The critical step in lysosomal targeting of soluble lysosomal enzymes is the recognition by an UDP-N-acetylglucosamine:lysosomal enzyme-N-acetylglucosamine-1-phosphotransferase. The structure of the determinant common to all lysosomal enzymes for proper recognition by the phosphotransferase is not completely understood. Our current knowledge is largely based on the introduction of targeted amino acid substitutions into lysosomal enzymes and analysis of their effects on phosphotransferase recognition. We have investigated the effect of eight anti-arylsulfatase A monoclonal antibodies on the interaction of arylsulfatase A with the lysosomal enzyme phosphotransferase in vitro. We also show that a lysine-rich surface area of arylsulfatases A and B is essential for proper recognition by the phosphotransferase. Monoclonal antibodies bind to at least six different epitopes at different locations on the surface of arylsulfatase A. All antibodies bind outside the lysine-rich recognition area, but nevertheless Fab fragments of these antibodies prevent interaction of arylsulfatase A with the phosphotransferase. Our data support a model in which binding of arylsulfatase A to the phosphotransferase is not restricted to a limited surface area but involves the simultaneous recognition of large parts of arylsulfatase A.

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Arylsulfatase A (ASA)-deficient mice represent an animal model for the fatal lysosomal storage disease metachromatic leukodystrophy, which is characterized by widespread intralysosomal deposition of sulfatide. Bone marrow stem cell gene therapy in mice, using a retroviral vector mediating expression of wild-type human ASA, has the potential to ameliorate the visceral pathology, but improves the prevailing brain disease and neurologic symptoms only marginally. One factor that influences the efficacy of bone marrow transplantation therapy in lysosomal storage diseases is the secretion level of the therapeutic enzyme from donor-type cells. Here we test the potential of a hypersecreted glycosylation variant of ASA. Although this mutant lacks mannose 6-phosphate residues it is taken up by cells by a mannose 6-phosphate receptor-independent pathway and causes partial metabolic correction of ASA-deficient mouse cells. Retrovirally mediated transfer of the mutant cDNA into ASA-deficient mice results in the sustained expression of the transgene. Serum levels argue for an increased secretion of the glycosylation mutant also in vivo. Tissue levels were reduced to 2% in liver and up to 40% in kidney compared with animals treated with the wild-type enzyme, indicating reduced endocytosis. Thus, the limited uptake of the variant enzyme outweighs the putative advantageous effect of improved supply. Although the mutant enzyme is able to correct the metabolic defect partially, histological examinations did not reveal any reduction of sulfatide storage in treated animals. Surprisingly, analysis of neurologic symptoms indicated a significant improvement of the gait pattern.

Metachromatic leukodystrophy is a lysosomal storage disease caused by the deficiency of arylsulfatase A. Here we describe a hitherto unknown arylsulfatase A allele carrying a E312D missense mutation and characterize the effects of this and three previously described missense mutations, G86D, Y201C, and D255H, on arylsulfatase A. In transfection experiments no enzyme activity can be expressed from arylsulfatase A cDNAs coding for the D255H substituted enzyme, whereas Y201C and E312D mutations were associated with low amounts of residual enzyme activity. All amino acid substitutions lead to a decreased stability of the mutant enzyme, and metabolic labeling experiments indicated that except for the E312D substitution the mutations cause arrest of the mutant arylsulfatase A polypeptides in a prelysosomal compartment.

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.