In vitro mutagenesis of potential N-glycosylation sites of arylsulfatase A. Effects on glycosylation, phosphorylation, and intracellular sorting.
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The correct intracellular sorting of lysosomal enzymes such as arylsulfatase A depends on the presence of mannose 6-phosphate residues on high mannose type oligosaccharides. The arylsulfatase A cDNA contains three potential N-glycosylation sites, two of which are utilized. We have mutated one or two of the N-glycosylation sites and analyzed the glycosylation, phosphorylation, and intracellular sorting of the mutant arylsulfatase A polypeptides. The results show that each of the three glycosylation sites (I, II, and III) can be glycosylated, but glycosylation at sites I and II is mutually exclusive. In mutants with one oligosaccharide side chain at positions I, II, or III all side chains can acquire mannose 6-phosphate residues irrespective of their location. This demonstrates spatial flexibility of the phosphotransferase, which specifically recognizes lysosomal enzymes and initiates the addition of mannose 6-phosphate residues on oligosaccharide side chains. However, these mutants have different intracellular sorting efficiencies and seem to use different (mannose 6-phosphate receptor-dependent and -independent) sorting pathways. (+info)
Terminal 22q deletion associated with a partial deficiency of arylsulphatase A.
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A 7 month old girl with psychomotor retardation, hypotonia, and minor malformations was found to have a terminal deletion of the long arm of chromosome 22, del(22)(q13.31). The partial deficiency of arylsulphatase A (ARSA) and the normal level of NADH diaphorase 1 (DIA1) suggests that the ARSA locus can be regionally assigned to 22q13.31----qter and the DIA1 locus can be excluded from the same segment. This report is the third published case with a terminal 22q deletion. (+info)
The structural basis for the electrophoretic isoforms of normal and variant human platelet arylsulphatase A.
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Human platelet arylsulphatase A (ASA) exhibits a multiple banding pattern when examined by PAGE. The isoform pattern (IVa) of the enzyme obtained from normal subjects differs from variants (IIIa, IIIb, IVb) which are primarily found in alcoholic patients. Alkaline phosphatase and endo-N-acetylglucosaminidase H treatments, as well as ion-exchange chromatography, demonstrate that the isoforms of ASA arise because of charge heterogeneity caused by phosphoglycan moieties. The isoform with the slowest mobility in PAGE lacks phosphate substituents. Based upon mannose-6-phosphate-receptor affinity chromatography it can be concluded that most, if not all, of the enzyme-linked phosphate is in the form of 6-O-phospho-D-mannosyl units. Lectin affinity chromatography and peptide-N-glycosidase F treatment followed by SDS/PAGE and Western-blot analysis indicate that normal platelet ASA contains two oligomannose and/or hybrid glycan moieties of which one, or both, possess a 6-O-L-fucosyl substituent on the asparagine-linked N-acetylglucosamine residue. Comparative analysis indicates that the variant IIIa and IIIb types of ASA differ from the IVa ASA with regard to the level of glycan phosphorylation and glycan structure, as well as the polypeptide structure. (+info)
Purification of rat liver arylsulfatase A and its microheterogeneity assayed by crossed affinity-immunoelectrophoresis.
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Arylsulfatase A (arylsulfatase sulfohydrolase) EC 3.1.6.1 was purified from rat liver by a procedure consisting of differential centrifugation, Con A-Sepharose and Blue Sepharose chromatography, PBE 94 chromatofocusing, DEAE-cellulose and gel filtration chromatography followed by preparative electrophoresis. A molecular mass of 132,000 was estimated by gradient PAGE. Particular proteins were detected by immunoelectrophoresis. Isoelectric focusing combined with immunoelectrophoresis gave two peaks of arylsulfatase A, with isoelectric points of pH 3.9 and 4.5. Microheterogeneity of rat liver arylsulfatase A was studied by affinity immunoelectrophoresis with 9 different lectins. The presence of concanavalin A-, Lens culinaris agglutinin-, Lotus tetragonolobus agglutinin- and wheat germ agglutinin-reactive forms permitted assessment of the types of carbohydrate moieties in arylsulfatase A. (+info)
Biochemical characteristics of a Korean patient with mucolipidosis III (pseudo-Hurler polydystrophy).
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We performed a biochemical study on the patient with mucolipidosis III (ML-III, pseudo-Hurler polydystrophy) in Korea. Confluent fibroblasts from the patient and from normal controls were cultured for 4, 12, 24, 48, and 72 hr, respectively. Lysosomal enzyme activities in culture media after different incubation times and in plasma, leuko-cytes, and fibroblasts were determined. Most of the leukocyte lysosomal enzymes were within normal limits or slightly lowered; however, plasma lysosomal enzyme activities such as those of hexosaminidase and arylsulfatase A were markedly increased. Numerous phase-dense inclusions were present in the cytoplasm of cultured fibroblasts. Lysosomal enzyme activities of fibroblasts were markedly decreased except for beta-glucosidase. The rates of increase of the lysosomal enzyme activities with incubation time were greater in the culture medium of the patient than in normal control, whereas no difference in the beta-glucosidase activity of the culture media of the patient and the control was found. This study describes the first case of ML-III in Korea, with its typical biochemical characteristics, i.e., a problem with targeting and transporting of lysosomal enzymes which results in a marked increase in plasma lysosomal enzyme activities and a high ratio of extracellular to intracellular lysosomal enzyme activities in cultured fibroblasts. (+info)
An unusual arylsulfatase A pseudodeficiency allele carrying a splice site mutation in a metachromatic leukodystrophy patient.
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A late infantile metachromatic leukodystrophy patient was found to be heterozygous for the arylsulfatase A (ARSA) pseudodeficiency (pd) polyadenylation site variant ((*)96A>G) in the absence of the commonly associated N-glycosylation site variant (N350S). ARSA alleles were sequenced and the genotype completely defined. Six sequence variations were identified, among which two resulted as severe disease-causing mutations, both leading to the loss of the reading frame: a splice acceptor site mutation in intron 4 (849-1G>A), located on the (*)96A>G allele and a mononucleotide deletion (258delC) in exon 2, located on the other allele. The altered splicing caused by the 849-1G>A mutation was shown by in vitro expression of a recombinant gene containing the genomic region surrounding the mutation. Haplotype analysis of the unusual pd allele was performed in order to investigate its possible origin. (+info)
Defective endoplasmic reticulum-resident membrane protein CLN6 affects lysosomal degradation of endocytosed arylsulfatase A.
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Variant late infantile neuronal ceroid lipofuscinosis, a lysosomal storage disorder characterized by progressive mental deterioration and blindness, is caused by mutations in a polytopic membrane protein (CLN6) with unknown intracellular localization and function. In this study, transient transfection of BHK21 cells with CLN6 cDNA and immunoblot analysis using peptide-specific CLN6 antibodies demonstrated the expression of a approximately 27-kDa protein that does not undergo proteolytic processing. Cross-linking experiments revealed the presence of CLN6 dimers. Using double immunofluorescence microscopy, epitope-tagged CLN6 was shown to be retained in the endoplasmic reticulum (ER) with no colocalization with the cis-Golgi or lysosomal markers. The translocation into the ER and proper folding were confirmed by the N-linked glycosylation of a mutant CLN6 polypeptide. Pulse-chase labeling of fibroblasts from CLN6 patients and from sheep (OCL6) and mouse (nclf) models of the disease followed by immunoprecipitation of cathepsin D indicated that neither the synthesis, sorting nor the proteolytic processing of this lysosomal enzyme was affected in CLN6-defective cells. However, the degradation of the endocytosed index protein arylsulfatase A was strongly reduced in all of the mutant CLN6 cell lines compared with controls. These data suggest that defects in the ER-resident CLN6 protein lead to lysosomal dysfunctions, which may result in lysosomal accumulation of storage material. (+info)
Late onset MLD with normal nerve conduction associated with two novel missense mutations in the ASA gene.
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Metachromatic leukodystrophy (MLD) rarely has its clinical onset in young adults, with a combination of cognitive and behavioural symptoms and peripheral neuropathy. Here we present an exceptional case with very late onset at 42 years of age and no clinical or neurophysiological sign of peripheral neuropathy. Molecular analysis revealed compound heterozygosity for two novel missense mutations affecting conserved residues in the arylsulphatase A (ASA) sulphatase and carboxyterminal domains, resulting in an 89% loss of enzymatic activity. This case indicates that MLD needs to be considered in the differential diagnosis of very late onset white matter diseases, even if not accompanied by peripheral nerve involvement. (+info)