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Structural studies of gangliosides from the YAC-1 mouse lymphoma cell line by immunological detection and fast atom bombardment mass spectrometry
Authors:Johannes Müthing  Jasna Peter-Katalini?  Franz-Georg Hanisch  Ulrich Neumann
Institution:(1) Institut für Zellkulturtechnik, Universität Bielefeld, Germany;(2) Institut für Physiologische Chemie der Universität, Bonn, Germany;(3) Abt. Immunobiologie der Universitätsklinik, Köln, Germany;(4) Klinik für Geflügel der Tierärztlichen Hochschule, Hannover, Germany
Abstract:YAC-1 cells were propagated in bioreactors in 11 and 7.51 volumes. The cells were metabolically labelled withd-1-14C]galactose andd-1-14C]glucosamine. The ganglioside fraction, purified by DEAE-Sepharose and silica gel column chromatography, showed on thin layer chromatography four major bands with mobilities between GM1 and GD1a. Gangliosides, obtained by further purification steps including high performance liquid chromatography on silica gel 60 columns with a gradient system of isopropanol:hexane:water, and preparative high performance TLC were characterized by (1) immunostaining of corresponding asialogangliosides obtained by mild acid hydrolysis and neuraminidase treatment and (2) fast atom bombardment mass spectrometry of native and permethylated samples and methylation analysis of GM1b ganglioside. As well as small amounts of GM2 and GM1, the major gangliosides found in the complex mixture were GM1b and GalNAc-GM1b. The structural heterogeneity of these gangliosides was cased by (a) substitution of the ceramide moiety by fatty acids of different chain length and degree of unsaturation (C16:0, C24:0, C24:1) and (b) N-substitution of the sialic acid moieties with either acetyl or glycolyl groups. Disialogangliosides were detected only in low amounts and will be the subject of further investigation. A polyclonal chicken antiserum was raised against IVNeuAc-GgOse5Cer. The antiserum was highly specific for gangliosides (IVNeuAc and IVNeuGc) and asialogangliosides with a GgOse5Cer backbone. No cross-reaction with GM1b or GgOse4Cer was observed. Abbreviations: FAB-MS, fast atom bombardment mass spectrometry; GSL(s), glycosphingolipid(s); HPLC, high performance liquid chromatography, HPTLC, high performance thin layer chromatography; NK, natural killer; SIM, selective ion monitoring; TIC, total ion current. NeuAc,N-acetylneuraminic acid; NeuGc,N-glycolylneuraminic acid. The designation of the following glycosphingolipids follows the IUB-IUPAC recommendations. GgOse3Cer or gangliotriaosylceramide or asialo GM2, GalNAcbeta1-4Galbeta1-4GlcCer; GgOse4Cer or gangliotetraosylceramide or asialo GM1, Galbeta1-3GalNAcbeta1-4Galbeta1-4GlcCer; GgOse5Cer organgliopentaosylceramide, GalNAcbeta1-4Galbeta1-3GalNAcbeta1-4Galbeta1-4GlcCer; II3NeuAc-GgOse4Cer or GM1; IV3NeuAcGgOse4Cer or GM1b; IV3NeuAc-GgOse5Cer or GalNAc-GM1b; IV3NeuAc, II3NeuAc-GgOse4Cer or GD1a; II3(NeuAc)2-GgOse4Cer or GD1b; IV3(NeuAc)2-GgOse4Cer or GD1c; IV3NeuAc,III6NeuAc-GgOse4Cer or GD1a; IV3NeuAc,II3(NeuAc)2-GgOse4Cer or GT1b;Vibrio cholerae and Arthrobacter ureafaciens neuraminidase (EC 3.2.1.18).
Keywords:gangliosides  YAC-1 T lymphoma  antibodies  FAB MS
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