Carbohydrate specificity of sea urchin sperm bindin: a cell surface lectin mediating sperm-egg adhesion.

We have examined the carbohydrate specificity of bindin, a sperm protein responsible for the adhesion of sea urchin sperm to eggs, by investigating the interaction of a number of polysaccharides and glycoconjugates with isolated bindin. Several of these polysaccharides inhibit the agglutination of eggs by bindin particles. An egg surface polysaccharide was found to be the most potent inhibitor of bindin-mediated egg agglutination. Fucoidin, a sulfated fucose heteropolysaccharide, was the next most potent inhibitor, followed by the egg jelly fucan, a sulfated fucose homopolysaccharide, and xylan, a beta(1 leads to 4) linked xylose polysaccharide. A wide variety of other polysaccharides and glycoconjugates were found to have no effect on egg agglutination. We also report that isolated bindin has a soluble lectinlike activity which is assayed by agglutination of erythrocytes. The bindin lectin activity is inhibited by the same polysaccharides that inhibit egg agglutination by particulate bindin. This suggests that the egg adhesion activity of bindin is directly related to its lectin activity. We have established that fucoidin binds specifically to bindin particles with a high apparent affinity (Kd = 5.5 X 10(-8) M). The other polysaccharides that inhibit egg agglutination also inhibit the binding of 125I-fucoidin to bindin particles, suggesting that they compete for the same site on bindin. The observation that polysaccharides of different composition and linkage type interact with bindin suggests that the critical structural features required for binding may reside at a higher level of organization. Together, these findings strengthen the hypothesis that sperm-egg adhesion in sea urchins is mediated by a lectin-polysaccharide type of interaction

Monoclonal Antibodies of a Diverse Isotype Induced by an O-Antigen Glycoconjugate Vaccine Mediate In Vitro and In Vivo Killing of African Invasive Nontyphoidal Salmonella.

Nontyphoidal Salmonella (NTS), particularly Salmonella enterica serovars Typhimurium and Enteritidis, is responsible for a major global burden of invasive disease with high associated case-fatality rates. We recently reported the development of a candidate O-antigen-CRM197 glycoconjugate vaccine against S. Typhimurium. Here, using a panel of mouse monoclonal antibodies generated by the vaccine, we examined the relative efficiency of different antibody isotypes specific for the O:4 antigen of S. Typhimurium to effect in vitro and in vivo killing of the invasive African S. Typhimurium strain D23580. All O:4-specific antibody isotypes could mediate cell-free killing and phagocytosis of S. Typhimurium by mouse blood cells. Opsonization of Salmonella with O:4-specific IgA, IgG1, IgG2a, and IgG2b, but not IgM, resulted in cell-dependent bacterial killing. At high concentrations, O:4-specific antibodies inhibited both cell-free complement-mediated and cell-dependent opsonophagocytic killing of S. Typhimurium in vitro. Using passive immunization in mice, the O:4-specific antibodies provided in vivo functional activity by decreasing the bacterial load in the blood and tissues, with IgG2a and IgG2b being the most effective isotypes. In conclusion, an O-antigen-CRM197 glycoconjugate vaccine can induce O-antigen-specific antibodies of different isotypes that exert in vitro and in vivo killing of S. Typhimurium

Optimisation of the enzyme-linked lectin assay for enhanced glycoprotein and glycoconjugate analysis

Lectin’s are proteins capable of recognising and binding to specific oligosaccharide tructures found on glycoproteins and other biomoloecules. As such they have found tility for glycoanalytical applications. One common difficulty encountered in the pplication of these proteins, particularly in multi-well plate assay formats known as Enzyme Linked Lectin Assays (ELLA’s), is in finding appropriate blocking solutions to prevent non-specific binding with plate surfaces. Many commonly used blocking agents contain carbohydrates and generate significant background signals in ELLA’s, limiting the utility of the assay. In this study we examined the suitability of a range of blocking reagents, including rotein based, synthetic and commercially available carbohydrate free blocking eagents, for ELLA applications. Each blocking reagent was assessed against a panel f 19 commercially available biotinylated lectins exhibiting diverse structures and arbohydrate specificities. We identified the synthetic polymer Polyvinyl Alcohol PVA) as the best global blocking agent for performing ELLA’s. We ultimately present n ELLA methodology facilitating broad spectrum lectin analysis of glycoconjugates nd extending the utility of the ELLA

Identification and purification of an endogenous receptor for the lectin pallidin from Polysphondylium pallidum.

We report the identification and purification of an endogenous carbohydrate-containing receptor of pallidin, the cell surface lectin implicated in mediating cell-cell adhesion in the cellular slime mold Polysphondylium pallidum. The receptor is identified in an aqueous extract of crude P. pallidum membranes as a potent inhibitor of the hemagglutination activity of pallidin. The inhibitor is purified to apparent homogeneity by affinity precipitation with pallidin followed by fractionation of the solubilized precipitate on Sepharose 4B. The hemagglutination inhibitor (HAI) is metabolically radiolabeled, indicating that it is a biosynthetic product of the amoebae and not an ingested food substance. The HAI is released into the extracellular medium by living, differentiated amoebae. This release is markedly facilitated by the addition of D-galactose, a specific saccharide that binds to pallidin. Hence, the HAI appears to have an in situ association with pallidin at the cell surface. Exogenously added HAI promotes the agglutination of differentiated amoebae in a gyrated suspension at very low concentrations. The results are consistent with a model of cell-cell adhesion in which the HAI is a multivalent, extracellular aggregation factor that is recognized by pallidin molecules on adjacent cells. The HAI would then be analogues to the aggregation factors identified in marine sponges

Neuroprotective effects of minocycline on double-stranded RNA-induced neurotoxicity in cultured cortical neurons

1. Minocycline, memantine,and glycoconjugate were assessed for their ability to protect cultured primary cortical neurons against double-stranded RNA-induced neurotoxicity. 2. Minocycline but not memantine or glycoconjugate protected cultured cells and warrants further investigation.published_or_final_versio

Nanobody mediated inhibition of attachment of F18 fimbriae expressing Escherichia coli

Post-weaning diarrhea and edema disease caused by F18 fimbriated E. coli are important diseases in newly weaned piglets and lead to severe production losses in farming industry. Protective treatments against these infections have thus far limited efficacy. In this study we generated nanobodies directed against the lectin domain of the F18 fimbrial adhesin FedF and showed in an in vitro adherence assay that four unique nanobodies inhibit the attachment of F18 fimbriated E. coli bacteria to piglet enterocytes. Crystallization of the FedF lectin domain with the most potent inhibitory nanobodies revealed their mechanism of action. These either competed with the binding of the blood group antigen receptor on the FedF surface or induced a conformational change in which the CDR3 region of the nanobody displaces the D ''-E loop adjacent to the binding site. This D ''-E loop was previously shown to be required for the interaction between F18 fimbriated bacteria and blood group antigen receptors in a membrane context. This work demonstrates the feasibility of inhibiting the attachment of fimbriated pathogens by employing nanobodies directed against the adhesin domain

How do nematodes transfer phosphorylcholine to carbohydrates?

An unusual aspect of the biology of nematodes is the attachment of phosphorylcholine (PC) to carbohydrate. The attachment appears to play an important role in nematode development and, in some parasitic species, in immunomodulation. This article considers the nature of the biosynthetic pathway of nematode PC-containing glycoconjugates and, in particular, the identity of the final component in the pathway - the enzyme that transfers PC to carbohydrate (the 'PC transferase'). We offer the opinion that the PC transferase could be a member of the fukutin family (fukutin refers to the mutated gene product that causes Fukuyama congenital muscular dystrophy), a group of enzymes with apparent phosphoryl-ligand transferase activity that are found in organisms ranging from bacteria to humans

A novel mechanism for glycoconjugate vaccine activation of the adaptive immune system

Although glycoconjugate vaccines have provided enormous health benefits globally, they have been less successful in significant high-risk populations. Exploring novel approaches to the enhancement of glycoconjugate effectiveness, we investigated molecular and cellular mechanisms governing the immune response to a prototypical glycoconjugate vaccine. In antigen-presenting cells, a carbohydrate epitope is generated upon endolysosomal processing of group B streptococcal type III polysaccharide coupled to a carrier protein. In conjunction with a carrier protein-derived peptide, this carbohydrate epitope binds to major histocompatibility class II (MHCII) and stimulates carbohydrate-specific CD4+ T-cell clones to produce interleukins 2 and 4—cytokines essential for providing T-cell help to antibody-producing B cells. An archetypical glycoconjugate vaccine constructed to maximize the presentation of carbohydrate epitopes recognized by T cells is 50–100 times more potent and significantly more protective in an animal model of infection than is a currently used vaccine construct

A Vaccine Against Group B Streptococcus: Recent Advances

Group B streptococcus (GBS) causes a high burden of neonatal and infant disease globally. Implementing a vaccine for pregnant women is a promising strategy to prevent neonatal and infant GBS disease and has been identified as a priority by the World Health Organisation (WHO). GBS serotype-specific polysaccharide – protein conjugate vaccines are at advanced stages of development, but a large number of participants would be required to undertake Phase III clinical efficacy trials. Efforts are therefore currently focused on establishing serocorrelates of protection in natural immunity studies as an alternative pathway for licensure of a GBS vaccine, followed by Phase IV studies to evaluate safety and effectiveness. Protein vaccines are in earlier stages of development but are highly promising as they might confer protection irrespective of serotype. Further epidemiological, immunological and health economic studies are required to enable the vaccine to reach its target population as soon as possible

Anomeric O-Functionalization of Carbohydrates for Chemical Conjugation to Vaccine Constructs.

Carbohydrates mediate a wide range of biological interactions, and understanding these processes benefits the development of new therapeutics. Isolating sufficient quantities of glycoconjugates from biological samples remains a significant challenge. With advances in chemical and enzymatic carbohydrate synthesis, the availability of complex carbohydrates is increasing and developing methods for stereoselective conjugation these polar head groups to proteins and lipids is critically important for pharmaceutical applications. The aim of this review is to provide an overview of commonly employed strategies for installing a functionalized linker at the anomeric position as well as examples of further transformations that have successfully led to glycoconjugation to vaccine constructs for biological evaluation as carbohydrate-based therapeutics