Robert A. Childs

Receptor-binding specificity of pandemic influenza A (H1N1) 2009 virus determined by carbohydrate microarray

Receptor-binding specificity of pandemic influenza A (H1N1) 2009 virus determined by carbohydrate microarray

Altered Receptor Specificity and Cell Tropism of D222G Hemagglutinin Mutants Isolated from Fatal Cases of Pandemic A(H1N1) 2009 Influenza Virus

ABSTRACT Mutations in the receptor-binding site of the hemagglutinin of pandemic influenza A(H1N1) 2009 viruses have been detected sporadically. An Asp222Gly (D222G) substitution has been associated with severe or fatal disease. Here we show that 222G variants infected a higher proportion of ciliated cells in cultures of human airway epithelium than did viruses with 222D or 222E, which targeted mainly nonciliated cells. Carbohydrate microarray analyses showed that 222G variants bind a broader range of α2-3-linked sialyl receptor sequences of a type expressed on ciliated bronchial epithelial cells and on epithelia within the lung. These features of 222G mutants may contribute to exacerbation of disease.

Blood group i and I activities of "lacto-N-norhexaosylceramide" and its analogues: the structural requirements for i-specificities.

Abstract A pure straight chain ceramide hexasaccharide (“lacto-N- nor hexaosylceramide” Galβ1→4GlcNAcβ1→3Galβ1→4GlcNAcβ1→3Galβ1→4Glc→Ceramide) showed strong i-activity determined by hemagglutination inhibition and by radioimmunoassay with five out of six anti-i antisera. Two repeating Galβ1→4GlcNAc residues and GlcNAcβ1→3Gal residues could be essential for the full expression of this activity; eleven closely related analogues including those derived by chemical modification had lower or no detectable activity. The same structure reacted also with some anti-I antisera. The strong i-activity and the moderate I-activity were both abolished by elimination of the terminal Gal or by removal of the N-acetyl groups of the two GlcNAc residues.

N-Glycolyl GM1 Ganglioside as a Receptor for Simian Virus 40

ABSTRACT Carbohydrate microarrays have emerged as powerful tools in analyses of microbe-host interactions. Using a microarray with 190 sequence-defined oligosaccharides in the form of natural glycolipids and neoglycolipids representative of diverse mammalian glycans, we examined interactions of simian virus 40 (SV40) with potential carbohydrate receptors. While the results confirmed the high specificity of SV40 for the ganglioside GM1, they also revealed that N-glycolyl GM1 ganglioside [GM1(Gc)], which is characteristic of simian species and many other nonhuman mammals, is a better ligand than the N-acetyl analog [GM1(Ac)] found in mammals, including humans. After supplementing glycolipid-deficient GM95 cells with GM1(Ac) and GM1(Gc) gangliosides and the corresponding neoglycolipids with phosphatidylethanolamine lipid groups, it was found that GM1(Gc) analogs conferred better virus binding and infectivity. Moreover, we visualized the interaction of NeuGc with VP1 protein of SV40 by molecular modeling and identified a conformation for GM1(Gc) ganglioside in complex with the virus VP1 pentamer that is compatible with its presentation as a membrane receptor. Our results open the way not only to detailed studies of SV40 infection in relation to receptor expression in host cells but also to the monitoring of changes that may occur with time in receptor usage by the virus.

Oligosaccharide-mediated interactions of the envelope glycoprotein gp120 of HIV-1 that are independent of CD4 recognition.

In this study carbohydrate-mediated interactions of the envelope glycoprotein, gp120, of HIV-1 were investigated. Oligosaccharide probes (neoglycolipids), prepared from the N-glycosidically-linked chains of the natural and recombinant forms of gp120, were used in conjunction with the intact glycoprotein to investigate reactivities with a soluble carbohydrate-binding protein (lectin) known as mannose-binding protein in human serum. Evidence is presented that the high-mannose-type oligosaccharides with seven, eight and nine mannose residues from both forms of gp120 are recognized by the serum lectin, and that these reactivities are unrelated to CD4 recognition. Reactivities of the two forms of envelope glycoprotein with macrophages derived from human blood monocytes and with the mannose-specific macrophage endocytosis receptor isolated from human placental membranes were also investigated. Evidence is presented that both forms of gp120 bind to the macrophage surface by multiple interactions in addition to CD4 binding, and that among these interactions is a carbohydrate-mediated binding to the endocytosis receptor. We propose that such carbohydrate-mediated interactions could form the basis of viral attachment to a variety of healthy and diseased tissues.

Evidence for the occurrence of O-glycosidically linked oligosaccharides of poly-N-acetyllactosamine type on the human leucocyte common antigen

High molecular weight glycoproteins of human B and T lymphocytes known as leucocyte common antigen or T200 have been shown to carry O- and N-glycosidically linked, sialylated, carbohydrate chains. The O-linked chains are polydisperse and those of B rather than T cell type are highly susceptible to degradation by endo-beta-galactosidase. These differences among lymphocytes that are functionally distinct raise the possibility that the oligosaccharides may contribute to the functions of these differentiation molecules as well as to their electrophoretic diversity.

Atomic resolution insight into host cell recognition by Toxoplasma gondii

The obligate intracellular parasite Toxoplasma gondii, a member of the phylum Apicomplexa that includes Plasmodium spp., is one of the most widespread parasites and the causative agent of toxoplasmosis. Micronemal proteins (MICs) are released onto the parasite surface just before invasion of host cells and play important roles in host cell recognition, attachment and penetration. Here, we report the atomic structure for a key MIC, TgMIC1, and reveal a novel cell‐binding motif called the microneme adhesive repeat (MAR). Using glycoarray analyses, we identified a novel interaction with sialylated oligosaccharides that resolves several prevailing misconceptions concerning TgMIC1. Structural studies of various complexes between TgMIC1 and sialylated oligosaccharides provide high‐resolution insights into the recognition of sialylated oligosaccharides by a parasite surface protein. We observe that MAR domains exist in tandem repeats, which provide a highly specialized structure for glycan discrimination. Our work uncovers new features of parasite–receptor interactions at the early stages of host cell invasion, which will assist the design of new therapeutic strategies.

Differences in carbohydrate moieties of high molecular weight glycoproteins of human lymphocytes of T and B origins revealed by monoclonal autoantibodies with anti-I and anti-I specificities

Summary The expression of the I and i antigens on glycoproteins of galactose oxidase/NaB[3H]4 labelled human lymphocytes of T and B origins has been examined by immune precipitation with monoclonal anti-I and -i antibodies. SDS polyacrylamide gel electrophoresis of immune precipitates has revealed major differences in the expression of these carbohydrate antigens on cells of T and B origins. The ‘T200’ family of glycoproteins of lymphocytes of B rather than T origins strongly express I and i activities. In contrast, the major sialoglycoproteins (120–140K) of cells of T origin express strongly the determinant recognized by one anti-I antibody (Ma). From knowledge of their properties on erythrocyte membranes and epithelial secretions it can be predicted that Ii type oligosaccharides may serve as backbone structures for allo- or iso-antigenic determinants on lymphocytes.

Calf heart lectin reacts with blood group Ii antigens and other precursor chains of the major blood group antigens.

[4,5] and the i antigen is expressed predominantly on straight chain oligosaccharides containing the sequence G@l+4GlcNAcf31+3Gal [6]. Both are found as cryptic antigens on the core oligosaccharides of blood group ABH-active secreted glycoproteins [4,7], erythrocyte glycolipids and gangliosides [5,6,8] and as detectable surface antigens on human erythrocytes [9], leucocytes [lo] and a variety of animal cell types (R. C., T. F., unpublished observations). They are of special interest because of the developmental changes in their expression on human erythrocytes [9], cell cycle related changes on lymphocytes [ 1 l] and their increased expression in certain human adenocarcinoma tissues [ 121. Therefore we have investigated the possible role of the blood group I and i antigens as natural receptors for the calf heart lectin. Parallel studies with the /I-galactosyl-specific plant lectin, Ricinus communis agglutinin 120 [ 131 have shown that: (a) The calf heart lectin shows more specificity for the terminal disaccharide units of blood group antigen precursor chains than does the plant lectin; (b) Ii-active (blood group ABH-inactive) mucins are more potent inhibitors of both lectins than ABH active (Ii inactive) mucins; (c) Calf heart tissue contains I and i antigens which are potent inhibitors of both lectins.

Neoglycolipid-Based Oligosaccharide Microarray System: Preparation of NGLs and Their Noncovalent Immobilization on Nitrocellulose-Coated Glass Slides for Microarray Analyses

Carbohydrate microarrays, since their advent in 2002, are revolutionizing studies of the molecular basis of protein-carbohydrate interactions both in endogenous recognition systems and pathogen-host interactions. We have developed a unique carbohydrate microarray system based on the neoglycolipid (NGL) technology, a well-validated microscale approach for generating lipid-tagged oligosaccharide probes for use in carbohydrate recognition studies. This chapter provides an overview of the principles and key features of the NGL-based oligosaccharide microarrays, and describes in detail the basic techniques - from the preparation of NGL probes to the generation of microarrays using robotic arraying hardware, as well as a general protocol for probing the microarrays with carbohydrate-binding proteins.