The dendritic cell specific C-type lectin dendritic cell specific ICAM-3 grabbing non-integrin (DC-SIGN) binds to ''self'' glycan ligands found on human cells and to ''foreign'' glycans of bacterial or parasitic pathogens. Here, we investigated the binding properties of DC-SIGN to a large array of potential ligands in a glycan array format. Our data indicate that DC-SIGN binds with K d < 2 lM to a neoglycoconjugate in which Galb1-4(Fuca1-3)GlcNAc (Le x ) trisaccharides are expressed multivalently. A lower selective binding was observed to oligomannose-type N-glycans, diantennary N-glycans expressing Le x and GalNAcb1-4(Fuca1-3)GlcNAc (LacdiNAc-fucose), whereas no binding was observed to N-glycans expressing corefucose linked either a1-6 or a1-3 to the Asn-linked GlcNAc of N-glycans. These results demonstrate that DC-SIGN is selective in its recognition of specific types of fucosylated glycans and subsets of oligomannose-and complex-type N-glycans.

The new field of functional glycomics encompasses information about both glycan structure and recognition by carbohydrate-binding proteins (CBPs) and is now being explored through glycan array technology. Glycan array construction, however, is limited by the complexity of efficiently generating derivatives of free, reducing glycans with primary amines for conjugation. Here we describe a straightforward method to derivatize glycans with 2,6-diaminopyridine (DAP) to generate fluorescently labeled glycans (glycan-DAP conjugates or GDAPs) that contain a primary amine for further conjugation. We converted a wide variety of glycans, including milk sugars, N-glycans, glycosaminoglycans and chitin-derived glycans, to GDAPs, as verified by HPLC and mass spectrometry. We covalently conjugated GDAPs to N-hydroxysuccinimide (NHS)-activated glass slides, maleimide-activated protein, carboxylated microspheres and NHS-biotin to provide quantifiable fluorescent derivatives. All types of conjugated glycans were well-recognized by appropriate CBPs. Thus, GDAP derivatives provide versatile new tools for biologists to quantify and covalently capture minute quantities of glycans for exploring their structures and functions and generating new glycan arrays from naturally occurring glycans.

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A common terminal structure in glycans from animal glycoproteins and glycolipids is the lactosamine sequence Gal␤4GlcNAc-R (LacNAc or LN). An alternative sequence that occurs in vertebrate as well as in invertebrate glycoconjugates is GalNAc␤4GlcNAc-R (LacdiNAc or LDN). Whereas genes encoding ␤4GalTs responsible for LN synthesis have been reported, the ␤4GalNAcT(s) responsible for LDN synthesis has not been identified. Here we report the identification of a gene from Caenorhabditis elegans encoding a UDP-GalNAc:GlcNAc␤-R ␤1,4-N-acetylgalactosaminyltransferase (Ce␤4GalNAcT) that synthesizes the LDN structure. Ce␤4GalNAcT is a member of the ␤4GalT family, and its cDNA is predicted to encode a 383-amino acid type 2 membrane glycoprotein. A soluble, epitope-tagged recombinant form of Ce␤4GalNAcT expressed in CHOLec8 cells was active using UDP-GalNAc, but not UDPGal, as a donor toward a variety of acceptor substrates containing terminal ␤-linked GlcNAc in both N-and O-glycan type structures. The LDN structure of the product was verified by co-chromatography with authentic standards and 1 H NMR spectroscopy. Moreover, Chinese hamster ovary CHO-Lec8 and CHO-Lec2 cells expressing Ce␤4GalNAcT acquired LDN determinants on endogenous glycoprotein N-glycans, demonstrating that the enzyme is active in mammalian cells as an authentic ␤4GalNAcT. The identification and availability of this novel enzyme should enhance our understanding of the structure and function of LDN-containing glycoconjugates.