1,3-Dipolar cycloadditions between azides and alkynes were exploited to attach oligosaccharides to a C(14) hydrocarbon chain that noncovalently binds to the microtiter well surface. Synthesis of sugar arrays was performed on a micromolar scale in situ in the microtiter plate. As a model study, the beta-galactosyllipid 5 was displayed on a 4-micromol scale. Formation of product was confirmed via ESI-MS, and the yield was determined via chemical and biological assays. Several complex carbohydrates (6-16) were also displayed in microtiter plates and successfully screened with various lectins. Moreover, sialyl Lewis x (17) was synthesized via the enzymatic fucosylation of a precursor displayed in the plate. Studies on inhibition of this biotransformation have been carried out, and the IC(50) value found for the known inhibitor 20 was consistent with previous studies in solution.
CD22 (Siglec-2) is a key regulator of B cell signaling whose function is modulated by interaction with extracellular glycan ligands mediated through its N-terminal Ig domain. Its preferred ligand is the sequence Siaα2-6Gal that is abundantly expressed on N-linked glycans of B cell glycoproteins, and by binding to CD22 in cis causes CD22 to appear “masked” from binding to synthetic sialoside probes. Yet, despite the presence of cis ligands, CD22 redistributes to sites of cell contact by binding to trans ligands on neighboring cells. In this study, we demonstrate the dynamic equilibrium that exists between CD22 and its cis and trans ligands, using a high-affinity multivalent sialoside probe that competes with cis ligands and binds to CD22 on native human and murine B cells. Consistent with the constitutive endocytosis reported for CD22, the probes are internalized once bound, demonstrating that CD22 is an endocytic receptor that can carry ligand-decorated “cargo” to intracellular compartments. Conjugation of the sialoside probes to the toxin saporin resulted in toxin uptake and toxin-mediated killing of B lymphoma cell lines, suggesting an alternative approach for targeting CD22 for treatment of B cell lymphomas.
Nuclear magnetic resonance spectroscopy demonstrates that the rhesus rotavirus hemagglutinin specifically binds ␣-anomeric N-acetylneuraminic acid with a K d of 1.2 mM. The hemagglutinin requires no additional carbohydrate moieties for binding, does not distinguish 3 from 6 sialyllactose, and has approximately tenfold lower affinity for N-glycolylneuraminic than for N-acetylneuraminic acid. The broad specificity and low affinity of sialic acid binding by the rotavirus hemagglutinin are consistent with this interaction mediating initial cell attachment prior to the interactions that determine host range and cell type specificity.Rotavirus causes approximately 600,000 childhood deaths through dehydrating diarrhea annually (43) and is also an important veterinary pathogen. Understanding early events in cell entry by rotavirus could aid in the development of therapeutic and preventive measures against rotavirus and could help explain the host range and cell type specificity of the virus.
We have expressed the Neisseria meningitidis lgtA gene at a high level in Escherichia coli. The encoded beta-N-acetylglucosaminyltransferase, referred to as LgtA, which in the bacterium is involved in the synthesis of the lacto-N-neo-tetraose structural element of the bacterial lipooligosaccharide, was obtained in an enzymatically highly active form. This glycosyltransferase appeared to be unusual in that it displays a broad acceptor specificity toward both alpha- and beta-galactosides, whether structurally related to N- or O-protein-, or lipid-linked oligosaccharides. Product analysis by one- and two-dimensional 400 MHz 1H- and 13C-NMR spectroscopy reveals that LgtA catalyzes the introduction of GlcNAc from UDP-GlcNAc in a beta 1-->3-linkage to accepting Gal residues. The enzyme can thus be characterized as a UDP-GlcNAc:Gal alpha/beta-R beta 3-N-acetylglucosaminyltransferase. Although lactose is a highly preferred acceptor substrate the recombinant enzyme also acts efficiently on monomeric and dimeric N-acetyllactosamine revealing its potential value in the synthesis of polylactosaminoglycan structures in enzyme assisted procedures. Furthermore, LgtA shows a high donor promiscuity toward UDP-GalNAc, but not toward other UDP-sugars, and can catalyze the introduction of GalNAc in beta 1-->3-linkage to alpha- or beta-Gal in the acceptor structures at moderate rates. LgtA therefore shows promise to be a useful catalyst in the preparative synthesis of both GlcNAc beta 1-->3Gal and GalNAc beta 1-->3Gal linkages.
Tetra- and hexasaccharides were arrayed on the exterior surface of cowpea mosaic virus by using a copper-catalyzed azide-alkyne cycloaddition reaction. Inoculation of chickens with these virus conjugates gave rise to large quantities of polyclonal anti-glycan IgY antibodies that displayed excellent avidity and specificity on analysis with printed glycan microarrays. Avian IgY antibodies are produced in significantly higher yield than is possible for mouse or rabbit IgG, and exhibit reduced cross reactivity with native mammalian proteins. For a tri-LacNAc antigen, affinity purification against immobilized mono-LacNAc was necessary to provide a set of antibodies with specific binding properties. Comparable performance was observed for the virus-based polyclonal versus a commercial monoclonal antibody raised against the globo-H tetrasaccharide; this highlights the utility of the glycan microarray for both quality control and rapid in-depth analysis. Virus-carbohydrate conjugates are promising candidates for development in diagnostic and immunotherapeutic applications.
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