Misfolded endoplasmic reticulum retained subunits cause degradation of wild-type subunits of arylsulfatase A heteromers.

Arylsulfatase A is an oligomeric lysosomal enzyme. In the present study, we use this enzyme as a model protein to examine how heteromerization of wild-type and misfolded endoplasmic reticulum-degraded arylsulfatase A polypeptides affects the quality control of wild-type arylsulfatase A subunits. Using a conformation sensitive monoclonal antibody, we show that, within heteromers of misfolded and wild-type arylsulfatase A, the wild-type subunits are not fully folded. The results obtained show that arylsulfatase A polypeptide complexes, rather than the monomers, are subject to endoplasmic reticulum quality control and that, within a heteromer, the misfolded subunit exerts a dominant negative effect on the wild-type subunit. Although it has been shown that mature lysosomal arylsulfatase A forms dimers at neutral pH, the results obtained in the present study demonstrate that, in the early biosynthetic pathway, arylsulfatase A forms oligomers with more than two subunits.

Missense mutations as a cause of metachromatic leukodystrophy. Degradation of arylsulfatase A in the endoplasmic reticulum.

Metachromatic leukodystrophy is a lysosomal storage disorder caused by a deficiency of arylsulfatase A (ASA). Biosynthesis studies of ASA with various structure-sensitive monoclonal antibodies reveal that some epitopes of the enzyme form within the first minutes of biosynthesis whereas other epitopes form later, between 10 and 25 min. When we investigated 12 various ASAs, with amino acid substitutions according to the missense mutations found in metachromatic leukodystrophy patients, immunoprecipitation with monoclonal antibodies revealed folding deficits in all 12 mutant ASA enzymes. Eleven of the 12 mutants show partial expression of the early epitopes, but only six of these show, in addition, incomplete expression of late epitopes. In none of the mutant enzymes were the late forming epitopes found in the absence of early epitopes. Thus, data from the wild-type and mutant enzymes indicate that the enzyme folds in a sequential manner and that the folding of early forming epitopes is a prerequisite for maturation of the late epitopes. All mutant enzymes in which the amino acid substitution prevents the expression of the late forming epitopes are retained in the endoplasmic reticulum (ER). In contrast, all mutants in which a single late epitope is at least partially expressed can leave the ER. Thus, irrespective of the missense mutation, the expression of epitopes forming late in biosynthesis correlates with the ability of the enzyme to leave the ER. The degradation of ER-retained enzymes can be reduced by inhibitors of the proteasome and ER alpha1,2-mannosidase I, indicating that all enzymes are degraded via the proteasome. Inhibition of degradation did not lead to an enhanced delivery from the ER for any of the mutant enzymes.

The functional consequences of mis-sense mutations affecting an intra-molecular salt bridge in arylsulphatase A.

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.