Characterization of the non-sulphated highly anionic Kurloff cell glycoconjugates by lectin- and immunoblotting: evidence for the presence of alpha (2,8)-linked polysialic acid-bearing molecules.
Urea or guanidine hydrochloride-soluble extracts from highly purified Kurloff cells (KC) radiolabelled in vitro were subjected to DEAE-cellulose chromatography. Among the three anionic peaks obtained, a major and non-sulphated peak (designated as peak IV) strongly affected by glucosamine-labelling and eluted at about 0.3 M NaCl was analyzed. Gel filtration on Sepharose CL4B and 10% SDS-PAGE indicated its heterogeneous size. Peak IV consisted mainly of N-glycans as shown by its susceptibility to tunicamycin. Further insight into its chemical nature was obtained by examining its binding capacity to different lectins and by immunodot analysis. It strongly interacted with concanavalin A (Con A) after dot-blot or Western blotting. A large amount of these glycoproteins is not of the high-mannose type since Galanthus nivalis agglutinin reacted weakly with peak IV. Moreover, bindings to Phaseolus vulgaris and to wheat germ agglutinins suggest the presence of bisecting N-acetylglucosamine residues. Bindings to Sambucus nigra and to Ricinus communis agglutinins, dramatically lessened and increased respectively after desialylation, suggest the presence of Neu5Ac alpha 2,6Gal/GalNAc sequences. The absence of outer sialic acid residues linked alpha 2,3 to galactose was demonstrated following Maackia amurensis agglutinin negativity. The use of poly(alpha 2,8-sialyl) endo-N-acylneuraminidase combined with immunodot procedure with a monoclonal antibody that specifically recognizes alpha 2,8-linked polysialic chains revealed that peak IV contains oligosaccharidic epitopes common to polysialylated neural cell adhesion molecules.
Letaïef
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Laboratoire d'Histologie, Université de Caen, CHU Côte de Nacre, France.
Landemore
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G
Oulhaj
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N
Izard
J
J
D000818:Animals; D000838:Anions; D002236:Carbohydrate Conformation; D027801:Galanthus; D006001:Glycoconjugates / Q000032:analysis*; D006168:Guinea Pigs; D015151:Immunoblotting; D066298:In Vitro Techniques; D007694:Killer Cells, Natural / Q000737:chemistry*; D037102:Lectins / Q000378:metabolism*; D037121:Plant Lectins; D011135:Polysaccharides, Bacterial / Q000032:analysis*; D011485:Protein Binding; D012794:Sialic Acids / Q000032:analysis*; D013431:Sulfates / Q000032:analysis
D000838:Anions; D006001:Glycoconjugates; D037102:Lectins; D037121:Plant Lectins; D011135:Polysaccharides, Bacterial; D012794:Sialic Acids; D013431:Sulfates; C021319:polysialic acid