Structural characterization of gangliosides from resting and endotoxin-stimulated murine B lymphocytes

• Published in: Biochemistry (Easton) 1993-11, Vol.32 (47), p.12685-12693
• Main Authors: Poertner, Antje, Peter-Katalinic, Jasna, Brade, Helmut, Unland, Frank, Buentemeyer, Heino, Muething, Johannes
• Format: Article
• Language: English
• Subjects: Animals; B-Lymphocytes - drug effects; B-Lymphocytes - metabolism; Biological and medical sciences; Carbohydrate Sequence; Cells, Cultured; Citrobacter freundii; Endotoxins - pharmacology; Escherichia coli; Female; Fundamental and applied biological sciences. Psychology; Fundamental immunology; G(M1) Ganglioside - analogs & derivatives; Gangliosides - chemistry; Gangliosides - metabolism; Immunobiology; Lymphocyte Activation; Lymphoid cells: ontogeny, maturation, markers, receptors, circulation and recirculation; Mice; Mice, Inbred CBA; Molecular Sequence Data; Salmonella minnesota; Sialic Acids - isolation & purification; Spectrometry, Mass, Fast Atom Bombardment; Spleen - cytology
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00210a018

B lymphocytes from CBA/J mice were stimulated in splenocyte cultures for 72 h with various endotoxins. Bisphosphoryl lipid A from Escherichia coli had the highest stimulatory effect followed by LPS of Citrobacter freundii and Salmonella minnesota as measured by [3H]thymidine uptake. Gangliosides of stimulated B cells (metabolically labeled with D-[1-14C]galactose and D-[1-14C]glucosamine) and unlabeled gangliosides from resting B cells (prepared from spleens without stimulus) were analyzed by high-performance TLC, DEAE anion-exchange HPLC, and immunostaining procedures. Contents of ganglioside-derived sialic acids, quantified by HPLC as their fluorescent derivatives, decreased from stimulated to resident B lymphocytes in the following order: LPS S. minnesota > LPS C. freundii > bisphosphoryl lipid A E. coli > resting B cells. Gangliosides of resting B cells contained more N-glycolyl- than N-acetylneuraminic acid, whereas inverse ratios were found in activated cells, indicating a shift from N-glycolyl- to N-acetylneuraminic acid due to stimulation. Furthermore, a higher disialoganglioside content was characteristic for activated B cells. Fast atom bombardment mass spectrometry was performed with permethylated mono- and disialoganglioside fractions of LPS S. minnesota and LPS C. freundii stimulated B cells. Major gangliosides were GM1a and GD1a beside minute amounts of GD1b. The structural heterogeneity in the gangliosides was caused by (a) N-substitution of the sialic acids with either acetyl or glycolyl groups, (b) variation in the long-chain base (sphingosine, sphinganine), and (c) substitution of the ceramide moiety by fatty acids of different chain length and degree of unsaturation (C16:0, C24:0,24:1).2+ p6