ABSTRACT
The amphibian Xenopus tropicalis appears an increasingly appealing model for both genetic and developmental biology studies, compared to the related species Xenopus laevis. Study of the glycosylation pattern of its secreted glycoproteins revealed that this species synthesizes large amounts of Lewis(a) epitope, whereas this motif has previously only been identified in animals within the primate lineage. The use of (1)H-nuclear magnetic resonance spectroscopy enabled us to resolve the sequence of three Lewis(a)-bearing O-linked glycans associated with oviducal secretions, out of which one contained the novel sequence Gal(beta 1-3)GlcNAc(beta 1-6)GalNAc-ol. These structural data suggested the emergence of an alpha 1,4-fucosyltransferase activity in animals outside the primate lineage. On this basis, the screening of a X. tropicalis GenBank database with human Lewis-fucosyltransferase sequences revealed the occurrence of a putative fucosyltransferase gene that presented an unusual acceptor motif.
Subject(s)
Lewis Blood Group Antigens/chemistry , Lewis Blood Group Antigens/isolation & purification , Mucins/chemistry , Oviducts/chemistry , Xenopus/metabolism , Amino Acid Sequence , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Carbohydrate Conformation , Carbohydrate Sequence , Epitopes/chemistry , Epitopes/metabolism , Female , Fucosyltransferases/genetics , Glycoproteins/chemistry , Glycoproteins/immunology , Glycoproteins/isolation & purification , Glycosylation , Lewis Blood Group Antigens/immunology , Magnetic Resonance Spectroscopy , Models, Animal , Molecular Sequence Data , Mucins/immunology , Mucins/metabolism , Oligosaccharides/chemistry , Oligosaccharides/immunology , Oligosaccharides/metabolism , Oviducts/metabolism , Xenopus/immunologyABSTRACT
The neutrophil surface carbohydrate groups LewisX and sialyl LewisX have previously been shown to interact with the vascular selectins E- and P-selectin. However, the proteins expressing these carbohydrate groups have not been fully characterised. We show that these carbohydrate groups, and the structurally related Lewisa group, are all carried predominantly on a sub-population of the carcinoembryonic antigen (CEA) related NCA-160, which is also recognised by CD66 antibodies. We demonstrate that the related NCA-160, NCA-90/95 and CD67 all undergo an increase in surface expression in response to fMLP stimulation, and that this increase is greater than that observed for the sialyl and non-sialyl LewisX carbohydrate groups. These data suggest that the expression of differently glycosylated forms of NCA is independently regulated.
Subject(s)
Antigens, CD/blood , Antigens, Differentiation/blood , Cell Adhesion Molecules/metabolism , Lewis Blood Group Antigens/metabolism , Neutrophils/metabolism , Platelet Membrane Glycoproteins/metabolism , Antigens, CD/isolation & purification , Antigens, Differentiation/isolation & purification , Cell Membrane/metabolism , Cells, Cultured , E-Selectin , Electrophoresis, Polyacrylamide Gel , Humans , Immunoblotting , Lewis Blood Group Antigens/isolation & purification , Membrane Glycoproteins/blood , Membrane Glycoproteins/isolation & purification , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/drug effects , P-SelectinABSTRACT
Our efforts to determine the carbohydrate binding specificity of two myeloid-specific monoclonal antibodies (VIM-1 and VIM-10) resulted in the purification of three fucosylated glycosphingolipids from human chronic myelogenous leukemia cells. After repeated high-performance liquid chromatographic separations, two forms of fucosylated glycosphingolipids were resolved. VIM-1 and VIM-10 were found to bind to the heptaosylceramide Gal beta 1-4 (Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer with the Le(x) (Lewis X) epitope, but not to either the hexaosylceramide Fuc alpha 1-2Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1-Cer or the octaosylceramide Fuc alpha 1-2Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1Cer, each with the Le(y) (Lewis Y) epitope. The latter two glycosphingolipids are the first Le(y) antigens to be purified from human leukocytes and structurally characterized. Binding studies with a range of glycosphingolipids from myeloid cells and other biological sources demonstrated that VIM-1 and VIM-10 bind to Le(x) glycosphingolipids with five or more sugar residues, but not to a glycosphingolipid (III3Fuc-nLc6Cer) with an internal Le(x) trisaccharide.
Subject(s)
Fucose/analysis , Glycosphingolipids/chemistry , Carbohydrate Conformation , Carbohydrate Sequence , Chromatography, High Pressure Liquid , Chromatography, Thin Layer , Glycosphingolipids/isolation & purification , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Lewis Blood Group Antigens/chemistry , Lewis Blood Group Antigens/isolation & purification , Methylation , Molecular Sequence Data , Spectrometry, Mass, Fast Atom Bombardment , Tumor Cells, CulturedABSTRACT
Three acidic oligosaccharide-alditols carrying Lewis X, Lewis Y and A-Lewis Y determinants were isolated from the jelly coat of Pleurodeles waltl eggs. These compounds possess the following structures. Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3[2-oxo-3-deoxy-D- glycero-D-galactononulosonic acid (KDN)alpha 2-6] GalNAc-ol; Fuc alpha 1-2Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3(KDN alpha 2-6) GalNAc-ol and Fuc alpha 1-2(GalNAc alpha 1-3)Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3(KDN alpha 2-6)GalNAc-ol. The complete 1H-n.m.r.-spectrum assignment for the three compounds and the 13C-n.m.r. analysis of the A-Lewis Y determinant-containing heptasaccharide are reported.
