ABSTRACT
We report the preparation of radioactive GM3 ganglioside and its use in the study of sialic acid storage disorders. For the first time GM3 was isotopically radiolabeled in three positions of the molecule: at the sialic acid acetyl group, [3H-Neu5Ac]GM3, at the C1 of the fatty acid moiety, [14C-Stearoyl]GM3, and at C3 of sphingosine, [3H-Sph]GM3. The radioactive GM3 administered to cultured human fibroblasts from a patient suffering from Salla disease was taken up by the cells and metabolized. An analysis of the distribution of radioactivity within the ganglioside metabolic derivatives showed an accumulation of free sialic acid and ceramide in the pathological cells.
Subject(s)
Animals , Carbohydrate Sequence , Carbon Radioisotopes , Cattle , Cells, Cultured , G(M3) Ganglioside/chemistry , Humans , Lysosomal Storage Diseases/metabolism , Molecular Sequence Data , Molecular Structure , Sialic Acids/metabolism , TritiumABSTRACT
The aggregative and geometrical properties of gangliosides have been compared and discussed. It is shown that, due to the steric packing features of gangliosides, significative considerations can be made on the primary and secondary structures of their head group, starting from their cooperative behaviour, supporting NMR direct observations.
Subject(s)
Carbohydrate Conformation , Carbohydrate Sequence , Gangliosides/chemistry , Lasers , Light , Molecular Sequence Data , Oligosaccharides/chemistry , Scattering, Radiation , Structure-Activity RelationshipABSTRACT
Laser and neutron scattering experiments showed that in mixed micelles of ganglioside GM2 and GT1b, a membrane mimicking system, the segregation of gangliosides may occur spontaneously. Photolabeling experiments using nitrophenylazide containing ganglioside GM1 proved that gangliosides added to cells in culture enter the cell and bind to its membrane as components of microdomains, which specifically interact with a protein of about 30 kDa. This suggests that ganglioside segregation may be a natural phenomenon. Gangliosides when added to granule cells in culture led to increase in protein phosphorylation, the effect exerted being related to the amount of ganglioside molecules inserted stably into the cell lipid layer and an increase of 0.7% of the cell original ganglioside content promoted an increase of 57% in the incorporation of 32P into cell membrane proteins. From the above results a possible relationship between ganglioside segregation and involvement of ganglioside in enzyme activity control is suggested.