Studies on glycosphingolipids in infection, immunity and differentiation

Cell surface glycoconjugates play a role in many biological processes such as responses to microbial infections, cell-cell interactions, differentiation, and inflammatory responses. The present work is focused on structural characterization of glycosphingolipids with potential roles in adhesion of Helicobacter pylori and Vibrio cholerae, differentiation of human pluripotent stem cells, and as blood group determinants. In the first study, the structural binding requirements of Helicobacter pylori BabA adhesin revealed a different carbohydrate binding potential than previously defined. Adhesion of H. pylori generalist, specialist and BabA deletion mutant strains were examined using mixtures of glycosphingolipids. An unexpected binding by specialist and generalist H. pylori to the hexaosylceramide region of porcine intestinal non-acid glycosphingolipids was found. After isolation and characterization by mass spectrometry and proton NMR, the binding-active glycosphingolipid was determined as Globo H hexaosylceramide (H type 4). Further binding studies demonstrated that the generalist strain, but not the specialist strain, also recognized Globo A heptaosylceramide (A type 4). Non-secretors have an increased risk of peptic ulcer disease although they express little or no H type 1 sequences, and thus no Leb. However, these individuals have a functional FUT1 enzyme that may produce the Globo H sequence, suggesting that Globo H hexaosylceramide might have a role in H. pylori adhesion to the gastric epithelium of non-secretor individuals. In the second study the carbohydrate binding potential of Vibrio cholerae was investigated. Binding-active glycosphingolipids, detected by the thin-layer chromatogram binding assay, were isolated and characterized by antibody binding, mass spectrometry and proton NMR. Thereby, three different binding modes were identified; the first was complex glycosphingo-lipids with GlcNAcβ3Galβ3/4GlcNAc sequence, the second glycosphingolipids with terminal Galα3Galα3Gal sequence, and the third lactosylceramide and related glycosphingolipids. V. cholerae with non-functional chitin binding protein GbpA bound to glycosphingolipids in the same manner as the wild type bacteria, demonstrating that the GbpA is not involved in glycosphingolipid recognition. In the third study the non-acid glycosphingolipids of human embryonic stem cells were structurally characterized. Chromatogram binding assays, mass spectrometry and proton NMR demonstrated the presence of type 2 core chain glycosphingolipids (neolactotetraosyl-ceramide, H type 2 pentaosylceramide, Lex pentaosylceramide, and Ley hexaosylceramide), and blood group A type 1 hexaosylceramide, along with the previously characterized glycosphingolipids with type 1 and type 4 core chains. Thus, the glycosphingolipid diversity of human embryonic stem cells is more complex than previously appreciated. The PX2 antigen is assumed to belong to the GLOB blood group system and has until further notice been assigned to that blood group. However the enzymatic machinery involved in PX2 synthesis has not been determined. In the fourth study, glycosphingolipids isolated from blood group AP1k erythrocytes, App erythrocytes and B3GALNT1 transfected MEG-01 cells were characterized by antibody binding and mass spectrometry. The B3GALNT1 transfected MEG-01 cells had an increased expression of PX2. No P antigen or PX2 were found in the AP1k erythrocytes, while the App erythrocytes expressed PX2, but no P1 and P antigens. The conclusion from these experiments is that the P synthase also is responsible for synthesis of the PX2 antigen