Wengang Chai

Oligosaccharide microarrays for high-throughput detection and specificity assignments of carbohydrate-protein interactions

We describe microarrays of oligosaccharides as neoglycolipids and their robust display on nitrocellulose. The arrays are sourced from glycoproteins, glycolipids, proteoglycans, polysaccharides, whole organs, or from chemically synthesized oligosaccharides. We show that carbohydrate-recognizing proteins single out their ligands not only in arrays of homogeneous oligosaccharides but also in arrays of heterogeneous oligosaccharides. Initial applications have revealed new findings, including: (i) among O-glycans in brain, a relative abundance of the Lewisx sequence based on N-acetyllactosamine recognized by anti-L5, and a paucity of the Lewisx sequence based on poly-N-acetyllactosamine recognized by anti-SSEA-1; (ii) insights into chondroitin sulfate oligosaccharides recognized by an antiserum and an antibody (CS-56) to chondroitin sulfates; and (iii) binding of the cytokine interferon-γ (IFN-γ) and the chemokine RANTES to sulfated sequences such as HNK-1, sulfo-Lewisx, and sulfo-Lewisa, in addition to glycosaminoglycans. The approach opens the way for discovering new carbohydrate-recognizing proteins in the proteome and for mapping the repertoire of carbohydrate recognition structures in the glycome.

Adhesion of Plasmodium falciparum-infected erythrocytes to hyaluronic acid in placental malaria

Infection with Plasmodium falciparum during pregnancy leads to the accumulation of parasite-infected erythrocytes in the placenta, and is associated with excess perinatal mortality, premature delivery and intrauterine growth retardation in the infant, as well as increased maternal mortality and morbidity. P. falciparum can adhere to specific receptors on host cells, an important virulence factor enabling parasites to accumulate in various organs. We report here that most P. falciparum isolates from infected placentae can bind to hyaluronic acid, a newly discovered receptor for parasite adhesion that is present on the placental lining. In laboratory isolates selected for specific high-level adhesion, binding to hyaluronic acid could be inhibited by dodecamer or larger oligosaccharide fragments or polysaccharides, treatment of immobilized receptor with hyaluronidase, or treatment of infected erythrocytes with trypsin. In vitro flow-based assays demonstrated that high levels of adhesion occurred at low wall shear stress, conditions thought to prevail in the placenta. Our findings indicate that adhesion to hyaluronic acid is involved in mediating placental parasite accumulation, thus changing the present understanding of the mechanisms of placental infection, with implications for the development of therapeutic and preventative interventions.

GM1 structure determines SV40-induced membrane invagination and infection

Incoming simian virus 40 (SV40) particles enter tight-fitting plasma membrane invaginations after binding to the carbohydrate moiety of GM1 gangliosides in the host cell plasma membrane through pentameric VP1 capsid proteins. This is followed by activation of cellular signalling pathways, endocytic internalization and transport of the virus via the endoplasmic reticulum to the nucleus. Here we show that the association of SV40 (as well as isolated pentameric VP1) with GM1 is itself sufficient to induce dramatic membrane curvature that leads to the formation of deep invaginations and tubules not only in the plasma membrane of cells, but also in giant unilamellar vesicles (GUVs). Unlike native GM1 molecules with long acyl chains, GM1 molecular species with short hydrocarbon chains failed to support such invagination, and endocytosis and infection did not occur. To conceptualize the experimental data, a physical model was derived based on energetic considerations. Taken together, our analysis indicates that SV40, other polyoma viruses and some bacterial toxins (Shiga and cholera) use glycosphingolipids and a common pentameric protein scaffold to induce plasma membrane curvature, thus directly promoting their endocytic uptake into cells.

Ligands for the β-Glucan Receptor, Dectin-1, Assigned Using “Designer” Microarrays of Oligosaccharide Probes (Neoglycolipids) Generated from Glucan Polysaccharides

Dectin-1 is a C-type lectin-like receptor on leukocytes that mediates phagocytosis and inflammatory mediator production in innate immunity to fungal pathogens. Dectin-1 lacks residues involved in calcium ligation that mediates carbohydrate-binding by classical C-type lectins; nevertheless, it binds zymosan, a particulate β-glucan-rich extract of Saccharomyces cerevisiae, and binding is inhibited by polysaccharides rich in β1,3- or both β1,3- and β1,6-linked glucose. The oligosaccharide ligands on glucans recognized by Dectin-1 have not yet been delineated precisely. It is also not known whether Dectin-1 can interact with other types of carbohydrates. We have investigated this, since Dectin-1 shows glucan-independent binding to a subset of T-lymphocytes and is involved in triggering their proliferation. Here we assign oligosaccharide ligands for Dectin-1 using the neoglycolipid-based oligosaccharide microarray technology, a unique approach for constructing microarrays of lipid-linked oligosaccharide probes from desired sources. We generate “designer” microarrays from three glucan polysaccharides, a neutral soluble glucan isolated from S. cerevisiae and two bacterial glucans, curdlan from Alcaligenes faecalis and pustulan from Umbilicaria papullosa, and use these in conjunction with 187 diverse, sequence-defined, predominantly mammalian-type, oligosaccharide probes. Among these, Dectin-1 binding is detected exclusively to 1,3-linked glucose oligomers, the minimum length required for detectable binding being a 10- or 11-mer. Thus, the ligands assigned so far are exogenous rather than endogenous. We further show that Dectin-1 ligands, 11-13 gluco-oligomers, in clustered form (displayed on liposomes), mimic the macromolecular β-glucans and compete with zymosan binding and triggering of tumor necrosis factor-α secretion by a Dectin-1-expressing macrophage cell line.

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

Oligosaccharide microarrays to decipher the glyco code.

The oligosaccharide moieties of glycoproteins, glycolipids, proteoglycans and polysaccharides are highly diverse, the reason for this diversity is not yet understood. Neoglycolipid technology allows the generation of oligosaccharide probes with lipid tags from desired sources and is showing promise as a basis for oligosaccharide microarrays. Such microarrays would allow surveys of glycomes and proteomes to be carried out, which would enable the molecular definition of carbohydrate-recognition systems in whole organisms.

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.

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.

Interactions with heparin-like molecules during erythrocyte invasion by Plasmodium falciparum merozoites

During erythrocyte invasion, Plasmodium falciparum merozoites use multiple receptor-ligand interactions in a series of coordinated events, but current knowledge of these interactions is limited. Using real-time imaging of invasion, we established that heparin-like molecules block early, and essential, events in erythrocyte invasion by merozoites. All P falciparum isolates tested, and parasites using different invasion pathways were inhibited to comparable levels. Furthermore, it was not possible to select for heparin-resistant parasites. Heparin-like molecules occur naturally on the surface of human erythrocytes, where they may act as receptors for binding of merozoite surface proteins. Consistent with this, we demonstrated that MSP1-42, a processed form of merozoite surface protein 1 (MSP1) involved in invasion, bound heparin in a specific manner; furthermore, binding was observed with the secondary processing fragment MSP1-33, but not MSP1-19. We defined key structural requirements of heparin-like molecules for invasion inhibition and interactions with MSP1-42. Optimal activity required a degree of sulfation more than or equal to 2, disulfation of the N-acetylglucosamine or hexuronic acid residue, and a minimum chain length of 6 monosaccharides. These findings have significant implications for understanding P falciparum invasion of erythrocytes and the development of novel therapeutics and vaccines.

Neoglycolipids: probes of oligosaccharide structure, antigenicity, and function.

Publisher Summary Neoglycoproteins have found considerable use in studies of carbohydrate-binding proteins and monoclonal antibodies and are available as commercial products. The special feature of the neoglycolipid technology is that it is applicable not only to mixtures of oligosaccharides released from proteins by established procedures [hydrazinolysis ( N -linked chains) or alkaline borohydride degradation ( O -linked chains) or by various partial degradation procedures, including endoglycosidase digestions] but is also applicable to any desired oligosaccharide sequence, be it natural or synthetic. By a microconjugation procedure each oligosaccharide is joined to a lipid molecule. The resulting neoglycolipids serve as immobilized probes on silica gel chromatograms, on plastic plates, or as microparticulate probes after incorporation into liposomes, and they are readily amenable to assay procedures established for glycosphingolipids. In the form of neoglycolipids, the oligosaccharides are presented at the matrix surface in a clustered state. This is an important requirement for interactions with carbohydrate-binding proteins, including antibodies, which frequently have low affinities and give no detectable binding to monovalent carbohydrate ligands under conventional binding assay conditions. The lipid most extensively used for generating neoglycolipids has been the aminophospholipid L -1,2-dipalmitoyl- sn -glycero-3-phosphoethanolamine.