Recognition of arylsulfatase A and B by the UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-phosphotransferase.

The critical step for sorting of lysosomal enzymes is the recognition by a Golgi-located phosphotransferase. The topogenic structure common to all lysosomal enzymes essential for this recognition is still not well defined, except that lysine residues seem to play a critical role. Here we have substituted surface-located lysine residues of lysosomal arylsulfatases A and B. In lysosomal arylsulfatase A only substitution of lysine residue 457 caused a reduction of phosphorylation to 33% and increased secretion of the mutant enzyme. In contrast to critical lysines in various other lysosomal enzymes, lysine 457 is not located in an unstructured loop region but in a helix. It is not strictly conserved among six homologous lysosomal sulfatases. Based on three-dimensional structure comparison, lysines 497 and 507 in arylsulfatase B are in a similar position as lysine 457 of arylsulfatase A. Also, the position of oligosaccharide side chains phosphorylated in arylsulfatase A is similar in arylsulfatase B. Despite the high degree of structural homology between these two sulfatases substitution of lysines 497 and 507 in arylsulfatase B has no effect on the sorting and phosphorylation of this sulfatase. Thus, highly homologous lysosomal arylsulfatases A and B did not develop a single conserved phosphotransferase recognition signal, demonstrating the high variability of this signal even in evolutionary closely related enzymes.

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.

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.