Normal-phase nanoscale liquid chromatography-mass spectrometry of underivatized oligosaccharides at low-femtomole sensitivity.

We here describe the online liquid chromatography (LC) electrospray ionization mass spectrometry (MS) of underivatized glycans using a nanoscale normal-phase amide column at a flow rate of 300 nL/min. Retention on the amide column is based on polar interactions of the oligosaccharide hydroxyl groups with the stationary phase, and thus, the retention time predictably increases with elongation of the oligosaccharide chain. The system is characterized by its high chromatographic resolution, which routinely allows the separation of isobaric structures.

Furin is a chemokine-modifying enzyme: in vitro and in vivo processing of CXCL10 generates a C-terminally truncated chemokine retaining full activity.

Chemokines comprise a class of structurally related proteins that are involved in many aspects of leukocyte migration under basal and inflammatory conditions. In addition to the large number of genes, limited processing of these proteins by a variety of enzymes enhances the complexity of the total spectrum of chemokine variants. We have recently shown that the native chemokine CXCL10 is processed at the C terminus, thereby shedding the last four amino acids.

A new approach for fluorescence correlation spectroscopy (FCS) based immunoassays.

Fluorescence correlation spectroscopy (FCS) is a powerful technique for measuring physicochemical properties, such as concentration and diffusion constant, of bio-molecules in complex mixtures. Although, as such, FCS is well suited for development of homogeneous immunoassays, a major obstacle lies in the relatively high molecular weight of antibodies. This is because in FCS discrimination between unbound fluorescently-labelled antibodies and the same antibodies bound to immune complexes is based on the difference of their respective diffusion coefficients.

A novel Gal(beta1-4)Gal(beta1-4)Fuc(alpha1-6)-core modification attached to the proximal N-acetylglucosamine of keyhole limpet haemocyanin (KLH) N-glycans.

KLH (keyhole limpet haemocyanin), the oxygen-carrying molecule of the marine snail Megathura crenulata, is often used as an adjuvant or as a hapten carrier for immunizations with peptides, oligosaccharides or other low-molecular-mass organic compounds. KLH exhibits several carbohydrate determinants, at least some of which are immunogenic: it shares an antigenic Fuc(alpha1-3)GalNAc-determinant with schistosomes and contains unique Gal-(beta1-6)Man-structural motifs on its N-glycans. This study reveals the presence of N-glycans with unusual +/-Gal(beta1-4)Gal(beta1-4)Fuc- units (alpha1-6)-linked to the reducing end N -acetylglucosamine residue.

Specific antibody responses to three schistosome-related carbohydrate structures in recently exposed immigrants and established residents in an area of Schistosoma mansoni endemicity.

By the use of surface plasmon resonance spectroscopy, immunoglobulin G (IgG) subclass and IgM antibodies against three schistosome-derived carbohydrate structures, FLDN (Fucalpha1-3GalNAcbeta1-4GlcNAcbeta1-3Galalpha1), LDN-DF [GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAcbeta1], and LDNF [GalNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galalpha1], were measured in 184 previously unexposed Kenyan immigrants who moved into the Masongaleni area, where Schistosoma mansoni is endemic. They were sampled within their first year of exposure and again 2 years later. A cohort selected out of the original residents of the area, who had been exposed for many years, served as controls.

Crystallization and preliminary X-ray analysis of an anti-LewisX Fab fragment with and without its LewisX antigen.

LewisX-containing glycoconjugates are abundantly expressed by schistosomes and are assumed to be of prime importance for the survival of the parasite within the human host. Monoclonal antibody 291-2G3-A, which was generated from mice infected with schistosomes, was found to interact with monomers, dimers and trimers of the LewisX trisaccharide. The Fab fragment of monoclonal antibody 291-2G3-A has been crystallized and soaked with its LewisX antigen.

Juvenile rhesus monkeys have lower type 2 cytokine responses than adults after primary infection with Schistosoma mansoni.

Adults and children have differences in their susceptibility to schistosomiasis. The relative influences of age-dependent innate resistance and acquired immunity in the differences between susceptibility to schistosomiasis are difficult to assess in humans. Therefore, we exposed juvenile and adult female rhesus monkeys to primary infection with Schistosoma mansoni.

Schistosoma mansoni-infected mice produce antibodies that cross-react with plant, insect, and mammalian glycoproteins and recognize the truncated biantennaryN-glycan Man3GlcNAc2-R.

To reveal the role of cross-reactive carbohydrate determinants in the host immune response in helminth infections and allergenicity, we developed monoclonal antibodies (mAbs) that recognize glycan epitopes present on glycoconjugates from both helminths and plants. An IgM mAb (100-4G11-A) was selected from a panel of anti-glycan mAbs generated from Schistosoma-infected or immunized mice because it recognized both a plant glycoprotein horseradish peroxidase and phospholipase A2 from honeybee venom. On further characterization, it was shown that mAb 100-4G11-A recognizes the truncated biantennary N-glycan Man3GlcNAc2-R.

Self-association of the spindle pole body-related intermediate filament protein Fin1p and its phosphorylation-dependent interaction with 14-3-3 proteins in yeast.

The Fin1 protein of the yeast Saccharomyces cerevisiae forms filaments between the spindle pole bodies of dividing cells. In the two-hybrid system it binds to 14-3-3 proteins, which are highly conserved proteins involved in many cellular processes and which are capable of binding to more than 120 different proteins. Here, we describe the interaction of the Fin1 protein with the 14-3-3 proteins Bmh1p and Bmh2p in more detail.

Novel lipochitin oligosaccharide structures produced by Rhizobium etli KIM5s.

The novel lipochitin oligosaccharide (LCOs) structures produced by Rhizobium etli KIM5s were characterized using a nanoHPLC reverse-phase system coupled to an ion-trap mass spectrometer. This technique was shown to be more sensitive for structural elucidation of LCOs than previously used mass spectrometric methods. The structures of the LCOs of R.

Triggering of innate immune responses by schistosome egg glycolipids and their carbohydrate epitope GalNAc beta 1-4(Fuc alpha 1-2Fuc alpha 1-3)GlcNAc.

To investigate the interactions of glycoconjugates with the innate immune system, peripheral blood mononuclear cells were stimulated with glycolipids derived from Schistosoma mansoni eggs and worms and with biochemically synthesized neoglycoconjugates. Egg glycolipids stimulated the production of interleukin (IL)--10, IL-6, and tumor necrosis factor--alpha in monocytes, whereas worm glycolipids failed to do so. When monoclonal antibodies that specifically recognize defined carbohydrate epitopes were used, the binding of a GalNAc beta 1-4(Fuc alpha 1-2Fuc alpha 1-3)GlcNAc (LDN-DF) reactive antibody was pronounced on egg glycolipids but was absent on worm glycolipids.