GLYCOSPHINGOLIPID EXPRESSION IN HUMAN SKELETAL AND HEART-MUSCLE ASSESSED BY IMMUNOSTAINING THIN-LAYER CHROMATOGRAPHY

In this study the comparative TLC immunostaining investigation of neut ral GSLs and gangliosides from human skeletal and heart muscle is desc ribed. A panel of specific polyclonal and monoclonal antibodies as wel l as the G(M1)-specific choleragenoid were used for the overlay assays , combined with preceding neuraminidase treatment of gangliosides on T LC plates. This approach proved homologies but also quantitative and q ualitative differences in the expression of ganglio-, globo- and neola cto-series neutral GSLs and gangliosides in these two types of striate d muscle tissue within the same species. The main neutral GSL in skele tal muscle was LacCer, followed by GbOse(3)Cer, GbOse(4)Cer, nLcOse(4) Cer and monohexosylceramide, whereas in heart muscle GbOse(3)Cer and G bOse(4)Cer were the predominant neutral GSLs beside small quantities o f LacCer, nLcOse(4)Cer and monohexosylceramide. No ganglio-series neut ral GSLs and no Forssman GSL were found in either muscle tissue. G(M3) (Neu5Ac) was the major ganglioside, comprising almost 70% in skeletal and about 50% in cardiac muscle total gangliosides. G(M2) was found in skeletal muscle only, while G(D3) and G(M1b)-type gangliosides (G(M1b ) and G(D1 alpha)) were undetectable in both tissues. G(M1a)-core gang liosides (G(M1), G(D1a), G(D1b) and G(T1b)) showed somewhat quantitati ve differences in each muscle; lactosamine-containing IV(3)Neu5Ac-nLcO se(4),Cer was detected in both specimens. Neutral GSLs were identified in TLC runs corresponding to e.g. 0.1 g muscle wet weight (GbOse(3)Ce r, GbOse(4)Cer), and gangliosides G(M3) and G(M2) were elucidated in r uns which corresponded to 0.2 g muscle tissue. Only 0.02 g and 0.004 g wet weight aliquots were necessary for unequivocal identification of neolacto-type and G(M1)-core gangliosides, respectively. Muscle is kno wn for the lowest GSL concentration from all vertebrate tissues studie d so far. Using the overlay technique, reliable GSL composition could be revealed, even from small muscle probes on a sub-orcinol and sub-re sorcinol detection level.