Expression of neutral glycosphingolipids and gangliosides in human skeletal and heart muscle determined by indirect immunofluorescence staining

The expression of neutral glycosphingolipids and gangliosides has been studied in human skeletal and heart muscle using indirect immunofluorescence microscopy. Transversal and longitudinal cryosections were immunostained with specific monoclonal and polyclonal antibodies against the neutral glycosphingolipids lactosylceramide, globoside, Forssman glycosphingolipid, gangliotetraosylceramide, lacto-N-neotetraosylceramide and against the gangliosides G_M3(Neu5Ac) and G_M1(Neu5Ac). To confirm the lipid nature of positive staining, control sections were treated with methanol and chloroform:methanol (1:1) before immunostaining. These controls were found to be either negative or strongly reduced in fluorescence intensity, suggesting that lipid bound oligosaccharides were detected. In human skeletal muscle, lactosylceramide was found to be the main neutral glycosphinogolipid. Globoside was moderately expressed, lacto-N-neotetraosylceramide and gangliotetraosylceramide were minimally expressed and Forssman glycosphingolipid was not detected in human skeletal muscle. The intensities of the immunohistological stains of G_M3 and G_M1 correlated to the fact that G_M3 is the major ganglioside in skeletal muscle whereas G_M1 is expressed only weakly. In human heart muscle globoside was the major neutral glycosphingolipid. Lactosylceramide and lacto-N-neotetraosylceramide were moderately expressed, gangliotetraosylceramide was weakly expressed and the Forssman glycosphingolipid was not expressed at all in cardiac muscle. G_M3 and G_M1 were detected with almost identical intensity. All glycosphingolipids were present in plasma membranes as well as at the intracellular level. Abbreviations used: BSA, bovine serum albumin; DAPI, 4,6-diamidine-2-phenylindole-dihydrochloride; DTAF, fluorescein isothiocyanate derivative; GSL(s), glycosphingolipid(s); Neu5Ac,N-acetylneuraminic acid 50]; PBS, phosphate buffered saline. The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations 51] and the nomenclature of Svennerholm 52]. Lactosylceramide or LacCer, Gal1-4Glc1-1Cer; gangliotriaosylceramide or GgOse₃Cer, GalNAc1-4Gal1-4Glc1-1Cer; globotriaosylceramide or GbOse₃Cer, Gall-4Gall-4Glcl-1Cer; gangliotetraosylceramide or GgOse₄Cer, Gal1-3GalNAc1-4Gal1-4Glc1-1Cer; globotetraosylceramide or GbOse₄Cer, GalNAc1-3Gal1-4Gal1-4Glc1-1Cer; lacto-N-neotetraosylceramide or nLcOse₄Cer, Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer; Forssman GSL or GbOse₃Cer, GalNAc1-3GalNAc1-3Gal1-4Gal1-4Gle1-1Cer; G_M3, II³Neu5Ac-LacCer; G_M2, II³Neu5Ac-GgOse₃Cer; G_M1, II³Neu5Ac-GgOse₄Cer; G_D3 II³(Neu5Ac)₂-LacCer; G_D2, II³(Neu5Ac)₂-GgOse₃Cer; G_D1a, IV³Neu5Ac, II³Neu5Ac-GgOse₄Cer; G_D1b, II³(Neu5Ac)₂-GgOse₄Cer.