Correction of Abnormal Cerebroside Sulfate Metabolism in Cultured Metachromatic Leukodystrophy Fibroblasts

Porter, Myna T.; Fluharty, Arvan L.; Kihara, Hayato

doi:10.1126/science.172.3989.1263

Cited by 100 publications

(22 citation statements)

References 15 publications

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“…For example, a-Gal A constitutes approximately 0.002% of total cellular protein in human lung, the source used for enzyme purification. Furthermore, the fact that lysosomal hydrolases turn over slowly in tissues (33,34) leads one to predict that the corresponding mRNAs will be present at disproportionately lower concentrations in poly(A)+ mRNA preparations used to make cDNA libraries. Indeed, the cDNA clone corresponding to another lysosomal hydrolase, a-fucosidase, is among the rarest cloned to date, with an mRNA abundance of 0.002% and a protein concentration of 0.01% of liver protein (27).…”

Section: Resultsmentioning

confidence: 99%

Fabry disease: isolation of a cDNA clone encoding human alpha-galactosidase A.

Calhoun¹,

Bishop²,

Bernstein³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

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Fabry disease is an X-linked inborn error of metabolism resulting from the deficient activity of the lysosomal hydrolase, a-galactosidase A (a-Gal A; a-Dgalactoside galactohydrolase, EC 3.2.1.22). To investigate the structure, organization, and expression of a-Gal A, as well as the nature of mutations in Fabry disease, a clone encoding human a-Gal A was isolated from a Agtll human liver cDNA expression library. To facilitate screening, an improved affinity purification procedure was used to obtain sufficient homogeneous enzyme for production of monospecific antibodies and for amino-terminal and peptide microsequencing. On the basis of an amino-terminal sequence of 24 residues, two sets of oligonucleotide mixtures were synthesized corresponding to adjacent, but not overlapping, amino acid sequences. In addition, an oligonucleotide mixture was synthesized based on a sequence derived from an a-Gal A internal tryptic peptide isolated by reversed-phase HPLC. Four positive clones were initially identified by antibody screening of 1.4 X 107 plaques. Of these, only one clone (designated XAG18) demonstrated both antibody binding specificity by competition studies using homogeneous enzyme and specific hybridization to synthetic oligonucleotide mixtures corresponding to amino-terminal and internal amino acid sequences. Nucleotide sequencing of the 5' end of the 1250-base-pair EcoRI insert ofclone XAG18 revealed an exact correspondence between the predicted and known amino-terminal amino acid sequence. The insert of clone XAG18 appears to contain the full-length coding region of the processed, enzymatically active a-Gal A, as well as sequences coding for five amino acids of the amino-terminal propeptide, which is posttranslationally cleaved during enzyme maturation.Fabry disease is an inborn error of glycosphingolipid metabolism that results from the defective activity of the lysosomal hydrolase a-galactosidase A (a-Gal A; a-D-galactoside galactohydrolase, EC 3.2.1.22) (1). The mature, active human enzyme is a homodimeric protein (subunit Mr -49,800) (2, 3), which is encoded by a structural gene localized to a narrow region (q21-q22) on the X chromosome (4). Deficient a-Gal A activity results in the accumulation of its major glycosphingolipid substrate, globotriaosylceramide and related glycolipids with terminal a-galactosidic linkages (1, 5). Progressive substrate deposition, particularly in the plasma and vascular endothelium, leads to ischemia and infarction with early demise due to vascular disease of the heart, kidney, and/or brain (1).Since the availability of a cDNA for a-Gal A would facilitate studies of the molecular basis of the disease, provide specific probes for heterozygote identification, and permit expression of large amounts of the enzyme for further structural characterization and therapeutic trials of enzyme replacement (6, 7), efforts were undertaken to isolate a cDNA encoding human a-Gal A. In this communication, we report the molecular cloning of a cDNA that apparently encodes the entire amino ...

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Section: Resultsmentioning

confidence: 99%

Fabry disease: isolation of a cDNA clone encoding human alpha-galactosidase A.

Calhoun¹,

Bishop²,

Bernstein³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

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“…Analyses of Fabry fibroblasts after exposure to enzyme revealed that under the conditions described, a-galactosidase activity rose from near 0 to the hydrolysis of 0.2 pxaol galactose/mg protein/hr, which is the mean activity of the native a-galactosidase in fibroblasts (Table I). In contrast to studies with arylsulfatase A [19], the ficin a-galactosidase was fairly rapidly inactivated within the (Fig. 3) indicated a slightly higher stability for the intracellular a-galactosidase but over 50% of the enzymic activity was lost between 6 and 24 hr after addition of a-galactosidase.…”

Section: Uptake Of Argalactosidase By Fibroblastsmentioning

confidence: 54%

“…Fibroblasts have been used as a model for enzyme replacement in the severe neurologic disease, metachromatic leukodystrophy (MLD) [19,25], and arylsulfatase A activity was then measurable for several weeks in culture. However, MLD fibroblasts do not normally accumulate sulfatide, necessitating the addition of sulfatide to the medium before the enzyme replacement.…”

Section: Introductionmentioning

confidence: 99%

Correction of the Enzymic Defect in Cultured Fibroblasts from Patients with Fabry's Disease: Treatment with Purified α-Galactosidase from Ficin

Dawson

Matalon

1973

Pediatr Res

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“…Significantly, only 1-5% of normal cellular enzymatic activity was needed to correct the abnormal accumulation of lysosomal substrate. 17,96,104 Starting in the 1970s, ERT clinical trials were performed by infusing normal human enzymes into individuals who had a variety of LSDs, including Sandhoff disease (␤-hexosaminidase A derived from urine), 61 Pompe disease (placental ␣-glucosidase), 28 Fabry disease (placental, plasma, and splenic ␣-galactosidase A), 14 and Gaucher disease Type 1 (placental acid ␤-glucosidase). 13 These studies showed both evidence of substrate degradation and rapid clearance of the intravenously delivered enzymes, but no significant clinical benefits.…”

Section: Enzyme Replacement Therapymentioning

confidence: 99%

Central nervous system therapy for lysosomal storage disorders

Enns

Huhn

2008

FOC

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✓ Most lysosomal storage disorders are characterized by progressive central nervous system impairment, with or without systemic involvement. Affected individuals have an array of symptoms related to brain dysfunction, the most devastating of which is neurodegeneration following a period of normal development. The blood–brain barrier has represented a significant impediment to developing therapeutic approaches to treat brain disease, but novel approaches—including enzyme replacement, small-molecule, gene, and cell-based therapies—have given children afflicted by these conditions and those who care for them hope for the future.

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Correction of Abnormal Cerebroside Sulfate Metabolism in Cultured Metachromatic Leukodystrophy Fibroblasts

Cited by 100 publications

References 15 publications

Fabry disease: isolation of a cDNA clone encoding human alpha-galactosidase A.

Fabry disease: isolation of a cDNA clone encoding human alpha-galactosidase A.

Correction of the Enzymic Defect in Cultured Fibroblasts from Patients with Fabry's Disease: Treatment with Purified α-Galactosidase from Ficin

Central nervous system therapy for lysosomal storage disorders

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