In vitro evolution of H5N1 avian influenza virus toward human-type receptor specificity.

Acquisition of α2-6 sialoside receptor specificity by α2-3 specific highly-pathogenic avian influenza viruses (H5N1) is thought to be a prerequisite for efficient transmission in humans. By in vitro selection for binding α2-6 sialosides, we identified four variant viruses with amino acid substitutions in the hemagglutinin (S227N, D187G, E190G, and Q196R) that revealed modestly increased α2-6 and minimally decreased α2-3 binding by glycan array analysis. However, a mutant virus combining Q196R with mutations from previous pandemic viruses (Q226L and G228S) revealed predominantly α2-6 binding.

Bifunctional CD22 ligands use multimeric immunoglobulins as protein scaffolds in assembly of immune complexes on B cells.

CD22 is a B cell-specific sialic acid-binding immunoglobulin-like lectin (Siglec) whose function as a regulator of B cell signaling is modulated by its interaction with glycan ligands bearing the sequence NeuAc alpha2-6Gal. To date, only highly multivalent polymeric ligands (n = 450) have achieved sufficient avidity to bind to CD22 on native B cells. Here we demonstrate that a synthetic bifunctional molecule comprising a ligand of CD22 linked to an antigen (nitrophenol; NP) can use a monoclonal anti-NP IgM as a decavalent protein scaffold to efficiently drive assembly of IgM-CD22 complexes on the surface of native B cells.

High-affinity ligand probes of CD22 overcome the threshold set by cis ligands to allow for binding, endocytosis, and killing of B cells.

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 Sia alpha2-6Gal 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.

ST6Gal-I restrains CD22-dependent antigen receptor endocytosis and Shp-1 recruitment in normal and pathogenic immune signaling.

The ST6Gal-I sialyltransferase produces Siglec ligands for the B-cell-specific CD22 lectin and sustains humoral immune responses. Using multiple experimental approaches to elucidate the mechanisms involved, we report that ST6Gal-I deficiency induces immunoglobulin M (IgM) antigen receptor endocytosis in the absence of immune stimulation. This coincides with increased antigen receptor colocalization with CD22 in both clathrin-deficient and clathrin-enriched membrane microdomains concurrent with diminished tyrosine phosphorylation of Igalpha/beta, Syk, and phospholipase C-gamma2 upon immune activation.

Sweet spots in functional glycomics.

Information contained in the mammalian glycome is decoded by glycan-binding proteins (GBPs) that mediate diverse functions including host-pathogen interactions, cell trafficking and transmembrane signaling. Although information on the biological roles of GBPs is rapidly expanding, challenges remain in identifying the glycan ligands and their impact on GBP function. Protein-glycan interactions are typically low affinity, requiring multivalent interactions to achieve a biological effect.

Homomultimeric complexes of CD22 in B cells revealed by protein-glycan cross-linking.

CD22 is a negative regulator of B-cell receptor signaling, an activity mediated by recruitment of SH2 domain-containing phosphatase 1 through a phosphorylated immunoreceptor tyrosine inhibitory motif in its cytoplasmic domain. As in other members of the sialic acid-binding immunoglobulin-like lectin, or siglec, family, the extracellular N-terminal immunoglobulin domain of CD22 binds to glycan ligands containing sialic acid, which are highly expressed on B-cell glycoproteins. B-cell glycoproteins bind to CD22 in cis and 'mask' the ligand-binding domain, modulating its activity as a regulator of B-cell signaling.

Ablation of CD22 in ligand-deficient mice restores B cell receptor signaling.

CD22 is a negative regulator of B cell signaling, an activity modulated by its interaction with glycan ligands containing alpha2-6-linked sialic acids. B cells deficient in the enzyme (ST6Gal I) that forms the CD22 ligand show suppressed BCR signaling. Here we report that mice deficient in both CD22 and its ligand (Cd22-/- St6gal1-/- mice) showed restored B cell receptor (BCR) signaling, suggesting that the suppressed signaling of St6gal1-/- cells is mediated through CD22.

Cell surface biology mediated by low affinity multivalent protein-glycan interactions.

Glycan-binding proteins mediate diverse aspects of cell biology including pathogen recognition of host cells, cell trafficking, endocytosis and modulation of cell signaling. This is accomplished despite the intrinsic low affinity for their ligands through multivalent interactions that increase effective affinity and adhesive force. Recent successes in the rational design of high-affinity ligands for glycan-binding proteins offer the promise to create well-defined tools for exploring the structure and functions of this class of receptors.

Peanut agglutinin high phenotype of activated CD8+ T cells results from de novo synthesis of CD45 glycans.

Following activation in the periphery, murine CD8+ T cells exhibit a characteristic increased binding of peanut agglutinin (PNA), reflecting an increased expression of hyposialylated O-linked glycans (Galbeta1-3GalNAcalpha-O-Thr/Ser) on the cell surface. In this report, we show that the majority of the PNA receptors expressed on activated CD8+ T cells are carried by CD45. Other glycoproteins (e.

Masking of CD22 by cis ligands does not prevent redistribution of CD22 to sites of cell contact.

CD22, a negative regulator of B cell signaling, is a member of the siglec family that binds to alpha2-6-linked sialic acids on glycoproteins. Previous reports demonstrated that binding of multivalent sialoside probes to CD22 is blocked, or "masked," by endogenous (cis) ligands, unless they are first destroyed by sialidase treatment. These results suggest that cis ligands on B cells make CD22 functionally unavailable for binding to ligands in trans.

Transitional and marginal zone B cells have a high proportion of unmasked CD22: implications for BCR signaling.

CD22, a B cell-specific member of the Siglec family, is an important inhibitor of B cell signaling. The first Ig-like domain of CD22 specifically binds to alpha2,6-linked sialic acids. Through these interactions CD22 can mediate adhesion to other cells in trans, but can also bind endogenous ligands on the B cell surface in cis.

Systematic synthesis and MAG-binding activity of novel sulfated GM1b analogues as mimics of Chol-1 (alpha-series) gangliosides: highly active ligands for neural siglecs.

Systematic synthesis and myelin-associated glycoprotein (MAG)-binding activity of novel sulfated GM1b analogues structurally related to Chol-1 (alpha-series) gangliosides, high-affinity ligands for neural siglecs, are described. The suitably protected gangliotriose derivatives, 2-(trimethylsilyl)ethyl 2-acetamido-2-deoxy-6-O-levulinoyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzyl-beta-D-glucopyranoside and 2-(trimethylsilyl)ethyl 2-acetamido-2-deoxy-6-O-levulinoyl-beta-D-galactopyranosyl-(1-->4)-2,6-di-O-benzyl-3-O-levulinoyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzyl-beta-D-glucopyranoside were each glycosylated with alpha-NeuAc-(2-->3)-galactose donor to give the corresponding pentasaccharides in 94% (beta1,3 glycoside only) and 90% (beta1,3:beta1,4 = 2:1), respectively. After proper manipulation of the protecting groups, the pentasaccharides were converted into three novel sulfated GM1b gangliosides by the successive introduction of the ceramide and sulfo groups, followed by complete deprotection.

Sialoside specificity of the siglec family assessed using novel multivalent probes: identification of potent inhibitors of myelin-associated glycoprotein.

Ten of the 11 known human siglecs or their murine orthologs have been evaluated for their specificity for over 25 synthetic sialosides representing most of the major sequences terminating carbohydrate groups of glycoproteins and glycolipids. Analysis has been performed using a novel multivalent platform comprising biotinylated sialosides bound to a streptavidin-alkaline phosphatase conjugate. Each siglec was found to have a unique specificity for binding 16 different sialoside-streptavidin-alkaline phosphatase probes.

Constitutively unmasked CD22 on B cells of ST6Gal I knockout mice: novel sialoside probe for murine CD22.

The interaction of CD22 with glycoprotein ligands bearing the Siaalpha2,6Gal-R sequence is believed to modulate its function as a regulator of B cell signaling. Although a commercial sialoside-polyacrylamide (PAA) probe, NeuAc- alpha2,6Gal-PAA, has facilitated studies on ligand binding by human CD22, murine CD22 binds instead with high affinity to NeuGcalpha2,6Gal-R. A multivalent probe with this sequence was constructed to facilitate investigations of ligand binding in CD22 function using genetically defined murine models.