Intracellular degradation of sulforhodamine-GM1: use for a fluorescence-based characterization of GM2-gangliosidosis variants in fibroblasts and white blood cells

Abstract 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 sulforhodamine-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.