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‣ Kidney Function and 24-Hour Proteinuria in Patients with Fabry Disease during 36 Months of Agalsidase Alfa Enzyme Replacement Therapy: A Brazilian Experience

THOFEHRN, Scheila; NETTO, Cristina; CECCHIN, Claudia; BURIN, Maira; MATTE, Ursula; BRUSTOLIN, Silvia; NUNES, Ane Claudia Fernandes; COELHO, Janice; TSAO, Marylin; JARDIM, Laura; GIUGLIANI, Roberto; BARROS, Elvino Jose Guardao
Fonte: TAYLOR & FRANCIS INC Publicador: TAYLOR & FRANCIS INC
Tipo: Artigo de Revista Científica
Português
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Background. Prior to the introduction of enzyme replacement therapy (ERT), management of Fabry disease (FD) consisted of symptomatic and palliative measures. ERT has been available for several years using recombinant human agalsidase alfa, an analogue of alpha-galactosidase A (GALA). However, the limitations of ERT in improving kidney function have not been established. This study evaluates the safety and therapeutic effect of agalsidase alfa replacement in terms of kidney function and reduction in 24-hour proteinuria. Methods. During the period between January 1, 2002, and August 1, 2005, nine Fabry patients (7 male, 2 female) were treated according to protocol, receiving 0.2 mg/kg agalsidase alfa IV every two weeks. Kidney function was evaluated by measuring the glomerular filtration rate (GFR) using chromium ethylene diamine tetra-acetate clearance ((51)Cr-EDTA mL/min/1.73 m(2)) at baseline, 12, 24, and 36 months. 24-hour proteinuria was measured at baseline, 3, 6, 12, 18, 24, and 36 months of ERT. Kidney disease was classified according to National Kidney Foundation Disease Outcome Quality Initiative (NKF/DOQI) Advisory Board criteria, which define stage I chronic kidney disease (CKD) as GFR >= 90mL/min/1.73 m(2), stage II as 60-89 mL/min/1.73m(2)...

‣ Debaryomyces hansenii UFV-1 Intracellular alpha-Galactosidase Characterization and Comparative Studies with the Extracellular Enzyme

VIANA, Pollyanna A.; REZENDE, Sebastiao T. de; PASSOS, Flavia Maria Lopes; OLIVEIRA, Jamil S.; TEIXEIRA, Kadima N.; SANTOS, Alexandre M. C.; BEMQUERER, Marcelo P.; ROSA, Jose C.; SANTORO, Marcelo M.; GUIMARAES, Valeia M.
Fonte: AMER CHEMICAL SOC Publicador: AMER CHEMICAL SOC
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
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Debaryomyces hansenii cells cultivated on galactose produced extracellular and intracellular alpha-galactosidases, which showed 54.5 and 54.8 kDa molecular mass (MALDI-TOF), 60 and 61 kDa (SDS-PAGE) and 5.15 and 4.15 pI values, respectively. The extracellular and intracellular deglycosylated forms presented 36 and 40 kDa molecular mass, with 40 and 34% carbohydrate content, respectively. The N-terminal sequences of the alpha-galactosidases were identical. Intracellular alpha-galactosidase showed smaller thermostability when compared to the extracellular enzyme. D. hansenii UFV-1 extracellular alpha-galactosidase presented higher k(cat) than the intracellular enzyme (7.16 vs 3.29 s(-1), respectively) for the p-nitrophenyl-alpha-D-galactopyranoside substrate. The K(m) for hydrolysis of pNP alpha Gal, melibiose, stachyose, and raffinose were 0.32, 2.12, 10.8, and 32.8 mM, respectively. The intracellular enzyme was acompetitively inhibited by galactose (K(i) = 0.70 mM), and it was inactivated by Cu(II) and Ag(I). Enzyme incubation with soy milk for 6 h at 55 degrees C reduced stachyose and raffinose amounts by 100 and 73%, respectively.; Fundacao de Amparo a Pesquisa do Estado de Minas Gerais-FAPEMIG[EDT24000]; Fundacao de Amparo a Pesquisa do Estado de Minas Gerais-FAPEMIG[EDT268/05]; Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior-CAPES...

‣ Purification of alpha-galactosidase from seeds of Sesbania marginata

Falco,A.L.P.; Durrant,L.R.; Franco,T.T.
Fonte: Brazilian Society of Chemical Engineering Publicador: Brazilian Society of Chemical Engineering
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/12/2000 Português
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Alpha-galactosidase taken from a raw extract of Sesbania marginata legume seeds was purified by partitioning in aqueous two-phase systems (ATPS). Initially, galactomannan/dextran 2,000,000 systems were used for the purification, and the partition coefficients of alpha -galactosidase varied from 1.5 to 4.0. However, mass transport in these systems was poor due to the high viscosity of the employed polymers. Therefore, partitioning in polyethyleneglycol (PEG)/ sodium phosphate systems and the effect of sodium chloride upon the enzyme purification and the yield of alpha -galactosidase were also investigated. The purification achieved in a single-step was 5.7 with a recovery of 144% of alpha -galactosidase, possibly due to the removal of materials which inhibited alpha -galactosidase activity before the purification. The removal of the main protein contaminants and the highest yields were achieved in PEG 4,000/ sodium phosphate + 6% NaCl system at pH 5.0. Further purification by preparative on-exchange chromatography was also developed.

‣ Correction in trans for Fabry disease: expression, secretion and uptake of alpha-galactosidase A in patient-derived cells driven by a high-titer recombinant retroviral vector.

Medin, J A; Tudor, M; Simovitch, R; Quirk, J M; Jacobson, S; Murray, G J; Brady, R O
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 23/07/1996 Português
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Fabry disease is an X-linked metabolic disorder due to a deficiency of alpha-galactosidase A (alpha-gal A; EC 3.2.1.22). Patients accumulate glycosphingolipids with terminal alpha-galactosyl residues that come from intracellular synthesis, circulating metabolites, or from the biodegradation Of senescent cells. Patients eventually succumb to renal, cardio-, or cerebrovascular disease. No specific therapy exists. One possible approach to ameliorating this disorder is to target corrective gene transfer therapy to circulating hematopoietic cells. Toward this end, an amphotropic virus-producer cell line has been developed that produces a high titer (>10(6) i.p. per ml) recombinant retrovirus constructed to transduce and correct target cells. Virus-producer cells also demonstrate expression of large amounts of both intracellular and secreted alpha-gal A. To examine the utility of this therapeutic vector, skin fibroblasts from Fabry patients were corrected for the metabolic defect by infection with this recombinant virus and secreted enzyme was observed. Furthermore, the secreted enzyme was found to be taken up by uncorrected cells in a mannose-6-phosphate receptor-dependent manner. In related experiments, immortalized B cell lines from Fabry patients...

‣ Enzyme therapy in Fabry disease: differential in vivo plasma clearance and metabolic effectiveness of plasma and splenic alpha-galactosidase A isozymes.

Desnick, R J; Dean, K J; Grabowski, G; Bishop, D F; Sweeley, C C
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /10/1979 Português
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A pilot trial of enzyme replacement with splenic and plasma alpha-galactosidase A (alpha-D-galactosidase; alpha-D-galactoside galactohydrolase, EC 3.2.1.22) isozymes was undertaken in two brothers with Fabry disease, an X-linked glycosphingolipid storage disease. Six unentrapped doses (2000 units/kg) of each isozyme were administered intravenously to the respective recipients during a 117-day period. The circulating half-life of the splenic isozyme was about 10 min, whereas that for the plasma isozyme was approximately 70 min. No immune response was detected by skin and immunodiffusion tests or by alterations in the maximal activity or clearance kinetics for either isozyme after successive administrations. After each dose of the splenic isozyme, the concentration of the accumulated circulating substrate, trihexosylceramide (globotriaosylceramide), decreased maximally (approximately 50% of initial values) in 15 min and returned to preinfusion levels by 2-3 hr. In marked contrast, injection of the plasma isozyme decreased the circulating substrate levels 50-70% by 2-6 hr; the concentrations gradually returned to preinfusion values by 36-72 hr.

‣ A carboxy-terminal truncation of human alpha-galactosidase A in a heterozygous female with Fabry disease and modification of the enzymatic activity by the carboxy-terminal domain. Increased, reduced, or absent enzyme activity depending on number of amino acid residues deleted.

Miyamura, N; Araki, E; Matsuda, K; Yoshimura, R; Furukawa, N; Tsuruzoe, K; Shirotani, T; Kishikawa, H; Yamaguchi, K; Shichiri, M
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/10/1996 Português
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Fabry disease is an X-linked disorder of glycosphingolipid metabolism caused by a deficiency of alpha-galactosidase A (alpha-Gal A). We identified a novel mutation of alpha-Gal A gene in a family with Fabry disease, which converted a tyrosine at codon 365 to a stop and resulted in a truncation of the carboxy (C) terminus by 65 amino acid (AA) residues. In a heterozygote of this family, although the mutant and normal alleles were equally transcribed in cultured fibroblasts, lymphocyte alpha-Gal A activity was approximately 30% of the normal control and severe clinical symptoms were apparent. COS-1 cells transfected with this mutant cDNA showed a complete loss of its enzymatic activity. Furthermore, those cotransfected with mutant and wildtype cDNAs showed a lower alpha-Gal A activity than those with wild type alone (approximately 30% of wild type alone), which suggested the dominant negative effect of this mutation and implied the importance of the C terminus for its activity. Thus, we generated mutant cDNAs with various deletion of the C terminus, and analyzed. Unexpectedly, alpha-Gal A activity was enhanced by up to sixfold compared with wild-type when from 2 to 10 AA residues were deleted. In contrast, deletion of 12 or more AA acid residues resulted in a complete loss of enzyme activity. Our data suggest that the C-terminal region of alpha-Gal A plays an important role in the regulation of its enzyme activity.

‣ Human alpha-galactosidase A: glycosylation site 3 is essential for enzyme solubility.

Ioannou, Y A; Zeidner, K M; Grace, M E; Desnick, R J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/06/1998 Português
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Human alpha-galactosidase A (EC 3.2.1.22; alpha-Gal A) is the homodimeric glycoprotein that hydrolyses the terminal alpha-galactosyl moieties from glycolipids and glycoproteins. The type, site occupancy and function of the N-linked oligosaccharide chains on this lysosomal hydrolase were determined. Endoglycosidase treatment of the purified recombinant enzyme and mutagenesis studies indicated that three (Asn-139, Asn-192 and Asn-215) of the four potential N-glycosylation consensus sequences were occupied by complex, high-mannose and hybrid-type oligosaccharides respectively. When expressed in COS-1 cells, glycoforms with glycosylation site 1 or 2 obliterated had more than 70% of wild-type activity, and both glycoforms were secreted. In contrast, the glycoform with only site 3 eliminated had decreased activity (less than 40%); little, if any, was secreted. Expressed mutant glycoforms in which site 3 and site 1 or 2 were obliterated had little, if any, intracellular or secreted enzymic activity, and immunofluorescence microscopy revealed that the expressed mutant glycoforms were retained in the endoplasmic reticulum, presumably where they were degraded. Thus glycosylation at site 3 was crucial to the formation of soluble, active enzyme...

‣ Nature and frequency of mutations in the alpha-galactosidase A gene that cause Fabry disease.

Eng, C M; Resnick-Silverman, L A; Niehaus, D J; Astrin, K H; Desnick, R J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1993 Português
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Fabry disease, an X-linked inborn error of glycosphingolipid catabolism, results from mutations in the alpha-galactosidase A (alpha-Gal A) gene at Xq22.1. To determine the nature and frequency of the molecular lesions causing the classical and milder-variant Fabry phenotypes, and for precise carrier detection in Fabry families, the alpha-Gal A transcripts or genomic sequences from unrelated Fabry hemizygotes were analyzed. In patients with the classical phenotype, 18 new mutations were identified: N34S, C56G, W162R, R227Q, R227X, D264V, D266V, S297F, D313Y, G328A, W340X, E398X, IVS2+2, IVS5 delta-2,3, 773 delta 2, 954 delta 5, 1016 delta 11, and 1123 delta 53. Unrelated asymptomatic or mildly affected patients with symptoms confined to the heart had a missense mutation, N215S, that expressed residual enzymatic activity. Related, moderately affected patients with late-onset cardiac and pulmonary manifestations had a small deletion, 1208 delta 3, that predicted the in-frame deletion of arginine 404 near the terminus of the 429 residue enzyme polypeptide. In addition, five small gene rearrangements involving exonic sequences were identified in unrelated classically affected patients. Two small deletions and one small duplication had short direct repeats at their respective breakpoint junctions and presumably resulted from slipped mispairing. A deletion occurred at a potential polymerase alpha arrest site...

‣ Identification of point mutations in the alpha-galactosidase A gene in classical and atypical hemizygotes with Fabry disease.

Sakuraba, H; Oshima, A; Fukuhara, Y; Shimmoto, M; Nagao, Y; Bishop, D F; Desnick, R J; Suzuki, Y
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1990 Português
Relevância na Pesquisa
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Efforts were directed to identify the specific mutations in the alpha-galactosidase A (alpha-Gal A) gene which cause Fabry disease in families of Japanese origin. By polymerase-chain-reaction-amplification of DNA from reverse-transcribed mRNA and genomic DNA, different point mutations were found in two unrelated Fabry hemizygotes. A hemizygote with classic disease manifestations and no detectable alpha-Gal A activity had a G-to-A transition in exon 1 (codon 44) which substituted a termination codon (TAG) for a tryptophan codon (TGG) and created an NheI restriction site. This point mutation would predict a truncated alpha-Gal A polypeptide, consistent with the observed absence of enzymatic activity and a classic Fabry phenotype. In an unrelated Japanese hemizygote who had an atypical clinical course characterized by late-onset cardiac involvement and significant residual alpha-Gal activity, a G-to-A transition in exon 6 (codon 301) resulted in the replacement of a glutamine for an arginine residue. This amino acid substitution apparently altered the properties of the enzyme such that sufficient enzymatic activity was retained to markedly alter the disease course. Identification of these mutations permitted accurate molecular heterozygote diagnosis in these families.

‣ Endocytosis of lysosomal alpha-galactosidase A by cultured fibroblasts from patients with Fabry disease.

Mayes, J S; Cray, E L; Dell, V A; Scheerer, J B; Sifers, R N
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1982 Português
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The endocytosis of alpha-galactosidase A was studied in cultured fibroblasts from patients with Fabry disease. Alpha-galactosidase A was purified from human placenta by chromatography on concanavalin A-Sepharose, DEAE-cellulose, and N-epsilon-aminocaproyl-alpha-D-galactosylamine-Sepharose. Separation of the high-uptake form of the enzyme from the low-uptake form was accomplished by chromatography on ECTEOLA-cellulose. With the high-uptake form of the enzyme, the uptake was linear at low concentrations of enzyme and had a Kuptake of 0.01 U/ml of medium that corresponds to a Km of 5.0 x 10(-9) M. At high concentrations of enzyme, it became saturated. The high-uptake form could be converted to the low-uptake form by treatment with acid phosphatase. Mannose-6-P strongly inhibited the active uptake of the enzyme. Once taken up into the lysosomes of Fabry disease fibroblasts, alpha-galactosidase A activity was rapidly lost in the first 2 days of incubation at 37 degrees C, but was fairly stable for the next 6 days. The half-life of internalized alpha-galactosidase A activity was calculated to be 4 days. Crosslinking of the enzyme with hexamethylene diisocyanate did not increase the intracellular stability of alpha-galactosidase A activity.

‣ Fabry disease: twenty novel alpha-galactosidase A mutations and genotype-phenotype correlations in classical and variant phenotypes.

Germain, Dominique P.; Shabbeer, Junaid; Cotigny, Sylvie; Desnick, Robert J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/2002 Português
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BACKGROUND: Fabry disease (OMIM 301500) is an X-linked inborn error of glycosphingolipid metabolism resulting from mutations in the alpha-galactosidase A (alpha-Gal A) gene. The disease is phenotypically heterogeneous with classic and variant phenotypes. To assess the molecular heterogeneity, define genotype/phenotype correlations, and for precise carrier identification, the nature of the molecular lesions in the alpha-Gal A gene was determined in 40 unrelated families with Fabry disease. MATERIALS AND METHODS: Genomic DNA was isolated from affected males or obligate carrier females and the entire alpha-Gal A coding region and flanking sequences were amplified by PCR and analyzed by automated sequencing. Haplotype analyses were performed with polymorphisms within and flanking the alpha-Gal A gene. RESULTS: Twenty new mutations were identified (G43R, R49G, M72I, G138E, W236X, L243F, W245X, S247C, D266E, W287C, S297C, N355K, E358G, P409S, g1237del15, g10274insG, g10679insG, g10702delA, g11018insA, g11185-delT), each in a single family. In the remaining 20 Fabry families, 18 previously reported mutations were detected (R49P, D92N, C94Y, R112C [two families], F113S, W162X, G183D, R220X, R227X, R227Q, Q250X, R301X, R301Q, G328R, R342Q, E358K...

‣ Twenty novel mutations in the alpha-galactosidase A gene causing Fabry disease.

Topaloglu, A. K.; Ashley, G. A.; Tong, B.; Shabbeer, J.; Astrin, K. H.; Eng, C. M.; Desnick, R. J.
Fonte: The Feinstein Institute for Medical Research Publicador: The Feinstein Institute for Medical Research
Tipo: Artigo de Revista Científica
Publicado em /12/1999 Português
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BACKGROUND: Fabry disease, an X-linked inborn error of glycosphingolipid catabolism, results from the deficient activity of the lysosomal exoglycohydrolase alpha-galactosidase A (EC 3.2.1.22; alpha-Gal A). The nature of the molecular lesions in the alpha-Gal A gene in 30 unrelated families was determined to provide precise heterozygote detection, prenatal diagnosis, and define genotype-phenotype correlations. MATERIALS AND METHODS: Genomic DNA was isolated from affected males and/or carrier females from 30 unrelated families with Fabry disease. The entire alpha-Gal A coding region and flanking intronic sequences were analyzed by PCR amplification and automated sequencing. RESULTS: Twenty new mutations were identified, each in a single family: C142R, G183D, S235C, W236L, D244H, P259L, M267I, I289F, Q321E, C378Y, C52X, W277X, IVS4(+4), IVS6(+2), IVS6(-1), 35del13, 256del1, 892ins1, 1176del4, and 1188del1. In the remaining 10 unrelated Fabry families, 9 previously reported mutations were detected: M42V, R112C, S148R, D165V, N215S (in 2 families), Q99X, C142X, R227X, and 1072del3. Haplotype analysis using markers closely flanking the alpha-Gal A gene indicated that the two patients with the N215S lesion were unrelated. The IVS4(+4) mutation was a rare intronic splice site mutation that causes Fabry disease. CONCLUSIONS: These studies further define the heterogeneity of mutations in the alpha-Gal A gene causing Fabry disease...

‣ Lyso-Gb3 Indicates that the Alpha-Galactosidase A Mutation D313Y is not Clinically Relevant for Fabry Disease

Niemann, Markus; Rolfs, Arndt; Giese, Anne; Mascher, Hermann; Breunig, Frank; Ertl, Georg; Wanner, Christoph; Weidemann, Frank
Fonte: Springer Berlin Heidelberg Publicador: Springer Berlin Heidelberg
Tipo: Artigo de Revista Científica
Publicado em 01/07/2012 Português
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The X-chromosomal-linked lysosomal storage disorder Fabry disease can lead to life-threatening manifestations. The pathological significance of the Fabry mutation D313Y is doubted, because, in general, D313Y patients do not present clinical manifestations conformable with Fabry disease. This is in contrast to the analysis of the alpha-galactosidase A activity, which is reduced in D313Y patients. We report a comprehensive clinical, biochemical and molecular genetic analysis of two patients with a D313Y mutation. The alpha-galactosidase A activity was reduced in both patients. No Fabry symptoms or Fabry organ involvement was detected in these patients. The new biomarker lyso-Gb3, severely increased in classical Fabry patients, was determined and in both patients lyso-Gb3 was below the average of a normal population.

‣ Cin?tica da enzima alfa-galactosidase a e investiga??o de doen?a de fabry em pacientes hemodialisados

ARAG?O, Camila de Britto Par? de
Fonte: Universidade Federal do Pará Publicador: Universidade Federal do Pará
Tipo: Dissertação de Mestrado
Português
Relevância na Pesquisa
77.53551%
A Alfa-galactosidase A (?-Gal A) humana ? uma enzima lisoss?mica que quando deficiente causa a doen?a de Fabry. A doen?a de Fabry ? uma esfingolipidose cuja principal causa de morbi-mortalidade ? a insufici?ncia renal cr?nica (IRC). O objetivo deste estudo foi a implanta??o de um protocolo laboratorial que permita o diagn?stico da doen?a de Fabry em plasma e leuc?citos, al?m da an?lise das caracter?sticas cin?ticas da enzima ?-Gal A em plasma e busca ativa da doen?a em 25 indiv?duos com IRC de causa desconhecida. Tamb?m foram avaliadas a reprodutibilidade e a estabilidade do m?todo enzim?tico. A padroniza??o dos ensaios foi realizada com o substrato fluorescente 4-metilumbeliferil-?-Dgalactopiranos?deo. A reprodutibilidade foi avaliada utilizando amostras de plasma aliquotadas a 4?C, -20?C e -70?C, analisadas uma vez ao m?s por 6 meses e a estabilidade da fluoresc?ncia por at? 24 horas ap?s o t?rmino do ensaio enzim?tico. A padroniza??o permitiu a implanta??o de valores de refer?ncia da ?-Gal A para o estado do Par?, de 4 a 28 nmoles/h/mL (plasma) e de 20 a 96 nmoles/h/mg prote?na (leuc?citos). A enzima ?-Gal A se mostrou termol?bil, visto que com apenas 1 minuto de pr?-incuba??o das amostras a 60?C, sua atividade decaiu 71,09%. Com rela??o ao tempo de incuba??o...

‣ Fabry disease: thirty-five mutations in the alpha-galactosidase A gene in patients with classic and variant phenotypes.

Eng, C. M.; Ashley, G. A.; Burgert, T. S.; Enriquez, A. L.; D'Souza, M.; Desnick, R. J.
Fonte: The Feinstein Institute for Medical Research Publicador: The Feinstein Institute for Medical Research
Tipo: Artigo de Revista Científica
Publicado em /03/1997 Português
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BACKGROUND: Fabry disease, an X-linked inborn error of glycosphingolipid catabolism, results from mutations in the alpha-galactosidase A (alpha-Gal A) gene located at Xq22.1. To determine the nature and frequency of the molecular lesions causing the classical and milder variant Fabry phenotypes and for precise carrier detection, the alpha-Gal A lesions in 42 unrelated Fabry hemizygotes were determined. MATERIALS AND METHODS: Genomic DNA was isolated from affected probands and their family members. The seven alpha-galactosidase A exons and flanking intronic sequences were PCR amplified and the nucleotide sequence was determined by solid-phase direct sequencing. RESULTS: Two patients with the mild cardiac phenotype had missense mutations, I9IT and F113L, respectively. In 38 classically affected patients, 33 new mutations were identified including 20 missense (MIT, A31V, H46R, Y86C, L89P, D92Y, C94Y, A97V, R100T, Y134S, G138R, A143T, S148R, G163V, D170V, C202Y, Y216D, N263S, W287C, and N298S), two nonsense (Q386X, W399X), one splice site mutation (IVS4 + 2T-->C), and eight small exonic insertions or deletions (304del1, 613del9, 777del1, 1057del2, 1074del2, 1077del1, 1212del3, and 1094ins1), which identified exon 7 as a region prone to gene rearrangements. In addition...

‣ Structural organization of the human alpha-galactosidase A gene: further evidence for the absence of a 3' untranslated region.

Bishop, D F; Kornreich, R; Desnick, R J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/1988 Português
Relevância na Pesquisa
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Human alpha-galactosidase A (alpha-D-galactoside galactohydrolase; EC 3.2.1.22) is a lysosomal hydrolase encoded by a gene localized to the chromosomal region Xq22. The deficient activity of this enzyme results in Fabry disease, an X chromosome-linked recessive disorder that leads to premature death in affected males. For studies of the structure and function of alpha-galactosidase A and for characterization of the genetic lesions in families with Fabry disease, the full-length cDNA was isolated, sequenced, and used to screen human genomic libraries. The 1393-base-pair full-length cDNA had a 60-nucleotide 5' untranslated region and encoded a precursor peptide of 429 amino acids including a signal peptide of 31 residues. Three overlapping lambda clones spanning 32 kilobases were identified that contained the entire approximately equal to 12-kilobase chromosomal gene as well as approximately equal to 9 and approximately equal to 11 kilobases of 5' and 3' flanking sequence, respectively. The gene had seven exons. The genomic exonic and full-length cDNA sequences were identical. All intron-exon splice junctions conformed to the GT/AT consensus sequence. The 5' flanking region of this lysosomal housekeeping gene contained Sp1 and CCAAT box promoter elements as well as sequences corresponding to the activator protein 1 (AP1)...

‣ Uneven X inactivation in a female monozygotic twin pair with Fabry disease and discordant expression of a novel mutation in the alpha-galactosidase A gene.

Redonnet-Vernhet, I; Ploos van Amstel, J K; Jansen, R P; Wevers, R A; Salvayre, R; Levade, T
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1996 Português
Relevância na Pesquisa
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We describe two female monozygotic (MZ) twins heterozygous for Fabry disease, an X linked disorder resulting from the deficient activity of alpha-galactosidase A. While one of the twins was clinically affected, the other was asymptomatic. Enzymatic assay of alpha-galactosidase in blood leucocytes, skin fibroblasts, Epstein-Barr virus transformed lymphoid cell lines, and hair follicles of the twins and their parents confirmed the heterozygous status of the twins and indicated that Fabry disease had occurred as a result of a de novo mutation. The son of the unaffected twin sister was shown to be hemizygous. Molecular analysis of the alpha-galactosidase A gene permitted the identification of an as yet undescribed point mutation at position 10182 of exon 5 which causes an Asp to Asn substitution at codon 231. Single strand conformation polymorphism (SSCP) analysis again showed the heterozygous status of the twins and a normal pattern in their parents. The basis for the discordant expression of this d novo mutation in the twins was investigated by studying their X inactivation status. Analysis of the inactive X specific methylation at the androgen receptor gene showed unbalanced inactivation in the twins' fibroblasts and in opposite directions. While the maternally derived X chromosome was preferentially active in the asymptomatic twin...

‣ Overexpression of human alpha-galactosidase A results in its intracellular aggregation, crystallization in lysosomes, and selective secretion

Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 01/12/1992 Português
Relevância na Pesquisa
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Human lysosomal alpha-galactosidase A (alpha-Gal A) was stably overexpressed in CHO cells and its biosynthesis and targeting were investigated. Clone AGA5.3-1000Mx, which was the highest enzyme overexpressor, produced intracellular alpha-Gal A levels of 20,900 U/mg (approximately 100 micrograms of enzyme/10(7) cells) and secreted approximately 13,000 U (or 75 micrograms/10(7) cells) per day. Ultrastructural examination of these cells revealed numerous 0.25-1.5 microns crystalline structures in dilated trans-Golgi network (TGN) and in lysosomes which stained with immunogold particles using affinity- purified anti-human alpha-Gal A antibodies. Pulse-chase studies revealed that approximately 65% of the total enzyme synthesized was secreted, while endogenous CHO lysosomal enzymes were not, indicating that the alpha-Gal A secretion was specific. The recombinant intracellular and secreted enzyme forms were normally processed and phosphorylated; the secreted enzyme had mannose-6-phosphate moieties and bound the immobilized 215-kD mannose-6-phosphate receptor (M6PR). Thus, the overexpressed enzyme's selective secretion did not result from oversaturation of the M6PR-mediated pathway or abnormal binding to the M6PR. Of note, the secreted alpha-Gal A was sulfated and the percent of enzyme sulfation decreased with increasing amplification...

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

Calhoun, D H; Bishop, D F; Bernstein, H S; Quinn, M; Hantzopoulos, P; Desnick, R J
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1985 Português
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Fabry disease is an X-linked inborn error of metabolism resulting from the deficient activity of the lysosomal hydrolase, alpha-galactosidase A (alpha-Gal A; alpha-D-galactoside galactohydrolase, EC 3.2.1.22). To investigate the structure, organization, and expression of alpha-Gal A, as well as the nature of mutations in Fabry disease, a clone encoding human alpha-Gal A was isolated from a lambda gt11 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 alpha-Gal A internal tryptic peptide isolated by reversed-phase HPLC. Four positive clones were initially identified by antibody screening of 1.4 X 10(7) plaques. Of these, only one clone (designated lambda AG18) 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 of clone lambda AG18 revealed an exact correspondence between the predicted and known amino-terminal amino acid sequence. The insert of clone lambda AG18 appears to contain the full-length coding region of the processed...

‣ Strategies for the production of difficult-to-express full-length eukaryotic proteins using microbial cell factories : production of human alpha-galactosidase A.

Unzueta Elorza, Ugutz; Vázquez Lima, Felícitas; Accardi, Giulia; Mendoza, Rosa; Toledo-Rubio, Verónica; Giuliani, Maria; Sannino, Filomena; Parrilli, Ermenegilda; Abasolo, Ibane; Schwartz, Simo; Villaverde Corrales, Antonio; Corchero Nieto, José Luis;
Fonte: Universidade Autônoma de Barcelona Publicador: Universidade Autônoma de Barcelona
Tipo: info:eu-repo/semantics/article; info:eu-repo/semantics/acceptedVersion
Publicado em //2015 Português
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This work was supported by ERANET-IB08-007 project from the European Union and its linked national project EUI2008- 03610 to AV. We also appreciate the support from EME2007-08 to NFM from Universitat Autonoma de Barcelona, from Antartide 2010 to MLT and EP, from MIUR Azioni Integrate Italia-Spagna 2010 Prot. IT10LECLM9 to MLT, from MINECO (IT2009-0021) to AV and LT, from AGAUR (2009SGR-108) to AV. AV is also supported by The Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN, Spain), an initiative funded by the VI National R&D&i Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. PS has received predoctoral fellowship from ISCIII, and AV has been distinguished with an ICREA ACADEMIA award (Catalonia, Spain).; Obtaining high levels of pure proteins remains the main bottleneck of many scientific and biotechnological studies. Among all the available recombinant expression systems, Escherichia coli facilitates gene expression by its relative simplicity, inexpensive and fast cultivation, well-known genetics and the large number of tools available for its biotechnological application. However...