INTRACELLULAR DEGRADATION OF SULFORHODAMINE-G(M1) - USE FOR A FLUORESCENCE-BASED CHARACTERIZATION OF G(M2)-GANGLIOSIDOSIS VARIANTS IN FIBROBLASTS AND WHITE BLOOD-CELLS

A novel fluorescent ganglioside, sulforhodamine-G(M1) was administered into cells derived from carriers and patients with different subtypes of G(M2) gangliosidosis, resulting from various mutations in the gene encoding the lysosomal enzyme hexosaminidase (Hex) A, The cells used were skin fibroblasts and white blood cells, i.e. lymphocytes, monocyt es and macrophages, In the severe infantile form of the G(M2) ganglios idosis, Tay-Sachs disease, the sulforhodamine-G(M1) was hydrolyzed wit hin the lysosomes to the corresponding sulforhodamine-G(M2) which, bec ause of lack of Hex A activity, was not further degraded. In compariso n, in the cells derived from G(M2) gangliosidoses carriers, as well as pseudodeficient and adult forms of G(M2) gangliosidosis, the sulforho damine-G(M2) was further processed and sequentially degraded by the ly sosomal glycosidases to sulforhodamine-ceramide. The latter was conver ted to sulforhodamine-sphingomyelin, which was secreted into the cultu re medium. The fluorescence of the sulforhodamine ceramide in cell ext racts and/or sulforhodamine-sphingomyelin in the culture medium was qu antified and related to parallel data obtained using cells of normal i ndividuals. This permitted distinguishing between the various G(M2) ga ngliosidoses subtypes and relating the intracellular hydrolysis of sul forhodamine-G(M1) to the genotypes of the respective G(M2) gangliosido ses variants.