SUMMARY The human JC polyomavirus (JCV) causes a fatal demyelinating disease, Progressive Multifocal Leukoencephalopathy (PML), in immunocompromised individuals. Current treatment options for PML are inadequate. Sialylated oligosaccharides and the serotonin receptor are known to be necessary for JCV entry, but the molecular interactions underlying JCV attachment remain unknown. Using glycan array screening and viral infectivity assays, we identify a linear sialylated pentasaccharide with the sequence NeuNAc-α2,6-Gal-β1,4-GlcNAc-β1,3-Gal-β1,4-Glc (LSTc) present on host glycoproteins and glycolipids as a specific JCV recognition motif. The crystal structure of the JCV capsid protein VP1 was solved alone and in complex with LSTc. It reveals extensive interactions with the terminal sialic acid of the LSTc motif and specific recognition of an extended conformation of LSTc. Mutations in the JCV oligosaccharide binding sites abolish cell attachment, viral spread and infectivity, further validating the importance of this interaction. Our findings provide a powerful platform for the development of antiviral compounds.

No abstract

N-Glycosylation starts in the endoplasmic reticulum (ER) where a 14-sugar glycan composed of three glucoses, nine mannoses, and two N-acetylglucosamines (Glc(3)Man(9)GlcNAc(2)) is transferred to nascent proteins. The glucoses are sequentially trimmed by ER-resident glucosidases. The Glc(3)Man(9)GlcNAc(2) moiety is the substrate for oligosaccharyltransferase; the Glc(1)Man(9)GlcNAc(2) and Man(9)GlcNAc(2) intermediates are signals for glycoprotein folding and quality control in the calnexin/calreticulin cycle. Here, we report a novel membrane-anchored ER protein that is highly conserved in animals and that recognizes the Glc(2)-N-glycan. Structure determination by nuclear magnetic resonance showed that its luminal part is a carbohydrate binding domain that recognizes glucose oligomers. Carbohydrate microarray analyses revealed a uniquely selective binding to a Glc(2)-N-glycan probe. The localization, structure, and binding specificity of this protein, which we have named malectin, open the way to studies of its role in the genesis, processing and secretion of N-glycosylated proteins.

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 ␤-glucan-rich extract of Saccharomyces cerevisiae, and binding is inhibited by polysaccharides rich in ␤1,3-or both ␤1,3-and ␤1,6-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 ␤-glucans and compete with zymosan binding and triggering of tumor necrosis factor-␣ secretion by a Dectin-1-expressing macrophage cell line.Dectin-1 is the main receptor on leukocytes that mediates innate immunity to fungal pathogens (1, 2). It is a germ line-encoded membrane-associated cell surface glycoprotein, first identified on a mouse dendritic cell line and cultured Langerhans cells (3) and on a murine macrophage cell line (4). It is now known to be also expressed at the surface of monocytes and neutrophils and at low levels on a subpopulation of T-cells (5). Studies of the tissue distribution of the murine Dectin-1 by immunohistochemistry have shown it to be expressed in the spleen, thymus, liver, lung, and gut and at low levels in the skin, but it is not detectable in the brain, heart, kidney, or eye (6). The human homologue of Dectin-1 has also been characterized (4, 7-9) and resembles the murine protein but differs in that the transcript is alternatively spliced, resulting in two major and several minor isoforms. The major isoforms appear to serve the same immune function as the murine receptor (10).The deduced amino acid sequence of Dectin-1 is that of a type II transmembrane protein, with an extracellular lectin-like domain at the C terminus followed by a stalk region and a short cyto...