Biochemical characterization of two (C300F, P425T) arylsulfatase a missense mutations

Metachromatic leukodystrophy (OMIM 250100) is a lysosomal storage disease caused by the deficiency of arylsulfatase A (ARSA, EC 3.1.6.8). This disease affects mainly the nervous system, because patients cannot degrade 3-O-sulfo-galactosylceramide (sulfatide), a major myelin lipid. Here we describe the characterization of the biochemical effects of two arylsulfatase A missense mutations, P425T and C300F. Transfection experiments demonstrate the expression of residual ARSA enzyme activity for P425T, but not for C300F substituted ARSA. Relative specific activity determination showed that the P425T substituted enzyme has retained about 12% of specific enzyme activity, whereas the C300F substituted enzyme is reduced to less than 1%. Pulse-chase experiments reveal that both mutant proteins are unstable, with a half life of less than 6 hr. Increased secretion upon addition of NH(4)Cl indicates that the mutant proteins can pass the Golgi apparatus and thus are not degraded in the endoplasmic reticulum (ER), but in the lysosomes. This is supported by experiments, which demonstrate the presence of mannose-6-phosphate residues on the oligosaccharide side chains of the mutant proteins. Addition of the cysteine protease inhibitor leupeptin increases the amount of ARSA activity in cells expressing the P425T substituted enzyme, whereas no increase in activity was seen with C300F substituted ARSA.