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, monocytes and macrophages. In the severe infantile form of the G_M2 gangliosidosis, Tay-Sachs disease, the sulforhodamine-G_M1 was hydrolyzed within the lysosomes to the corresponding sulforhodamine-G_M2 which, because of lack of Hex A activity, was not further degraded. In comparison, in the cells derived from G_M2 gangliosidoses carriers, as well as pseudodeficient and adult forms of G_M2 gangliosidosis, the sulforhodamine-G_M2 was further processed and sequentially degraded by the lysosomal glycosidases to sulforhodamme-ceramide. The latter was converted to sulforhodamine-sphingomyelin, which was secreted into the culture medium. The fluorescence of the sulforhodamine ceramide in cell extracts and/or sulforhodamine-sphingomyelin in the culture medium was quantified and related to parallel data obtained using cells of normal individuals. This permitted distinguishing between the various G_M2 gangliosidoses subtypes and relating the intracellular hydrolysis of sulforhodamine-G_M1 to the genotypes of the respective G_M2 gangliosidoses variants.