Inhibition of P-Selectin and PSGL-1 Using Humanized Monoclonal Antibodies Increases the Sensitivity of Multiple Myeloma Cells to Bortezomib.

Multiple myeloma (MM) is a plasma cell malignancy localized in the bone marrow. Despite the introduction of novel therapies majority of MM patients relapse. We have previously shown that inhibition of P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) play a key role in proliferation of MM and using small-molecule inhibitors of P-selectin/PSGL-1 sensitized MM cells to therapy.

PSGL-1 from the murine leukocytic cell line WEHI-3 is enriched for core 2-based O-glycans with sialyl Lewis x antigen.

Leukocyte trafficking involves specific recognition between P-selectin and L-selectin and PSGL-1 containing core 2-based O-glycans expressing sialyl Lewis x (SLe(x)) antigen. However, the structural identity of the glycan component(s) displayed by murine neutrophil PSGL-1 that contributes to its P-selectin counter-receptor activity has been uncertain, since these cells express little if any SLe(x) antigen, and because there have been no direct studies to examine murine PSGL-1 glycosylation. To address this uncertainty, we studied PSGL-1 glycosylation in the murine cell line WEHI-3 using metabolic-radiolabeling with (3)H-monosaccharide precursors to detect low-abundance O-glycan structures.

Molecular cloning and characterization of the Caenorhabditis elegans alpha1,3-fucosyltransferase family.

The genome of Caenorhabditis elegans encodes five genes with homology to known alpha1,3 fucosyltransferases (alpha1,3FTs), but their expression and functions are poorly understood. Here we report the molecular cloning and characterization of these C. elegans alpha1,3FTs (CEFT-1 through -5).

Specificity of DC-SIGN for mannose- and fucose-containing glycans.

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)<2muM to a neoglycoconjugate in which Galbeta1-4(Fucalpha1-3)GlcNAc (Le(x)) trisaccharides are expressed multivalently.

Versatile fluorescent derivatization of glycans for glycomic analysis.

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.

Novel poly-GalNAcbeta1-4GlcNAc (LacdiNAc) and fucosylated poly-LacdiNAc N-glycans from mammalian cells expressing beta1,4-N-acetylgalactosaminyltransferase and alpha1,3-fucosyltransferase.

Glycans containing the GalNAcbeta1-4GlcNAc (LacdiNAc or LDN) motif are expressed by many invertebrates, but this motif also occurs in vertebrates and is found on several mammalian glycoprotein hormones. This motif contrasts with the more commonly occurring Galbeta1-4GlcNAc (LacNAc or LN) motif. To better understand LDN biosynthesis and regulation, we stably expressed the cDNA encoding the Caenorhabditis elegans beta1,4-N-acetylgalactosaminyltransferase (GalNAcT), which generates LDN in vitro, in Chinese hamster ovary (CHO) Lec8 cells, to establish L8-GalNAcT CHO cells.

Antigenic glycans in parasitic infections: implications for vaccines and diagnostics.

Infections by parasitic protozoans and helminths are a major world-wide health concern, but no vaccines exist to the major human parasitic diseases, such as malaria, African trypanosomiasis, amebiasis, leishmaniasis, schistosomiasis, and lymphatic filariasis. Recent studies on a number of parasites indicate that immune responses to parasites in infected animals and humans are directed to glycan determinants within cell surface and secreted glycoconjugates and that glycoconjugates are important in host-parasite interactions. Because of the tremendous success achieved recently in generating carbohydrate-protein conjugate vaccines toward microbial infections, such as Haemophilus influenzae type b, there is renewed interest in defining parasite-derived glycans in the prospect of developing conjugate vaccines and new diagnostics for parasitic infections.

Molecular cloning and enzymatic characterization of a UDP-GalNAc:GlcNAc(beta)-R beta1,4-N-acetylgalactosaminyltransferase from Caenorhabditis elegans.

A common terminal structure in glycans from animal glycoproteins and glycolipids is the lactosamine sequence Gal(beta)4GlcNAc-R (LacNAc or LN). An alternative sequence that occurs in vertebrate as well as in invertebrate glycoconjugates is GalNAc(beta)4GlcNAc-R (LacdiNAc or LDN). Whereas genes encoding beta4GalTs responsible for LN synthesis have been reported, the beta4GalNAcT(s) responsible for LDN synthesis has not been identified.