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Molecular characterization of arylsulfatase G: expression, processing, glycosylation, transport, and activity.

ABSTRACT: Arylsulfatase G (ARSG) is a recently identified lysosomal sulfatase that was shown to be responsible for the degradation of 3-O-sulfated N-sulfoglucosamine residues of heparan sulfate glycosaminoglycans. Deficiency of ARSG leads to a new type of mucopolysaccharidosis, as described in a mouse model. Here, we provide a detailed molecular characterization of the endogenous murine enzyme. ARSG is expressed and proteolytically processed in a tissue-specific manner. The 63-kDa single-chain precursor protein localizes to pre-lysosomal compartments and tightly associates with organelle membranes, most likely the endoplasmic reticulum. In contrast, proteolytically processed ARSG fragments of 34-, 18-, and 10-kDa were found in lysosomal fractions and lost their membrane association. The processing sites and a disulfide bridge between the 18- and 10-kDa chains could be roughly mapped. Proteases participating in the processing were identified as cathepsins B and L. Proteolytic processing is dispensable for hydrolytic sulfatase activity in vitro. Lysosomal transport of ARSG in the liver is independent of mannose 6-phosphate, sortilin, and Limp2. However, mutation of glycosylation site N-497 abrogates transport of ARSG to lysosomes in human fibrosarcoma cells, due to impaired mannose 6-phosphate modification.

SUBMITTER: Kowalewski B

PROVIDER: S-EPMC4183830 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Molecular characterization of arylsulfatase G: expression, processing, glycosylation, transport, and activity.

Kowalewski Björn B Lübke Torben T Kollmann Katrin K Braulke Thomas T Reinheckel Thomas T Dierks Thomas T Damme Markus M

The Journal of biological chemistry 20140818 40

Arylsulfatase G (ARSG) is a recently identified lysosomal sulfatase that was shown to be responsible for the degradation of 3-O-sulfated N-sulfoglucosamine residues of heparan sulfate glycosaminoglycans. Deficiency of ARSG leads to a new type of mucopolysaccharidosis, as described in a mouse model. Here, we provide a detailed molecular characterization of the endogenous murine enzyme. ARSG is expressed and proteolytically processed in a tissue-specific manner. The 63-kDa single-chain precursor p ...[more]

PMID: 25135642

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Project description:BackgroundMetachromatic Leukodystrophy (MLD, OMIM 250100) is a neurodegenerative disease caused by mutations in the ARSA gene (OMIM 607574) that lead to deficiency in Arylsulfatase A (ASA). ASA pseudodeficiency (PD-ASA) is a biochemical condition that substantially diminishes ASA activity but is not associated with clinical manifestations. PD-ASA is associated with the c.1055A>G (p.Asn352Ser) (rs2071421) and c.*96A>G (rs6151429) variants, which have an estimated frequency of 2% in the population.ObjectiveTo determine the activity of Arylsulfatase A and to identify variants and haplotypes in the ARSA gene in Mexican individuals with pseudodeficiency.MethodsTwo-hundred apparently healthy individuals were included to determine the enzymatic activity of ASA in leukocytes by spectrophotometric analysis, and identification of the PD-ASA alleles was performed by PCR-RFLP assays. Genotypes were confirmed by semi-automated Sanger sequencing. Haplotypes were constructed using Arlequin v.10.04, and linkage disequilibrium analysis was performed with Cube X.ResultsThe enzymatic activity of ASA was determined to be 1.74-2.09 nmol/mg protein/min and later correlated with genotypes and haplotypes. For the (p.Asn352Ser) variant, we found 126 (0.63) individuals with the AA genotype, 62 with AG (0.31) and 12 with GG (0.06); the frequency of the polymorphic allele was 0.215 (86 alleles, 21.5%), and the variant was in HWE (p = .2484). The variant c.*96A>G was also in HWE (p = .2105): 185 individuals (0.925) with the AA genotype, 14 (0.07) with AG, and 1 (0.005) with (GG), with a frequency of 0.04 (4%) for the polymorphic allele. The inference of haplotypes resulted in 312 (0.78) AA, 72 (0.18) GA, and 16 (0.04) GG haplotypes. The AG haplotype was not found. The variants were found to be in linkage disequilibrium (D' = 1). Of the nine possible diplotypes, AA/AG, AA/GG, and AG/GG were not found, in concordance with the hypothesis that the G allele of c.*96A>G does not occur in the absence of the G allele of c.1055A>G. We found a slight correlation between ASA biochemical activity and variants, mainly due to the G allele of c.*96A>G in either genotypes or haplotypes.ConclusionsIn Northwestern Mexico, the presence of PD-ASA alleles was biochemically and molecularly determined, and the frequencies were found to be in HWE. The frequency of PD-ASA for the North Western Mexican mestizo is 8%.

Dataset Information

Molecular characterization of arylsulfatase G: expression, processing, glycosylation, transport, and activity.

Publications

Molecular characterization of arylsulfatase G: expression, processing, glycosylation, transport, and activity.

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