The functional consequences of mis-sense mutations affecting anintra-molecular salt bridge in arylsulphatase A

Abstract

Metachromatic leukodystrophy is a lysosomal storage disorder caused by the deficiency of arylsulphatase A. We describe the functional consequences of three mis-sense mutations in the arylsulphatase A gene (Asp-335-Val, Arg-370-Trp and Arg-370-Gln), affecting an apparent intramolecular Asp-335 to Arg-370 salt bridge, and interpret the effects and clinical consequences on the basis of the three-dimensional structure of arylsulphatase A. Asp-335-Val and Arg-370-Trp substitutions each cause a complete loss of enzyme activity and are associated with the most severe form of the human disease, whereas the Arg-370-Gln-substituted enzyme retains some residual activity, being found in a patient suffering from the milder juvenile form of the disease. Detailed analysis reveals that formation of the apparent salt bridge depends critically on the presence of aspartic acid and arginine residues at positions 335 and 370, respectively. Substitution by various other amino acids, including glutamic acid and lysine, affects enzyme function severely. Biosynthesis and immunoprecipitation studies indicate that the Asp-335-Val substitution affects folding of arylsulphatase A more severely than either the Arg-370-Trp or Arg-370-Gln substitutions. In vitro mutagenesis data show that clinical severity correlates with the space occupied by residue 370. The combination with structural data suggests that the bulky tryptophan residue broadens the cleft held together by the apparent salt bridge, whereas the smaller glutamine residue still allows the cleft to close, yielding a less severely affected enzyme. The position of residue 370 in the three-dimensional structure of the enzyme provides a plausible explanation for the differing severities in loss of enzyme function caused by the mutations and thus the clinical phenotype.

What can cell biology tell us about heterogeneity in lysosomal storage diseases?

“…In arylsulphatase A, the negatively charged amino acid residue Asp335 and the positively charged Arg370 form an intramolecular salt bridge. Several patients with missense mutations causing different substitutions of these residues were identified 14, 17. Substitutions Asp335Val and Arg370Trp cause severe metachromatic leukodystrophy, whereas Arg370Gln was found in a more mildly affected juvenile patient.…”

“…In accordance with the phenotype of the patients, expression of these defective enzymes in heterologous cells revealed a complete loss of enzyme activity for the Asp335Val and Arg370Trp enzymes, whereas the Arg370Gln enzyme is associated with some residual enzyme activity. In another experiment, cells were labelled with [ 32 P]orthophosphate 14, 17. As lysosomal enzymes receive mannose‐6‐phosphate residues in the Golgi apparatus, arylsulphatase A should become labelled with [ 32 P]orthophosphate.…”

Missense mutations as a cause of metachromatic leukodystrophy

“…1. + ⁄ -indicates whether or not the mutant enzymes according to previous publications (references in brackets; [2,9,10,[25][26][27][28]) are retained in the ER. Polypeptides of lower apparent molecular mass, which can be seen in some of the experiments are unrelated to ASA.…”

What can cell biology tell us about heterogeneity in lysosomal storage diseases?

“…In arylsulphatase A, the negatively charged amino acid residue Asp335 and the positively charged Arg370 form an intramolecular salt bridge. Several patients with missense mutations causing different substitutions of these residues were identified [14,17]. Substitutions Asp335Val and Arg370Trp cause severe metachromatic leukodystrophy, whereas Arg370Gln was found in a more mildly affected juvenile patient.…”