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

Abstract

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.

Missense mutations as a cause of metachromatic leukodystrophy

“…Our results suggest that enzymes have reached a folding state which suffices to pass the ER quality control, when they express at least epitope C partially (Arg370Gln and Arg370Trp). In a separate study we have identified two additional mutations (Phe219Val, Pro425Thr) that generally also fit into this pattern ([11] A Marcão, unpublished data). It should be mentioned that one of these mutations (Phe219Val) was found in a patient with an unusual phenotype and encodes an enzyme that, like Thr274Met, does not react with any of the mAbs.…”

“…The effects of these mutations have been shown to be rather uniform. Either the amino acid substitutions lead to an arrest of the mutant enzyme in the ER, or the enzyme is degraded in the lysosome after correct sorting [2,9–12]. Here we have investigated wild‐type and mutant ASAs by immunoprecipitation with six structure‐sensitive mAbs.…”

“…We have previously identified various missense mutations in the ASA gene and we have examined the biochemical effects of the corresponding amino acid substitutions on ASA. In a number of mutants, the amino acid substitution causes an arrest of ASA in the ER [2,9,10], whereas others can leave the ER [10–12]. We have expressed these mutant ASAs transiently in BHK cells.…”

“…According to these results two main mechanisms cause ASA deficiency. In about half of the examined cases the mutant enzymes are retained in the ER [2,9,10], in the other half, enzymes can leave the ER and be degraded after arrival in the lysosome [10–12]. Whereas the latter mechanism has been investigated thoroughly in view of potential therapeutic intralysosomal stabilization, nothing is known about the ER‐associated degradation as a cause of MLD.…”

Adult Onset Metachromatic Leukodystrophy Without Electroclinical Peripheral Nervous System Involvement

“…The P425T substitution has recently been shown to be associated with residual enzyme activity. 4 Almost no activity was expressed from the plasmid containing the F219V complementary DNA, whereas low levels of residual activity were found in cells expressing the P425T enzyme (Figure 4). Western blot analysis revealed the presence of ARSA polypeptides in all transfected cells, allowing the conclusion that F219V ARSA has severely reduced specific enzymatic activity (Figure 4, Table).…”