Structural characterization of gangliosides from murine T lymphocytes

Authors:
  • JMJohannes Müthing
    Johannes Müthing
  • HEHeinz Egge
    Heinz Egge
  • BKBernhard Kniep
    Bernhard Kniep
  • PMP F Mühlradt
    P F Mühlradt
  • PFPeter F.
    Peter F.
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Abstract
Mouse spleen cells were prepared from CBA/J mice, and T lymphocytes were selectively stimulated with the T cell mitogen concanavalin A and further propagated in the presence of the T cell growth factor interleukin‐2. The T cells were metabolically labeled with D‐[1‐14C]galactose and D[1‐14C]glucosamine, and the gangliosides were extracted and purified by DEAE‐Sepharose column chromatography. Carbohydrate backbone structures of the asialogangliosides, prepared by mild acid hydrolysis, were determined by (1) high‐performance liquid chromatography, (2) treatment with exoglycosidases and (3) immunostaining. Monosialylated gangliosides were isolated by gradient elution from DEAE‐Sepharose and further separated by preparative high‐performance thin‐layer chromatography in two solvent systems. Isolated fractions were characterized by (1) preparation of asialogangliosides by mild acid hydrolysis, (2) the action of Vibrio cholerae neuraminidase, and (3) fast‐atom‐bombardment mass spectrometry. The following structures were identified: IVNeuAc‐GgOse4Cer; IVNeuG‐GgOse4Cer; IVNeuAc‐GgOse5Cer; and IVNeu‐Gc‐GgOse5Cer. The latter two gangliosides were not detected on B lymphoblasts and may be T‐cell‐specific structures. All gangliosides were heterogeneous in their ceramide moieties, being substituted with C16:0, C24:0, and C24:1 fatty acids, A preliminary study of several other mouse strains showed no strain‐specific genetic variations in the T cell gangliosides. The possible role of these gangliosides is discussed.

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