Ganglioside GM2 N‐acetyl‐ß‐D‐gafactosaminidase and asialo GM2 (GA2) N‐acetyl‐ß‐D‐galactosaminidase; studies in human skin fibroblasts

Abstract

Ganglioside G_M2 and its asialo-derivative, G_A2 were radiolabeled in their N-acetyl-D-galactosaminyl moieties by oxidation with galactose oxidase and reduction with tritiated sodium borohydride. Specific activities of 6 × 10⁴ dpm/nmol (G_M2) and 1.8 × 10⁶ dpm/nmol (G_A2) were achieved. About 98% of the label was in N-acetyl-D-galactosamine. Using these substrates, an assay was developed for G_M2-N-acetyl-β-D-galactosaminidase (E.C.3.2.1.30) and G_A2-N-acetyl-β-D-galactosaminidase (E.C.3.2.1.30) activities in human cultured skin fibroblasts. The products of the G_M2 cleaving reaction were identified as N-acetylgalactosamine and ganglioside G_M3- Both G_M2 and G_A2 cleaving activities were stimulated about 5-fold by purified sodium taurocholate, and this stimulation was inhibited by neutral detergents, lipids and albumin at low concentrations. Addition of various salts, reducing agents and a protein activator factor from human liver of Li et al. (1973) did not stimulate G_M2-N-acetyl-β-D-galactosaminidase activity beyond that found with sodium taurocholate. Under optimal conditions, control fibroblast supernates cleaved ganglioside G_M2 at a rate of 3.7 nmol/mg protein/h compared to 1100 for G_A2-N-acetyl-β-D-galactosaminidase and 4700 for 4-methylumbelliferyl-N-acetyl-β-D-glucosaminidase. Supernates from two patients with Tay-Sachs disease had markedly reduced activity levels for G_M2-N-acetyl-β-D-galactosaminidase but not for the other two substrates. Supernates from two patients with Sandhoff's disease had reduced activities for all three substrates. A supernate from one patient with juvenile G_M2 gangliosidosis cleaved G_M2 at a somewhat faster rate than those from Tay-Sachs or Sandhoff's patients. Two healthy adult women with markedly reduced hexosaminidase A activities using 4MU-N-acetyl-β-D-glucosaminide as substrate had approximately half-normal activities using G_M2 as substrate. A patient with the Tay-Sachs phenotype but with a partial deficiency of hexosaminidase A using the 4-MU substrate had a profound deficiency using G_M2 as substrate. In such unusual hexosaminidase mutants, assays using G_M2 as substrate are better indicators of phenotype than those using synthetic substrates.