive capsid therapeutics. We successfully developed and characterized four norovirus nanobodies that bound at the HBGA pockets. Compared with previously developed norovirus nanobodies that inhibited HBGA through disrupted particle stability, these four novel nanobodies directly inhibited HBGA engagement and interacted with HBGA binding residues. Importantly, these new nanobodies specifically target two genotypes that have caused the majority of outbreaks worldwide and consequently would have an enormous benefit if they could be further developed as norovirus therapeutics. To date, we have structurally characterized 16 different GII nanobody complexes, a number of which block HBGA binding. These structural data could be used to design multivalent nanobody constructs with improved inhibition properties.

Identification of Potential Proteinaceous Ligands of GI.1 Norovirus in Pacific Oyster Tissues.

Human norovirus (HuNoV) is the leading foodborne pathogen causing nonbacterial gastroenteritis worldwide. The oyster is an important vehicle for HuNoV transmission, especially the GI.1 HuNoV. In our previous study, oyster heat shock protein 70 (oHSP 70) was identified as the first proteinaceous ligand of GII.4 HuNoV in Pacific oysters besides the commonly accepted carbohydrate ligands, a histo-blood group antigens (HBGAs)-like substance. However the mismatch of the distribution pattern between discovered ligands and GI.1 HuNoV suggests that other ligands may exist. In our study, proteinaceous ligands for the specific binding of GI.1 HuNoV were mined from oyster tissues using a bacterial cell surface display system. Fifty-five candidate ligands were identified and selected through mass spectrometry identification and bioinformatics analysis. Among them, the oyster tumor necrosis factor (oTNF) and oyster intraflagellar transport protein (oIFT) showed strong binding abilities with the P protein of GI.1 HuNoV. In addition, the highest mRNA level of these two proteins was found in the digestive glands, which is consistent with GI.1 HuNoV distribution. Overall the findings suggested that oTNF and oIFT may play important roles in the bioaccumulation of GI.1 HuNoV.

Long-term storage protocol of reagent red blood cells treated with 0.01M dithiothreitol (DTT) for pre-transfusion testing of patients receiving anti-CD38 therapy, daratumumab.

OBJECTIVE: Use red blood cell stabilizer to store the antibody screening and antibody identification reagent red blood cells (RBCs) treated with 0.01 mol/L DTT and investigate its value in the pre-transfusion examinations of patients treated with daratumumab. METHOD: Determined the optimal incubation time for the 0.01 mol/L DTT-treated RBCs method by evaluating the effect of treatment at different time points. Added ID-CellStab to store DTT-treated RBCs, determined the maximum shelf life of reagent RBCs by monitoring the hemolysis index, and assessed changes in the antigenicity of blood group antigens on the surface of RBCs during storage with antibody reagents. RESULT: A protocol for long-term storage of reagent red blood cells treated with the 0.01 mol/L DTT method was established. The optimal incubation time was 40-50 min. RBCs could be stored stably for 18 days after adding ID-CellStab. The protocol was able to eliminate pan-agglutination caused by daratumumab, with no significant changes in the antigens of most blood group systems, except for some attenuation of K antigen and Duffy blood group system antigens during the storage period. CONCLUSION: The storage protocol of reagent RBCs based on the 0.01 mol/L DTT method does not affect the detection of most blood group antibodies and retains a certain degree of detection ability for anti-K antibodies, allowing patients treated with daratumumab to quickly perform pre-transfusion examinations, making up for the shortcomings of currently commercial reagent RBCs.

Long-time scale simulations of virus-like particles from three human-norovirus strains.

The dynamics of the virus like particles (VLPs) corresponding to the GII.4 Houston, GII.2 SMV, and GI.1 Norwalk strains of human noroviruses (HuNoV) that cause gastroenteritis was investigated by means of long-time (about 30 µs in the laboratory timescale) molecular dynamics simulations with the coarse-grained UNRES force field. The main motion of VLP units turned out to be the bending at the junction between the P1 subdomain (that sits in the VLP shell) and the P2 subdomain (that protrudes outside) of the major VP1 protein, this resulting in a correlated wagging motion of the P2 subdomains with respect to the VLP surface. The fluctuations of the P2 subdomain were found to be more pronounced and the P2 domain made a greater angle with the normal to the VLP surface for the GII.2 strain, which could explain the inability of this strain to bind the histo-blood group antigens (HBGAs).

Differential enzymatic deglycosylation reveals attachment of red cell B antigen onto the carbohydrate moiety of glycophorin A and glycophorin B.

BACKGROUND AND OBJECTIVES: Early studies indicate that red cell A and B antigens are attached primarily onto band 3 and GLUT1 on the erythrocyte membrane and little onto glycophorin A (GPA) and glycophorin B (GPB). But as GPA and band 3 form stable protein complexes and GPA is much more heavily glycosylated than band 3, this study re-examined the association between ABO antigens and GPA/GPB. MATERIALS AND METHODS: Band 3/GPA-associated protein complexes were first immunoprecipitated, followed by differential enzymatic deglycosylation that removed sialic acids, N-glycans and O-glycans. Serological anti-A (BIRMA 1) and anti-B IgM (GAMA 110) could be used for western blot (WB); however, only the anti-B IgM showed significant reactivity for the immunoprecipitates isolated by anti-band 3. The expression of the B antigen in un-deglycosylated and differentially deglycosylated band 3 immunoprecipitates was thus compared. RESULTS: Besides attachment to band 3, red cell B antigen expressed substantially on GPA monomer and homodimer, GPA*GPB heterodimer, and GPB monomer and dimer via attachments through the N- and O-glycans. CONCLUSION: Immunoprecipitation (IP), as a means of protein separation and concentration, was used in combination with a WB to differentiate glycosylation on different proteins and oligomers. This study implemented differential enzymatic deglycosylation during IP of the band 3 complexes. This combined approach allowed separate identification of the B antigen on GPA/GPB monomer and dimer and GPA*GPB heterodimer, and band 3 on the WB and verified non-trivial expression of the B antigen on GPA and GPB on the erythrocyte surface.

Functional and structural characterization of Norovirus GII.6 in recognizing histo-blood group antigens.

Noroviruses (NoVs) are the primary cause of acute gastroenteritis worldwide. Histo-blood group antigens (HBGAs) are receptors or attachment factors that affect the prevalence and host susceptibility of NoVs. GII.6 NoV is one of the predominant genotypes in humans, which recognizes the type ABO secretor of HBGAs. However, the structural basis of GII.6 NoV's interaction with HBGAs receptors remains elusive. In this study, we investigated the binding features of the GII.6 strain to HBGAs using saliva- and glycan-ELISA assays and characterized the molecular basis of the GII.6 virus that recognizes H disaccharide. We showed that the GII.6 â€‹P domain recognized some A and O secretor's saliva samples, most B secretor's saliva samples, and H disaccharide antigen, but did not bind non-secretors' saliva. Further, we determined the crystal structures of GII.6 and its complex with H disaccharides at 1.7 â€‹Å, revealing that the P domain of GII.6 shares the conventional binding interface and mode of GII HBGAs. Single residue mutations at the GII.6-H binding sites could inhibit the binding of GII.6 to HBGAs, demonstrating that the interaction residues were crucial in maintaining NoV-glycan integrity. Finally, structural and sequence analyses showed that the major residues of the GII.6-H interaction were conserved among NoVs in the GII genogroup. Taken together, our study characterized the functional and structural features of GII.6 that allow it to interact with HBGAs, and shed light on NoV evolution, epidemiology, and anti-viral drug development.

Synthesis of a B-Antigen Hexasaccharide, a B-Lewis b Heptasaccharide and Glycoconjugates Thereof to Investigate Binding Properties of Helicobacter pylori.

Infecting the stomach of almost 50 % of people, Helicobacter pylori is a causative agent of gastritis, peptic ulcers and stomach cancers. Interactions between bacterial membrane-bound lectin, Blood group Antigen Binding Adhesin (BabA), and human blood group antigens are key in the initiation of infection. Herein, the synthesis of a B-antigen hexasaccharide (B6) and a B-Lewis b heptasaccharide (BLeb7) and Bovine Serum Albumin glycoconjugates thereof is reported to assess the binding properties and preferences of BabA from different strains. From a previously reported trisaccharide acceptor a versatile key Lacto-N-tetraose tetrasaccharide intermediate was synthesized, which allowed us to explore various routes to the final targets, either via initial introduction of fucosyl residues followed by introduction of the B-determinant or vice versa. The first approach proved unsuccessful, whereas the second afforded the target structures in good yields. Protein conjugation using isothiocyanate methodology allowed us to reach high glycan loadings (up to 23 per protein) to mimic multivalent displays encountered in Nature. Protein glycoconjugate inhibition binding studies were performed with H. pylori strains displaying high or low affinity for Lewis b hexasaccharide structures showing that the binding to the high affinity strain was reduced due to the presence of the B-determinant in the Bleb7-conjugates and further reduced by the absence of the Lewis fucose residue in the B6-conjugate.

Missense mutations in PIEZO1, which encodes the Piezo1 mechanosensor protein, define Er red blood cell antigens.

Despite the identification of the high-incidence red cell antigen Era nearly 40 years ago, the molecular background of this antigen, together with the other 2 members of the Er blood group collection, has yet to be elucidated. Whole exome and Sanger sequencing of individuals with serologically defined Er alloantibodies identified several missense mutations within the PIEZO1 gene, encoding amino acid substitutions within the extracellular domain of the Piezo1 mechanosensor ion channel. Confirmation of Piezo1 as the carrier molecule for the Er blood group antigens was demonstrated using immunoprecipitation, CRISPR/Cas9-mediated gene knockout, and expression studies in an erythroblast cell line. We report the molecular bases of 5 Er blood group antigens: the recognized Era, Erb, and Er3 antigens and 2 novel high-incidence Er antigens, described here as Er4 and Er5, establishing a new blood group system. Anti-Er4 and anti-Er5 are implicated in severe hemolytic disease of the fetus and newborn. Demonstration of Piezo1, present at just a few hundred copies on the surface of the red blood cell, as the site of a new blood group system highlights the potential antigenicity of even low-abundance membrane proteins and contributes to our understanding of the in vivo characteristics of this important and widely studied protein in transfusion biology and beyond.

Structural Basis for Rabbit Hemorrhagic Disease Virus Antibody Specificity.

Rabbit hemorrhagic disease virus (RHDV) typically causes a fatal disease in rabbits. In Australia, RHDV was imported to control the feral rabbit population, while it poses a severe threat to native rabbits in other countries. RHDV variants are genetically diverse and serological studies using antibodies isolated from infected rabbits or raised against RHDV virus-like particles (VLPs) have found RHDV variants antigenically distinct. In this study, we determined the X-ray crystal structure of an RHDV GI.2 (N11 strain) protruding (P) domain in complex with a diagnostic monoclonal antibody (2D9) Fab. We showed that 2D9 interacted with conserved and variable residues on top of the P domain with nanomolar affinity. To better illustrate 2D9 specificity, we determined the X-ray crystal structure of an RHDV GI.1b (Ast89 strain) that was a 2D9 non-binder. Structural analysis indicated that amino acid substitutions on the GI.1b P domain likely restricted 2D9 binding. Interestingly, a model of the GI.2 P domain-Fab complex superimposed onto a cryo-EM structure of an RHDV VLP revealed that 2D9 Fab molecules clashed with neighboring Fabs and indicated that there was a reduced antibody binding occupancy. Moreover, the RHDV GI.2 histo-blood group antigen (HBGA) co-factor binding site appeared obstructed when 2D9 was modeled on the VLP and suggested that 2D9 might also function by blocking HBGA attachment. Overall, this new data provides the first structural basis of RHDV antibody specificity and explains how amino acid variation at the binding site likely restricts 2D9 cross-reactivity. IMPORTANCE Isolated RHDV antibodies have been used for decades to distinguish between antigenic variants, monitor temporal capsid evolution, and examine neutralizing capacities. In this study, we provided the structural basis for an RHDV GI.2 specific diagnostic antibody (2D9) binding and reveal that a small number of amino acid substitutions at the binding site could differentiate between RHDV GI.2 and GI.1b. This novel structural information provides a framework for understanding how RHDV displays a specific antigenic epitope and engages an antibody at the atomic level. Importantly, part of the 2D9 binding region was earlier reported to contain a neutralizing epitope and our structural modeling as well as recent human norovirus antibody-mediated neutralization studies, suggest that the 2D9 antibody has the potential to block HBGA attachment. These new findings should aid in characterizing antigenic variants and advance the development of novel monoclonal antibodies for diagnostics and therapeutics.

Glycan-mediated interactions between bacteria, rotavirus and the host cells provide an additional mechanism of antiviral defence.

Limited efficacy of rotavirus (RV) vaccines in children in developing countries and in animals remains a significant problem necessitating further search for additional approaches to control RV-associated gastroenteritis. During cell attachment and entry events, RV interacts with cell surface O-glycans including histo-blood group antigens (HBGAs). Besides modulation of the protective immunity against RV, several commensal and probiotic bacteria were shown to express HBGA-like substances suggesting that they may affect RV attachment and entry into the host cells. Moreover, some beneficial bacteria have been shown to possess the ability to bind host HBGAs via sugar specific proteins called lectins. However, limited research has been done to evaluate the effects of HBGA-expressing and/or HBGA-binding bacteria on RV infection. The aim of this study was to investigate the ability of selected commensal and probiotic bacteria to bind different RV strains via HBGAs and to block RV infection of IPEC-J2 cells. Our data indicated that Gram-negative probiotic Escherichia coli Nissle 1917 (E. coli Nissle 1917) and commensal Gram-positive (Streptococcus bovis and Bifidobacterium adolescentis) and Gram-negative (Bacteroides thetaiotaomicron, Clostridium clostridioforme and Escherichia coli G58 (E. coli G58) bacteria of swine origin expressed HBGAs which correlated with their ability to bind group A and C RVs. Additionally, Gram-positive E. coli 1917 and E. coli G58 demonstrated the ability to block RV attachment onto IPEC-J2 cells. Taken together, our results support the hypothesis that physical interactions between RVs and HBGA-expressing beneficial bacteria may limit RV replication.