Sequence determinants directing conversion of cysteine to formylglycine in eukaryotic sulfatases.
Sulfatases carry at their catalytic site a unique post-translational modification, an alpha-formylglycine residue that is essential for enzyme activity. Formylglycine is generated by oxidation of a conserved cysteine or, in some prokaryotic sulfatases, serine residue. In eukaryotes, this oxidation occurs in the endoplasmic reticulum during or shortly after import of the nascent sulfatase polypeptide. The modification of arylsulfatase A was studied in vitro and was found to be directed by a short linear sequence, CTPSR, starting with the cysteine to be modified. Mutational analyses showed that the cysteine, proline and arginine are the key residues within this motif, whereas formylglycine formation tolerated the individual, but not the simultaneous substitution of the threonine or serine. The CTPSR motif was transferred to a heterologous protein leading to low-efficient formylglycine formation. The efficiency reached control values when seven additional residues (AALLTGR) directly following the CTPSR motif in arylsulfatase A were present. Mutating up to four residues simultaneously within this heptamer sequence inhibited the modification only moderately. AALLTGR may, therefore, have an auxiliary function in presenting the core motif to the modifying enzyme. Within the two motifs, the key residues are fully, and other residues are highly conserved among all known members of the sulfatase family. (+info)
Amino acid residues forming the active site of arylsulfatase A. Role in catalytic activity and substrate binding.
Arylsulfatase A belongs to the sulfatase family whose members carry a Calpha-formylglycine that is post-translationally generated by oxidation of a conserved cysteine or serine residue. The formylglycine acts as an aldehyde hydrate with two geminal hydroxyls being involved in catalysis of sulfate ester cleavage. In arylsulfatase A and N-acetylgalactosamine 4-sulfatase this formylglycine was found to form the active site together with a divalent cation and a number of polar residues, tightly interconnected by a net of hydrogen bonds. Most of these putative active site residues are highly conserved among the eukaryotic and prokaryotic members of the sulfatase family. To analyze their function in binding and cleaving sulfate esters, we substituted a total of nine putative active site residues of human ASA by alanine (Asp29, Asp30, Asp281, Asn282, His125, His229, Lys123, Lys302, and Ser150). In addition the Mg2+-complexing residues (Asp29, Asp30, Asp281, and Asn282) were substituted conservatively by either asparagine or aspartate. In all mutants Vmax was decreased to 1-26% of wild type activity. The Km was more than 10-fold increased in K123A and K302A and up to 5-fold in the other mutants. In all mutants the pH optimum was increased from 4.5 by 0.2-0.8 units. These results indicate that each of the nine residues examined is critical for catalytic activity, Lys123 and Lys302 by binding the substrate and the others by direct (His125 and Asp281) or indirect participation in catalysis. The shift in the pH optimum is explained by two deprotonation steps that have been proposed for sulfate ester cleavage. (+info)
Phosphorylation of arylsulphatase A occurs through multiple interactions with the UDP-N-acetylglucosamine-1-phosphotransferase proximal and distal to its retrieval site by the KDEL receptor.
Phosphorylation of oligosaccharides of the lysosomal enzyme arylsulphatase A (ASA), which accumulate in the secretions of cells that mis-sort most of the newly synthesized lysosomal enzymes due to a deficiency of mannose 6-phosphate receptors, was found to be site specific. ASA residing within the secretory route of these cells contains about one third of the incorporated [2-3H]mannose in phosphorylated oligosaccharides. Oligosaccharides carrying two phosphate groups are almost 2-fold less frequent than those with one phosphate group and only a few of the phosphate groups are uncovered. Addition of a KDEL (Lys-Asp-Glu-Leu) retention signal prolongs the residence time of ASA within the secretory route 6-fold, but does not result in more efficient phosphorylation. In contrast, more than 90% of the [2-3H]mannose incorporated into secreted ASA (with or without a KDEL retention signal) is present in phosphorylated oligosaccharides. Those with two phosphate groups are almost twice as frequent as those with one phosphate group and most of the phosphate groups are uncovered. Thus, ASA receives N-acetylglucosamine 1-phosphate groups in a sequential manner at two or more sites located within the secretory route proximal and distal to the site where ASA is retrieved by the KDEL receptor, i.e. proximal to the trans-Golgi. At each of these sites up to two N-acetylglucosamine 1-phosphate groups can be added to a single oligosaccharide. Of several drugs known to inhibit transit of ASA through the secretory route only the ionophore monensin had a major inhibitory effect on phosphorylation, uncovering and sialylation. (+info)
Arylsulfatase A pseudodeficiency in healthy Brazilian individuals.
Molecular alterations associated with arylsulfatase A pseudodeficiency (ASA-PD) were characterized by PCR and restriction endonuclease analysis in a sample of healthy individuals from Brazil. ASA activity was also assayed in all subjects. Two individuals homozygous for the N350S and 1524+95A<--G mutations were detected, corresponding to a frequency of 1.17% (4 of 324 alleles). The individual frequency of the N350S mutation was 20.7% (71 of 342 alleles) and 7.9% (27 of 342 alleles) for the 1524+95A<--G mutation. The frequency of the ASA-PD allele in our population was estimated to be 7.9%. This is the first report of ASA-PD allele frequency in a South American population. In addition, the methods used are effective and suitable for application in countries with limited resources. All patients with low ASA activity should be screened for ASA-PD as part of the diagnostic protocol for metachromatic leukodystrophy. (+info)
Measurements from normal umbilical cord blood of four lysosomal enzymatic activities: alpha-L-iduronidase (Hurler), galactocerebrosidase (globoid cell leukodystrophy), arylsulfatase A (metachromatic leukodystrophy), arylsulfatase B (Maroteaux-Lamy).
Umbilical cord blood (UCB) has received increasing attention as a source of unrelated hematopoietic stem cells for transplantation. Lysosomal diseases have been effectively treated and normal enzymatic activity has occurred subsequent to engraftment using UCB. The use of donor cells with normal amounts of enzyme, rather than those from carriers whose level may be 50% or less, is an obvious goal. The frequency of such heterozygotes varies from 1:10 to 1:140 or lower depending upon the disease at issue. We assayed the levels of lysosomal enzymes in normal UCB in random samples as well as those used for transplantation. We measured the following enzymatic activities: alpha-l-iduronidase (Hurler), galactocerebrosidase (globoid cell leuko- dystrophy) and arylsulfatase A (metachromatic leukodystrophy). For the latter, levels of activity in UCB are comparable to those found in adult blood. In the case of arylsulfatase B (Maroteaux-Lamy) a level lower than adult level was found. An informed choice by the transplanting physician based on the activity of the relevant enzyme in the UCB donor will provide a better opportunity for an improved prognosis for more complete correction of the recipient's primary disease. Bone Marrow Transplantation (2000) 25, 541-544. (+info)
High-mannose-type oligosaccharides from human placental arylsulfatase A are core fucosylated as confirmed by MALDI MS.
Despite numerous studies on arylsulfatase A, the structure of its glycans is not well understood. It has been shown that the concentration of arylsulfatase A increases in the body fluids of patients with some forms of cancer, and the carbohydrate component of arylsulfatase A synthesized in tumor tissues and transformed cells undergoes increased sialylation, phosphorylation and sulfation. To understand the significance of any changes in the glycosylation of arylsulfatase A in cancer, it is important to know the structure of its carbohydrate component in normal tissue. In the present study we have analyzed carbohydrate moieties of human placental arylsylfatase A using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting on Immobilon P and on-blot deglycosylation using PNGase F for glycan release. Profiles of N-glycans were obtained by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Oligosaccharides were sequenced using specific exoglycosidases, and digestion products were analyzed by MALDI MS and the computer matching of the resulting masses with those derived from a sequence database. Fifty picomoles (6 microg) of arylsulfatase A applied to the gel were sufficient to characterize its oligosaccharide content. The results indicated that human placental arylsulfatase A possesses only high-mannose-type oligosaccharides, of which almost half are core fucosylated. In addition, there was a minor species of high-mannose-type glycan bearing six mannose residues with a core fucose. This structure was not expected since high-mannose-type oligosaccharides basically have not been recognized as a substrate for the alpha1,6-fucosyltransferase. (+info)
Retrovirally expressed human arylsulfatase A corrects the metabolic defect of arylsulfatase A-deficient mouse cells.
A deficiency of arylsulfatase A (ASA) causes the lysosomal storage disease metachromatic leukodystrophy (MLD) which is characterized primarily by demyelination of the central nervous system. ASA-deficient mice develop a disease which resembles MLD in many respects and thus serve as an appropriate animal model for this disease. To establish gene therapy protocols for ASA-deficient mice, we constructed two retroviral vectors based on the murine stem cell virus. Both vectors harbor the human ASA cDNA controlled by the retroviral promoter/enhancer element, but differ by the presence or absence of a neomycin resistance gene driven by an internal promoter. A comparative analysis of the one- versus the two-gene vector and an amphotropic versus an ecotropic producer cell line revealed that the amphotropic producer cell line for the one-gene vector transfers ASA overexpression to the target cells most efficiently. The human ASA encoded by this vector is correctly expressed in heterologous mouse cells and corrects the metabolic defect of transduced ASA-deficient murine cells. The constructed one-gene vector might thus be a potentially useful tool for the development of a gene-based therapy for ASA-deficient mice. Gene Therapy (2000) 7, 805-812. (+info)
Effect of collection, transport, processing and storage of blood specimens on the activity of lysosomal enzymes in plasma and leukocytes.
This study was designed to evaluate the effect of different conditions of collection, transport and storage on the quality of blood samples from normal individuals in terms of the activity of the enzymes ss-glucuronidase, total hexosaminidase, hexosaminidase A, arylsulfatase A and ss-galactosidase. The enzyme activities were not affected by the different materials used for collection (plastic syringes or vacuum glass tubes). In the evaluation of different heparin concentrations (10% heparin, 5% heparin, and heparinized syringe) in the syringes, it was observed that higher doses resulted in an increase of at least 1-fold in the activities of ss-galactosidase, total hexosaminidase and hexosaminidase A in leukocytes, and ss-glucuronidase in plasma. When the effects of time and means of transportation were studied, samples that had been kept at room temperature showed higher deterioration with time (72 and 96 h) before processing, and in this case it was impossible to isolate leukocytes from most samples. Comparison of heparin and acid citrate-dextrose (ACD) as anticoagulants revealed that ss-glucuronidase and hexosaminidase activities in plasma reached levels near the lower normal limits when ACD was used. In conclusion, we observed that heparin should be used as the preferable anticoagulant when measuring these lysosomal enzyme activities, and we recommend that, when transport time is more than 24 h, samples should be shipped by air in a styrofoam box containing wet ice. (+info)