Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.20 (alpha-glucosidase)
4,237 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The substrate analogue conduritol B epoxide (CBE) is demonstrated to be an active site-directed inhibitor of human lysosomal alpha-glucosidase. A competitive mode of inhibition is obtained with glycogen as natural and 4-methylumbelliferyl-alpha-D-glucopyranoside as artificial substrate. The inactivation of the enzyme is time and concentration dependent and results in the covalent binding of CBE. Catalytic activity is required for binding to occur. CBE-labeled peptides containing the catalytic residue of lysosomal alpha-glucosidase were isolated and identified by microsequencing and amino acid analysis. The peptides appeared to originate from a protein domain which is highly conserved among alpha-amylases, maltase, glucoamylases, and transglucanosylases. Based on the sequence similarity and the mechanism of CBE binding, Asp-518 is predicted to be the essential carboxylate in the active site of lysosomal alpha-glucosidase. The functional importance of Asp-518 and other residues around the catalytic site was studied by expression of in vitro mutagenized alpha-glucosidase cDNA in transiently transfected COS cells. Substitution of Asp-513 by Glu-513 is shown to interfere with the posttranslational modification and the intracellular transport of the alpha-glucosidase precursor. The residues Trp-516 and Asp-518 are demonstrated to be critical for catalytic function.
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PMID:Human lysosomal alpha-glucosidase. Characterization of the catalytic site. 185 89

Glycogen storage disease type II (GSDII, Pompe's disease) is caused by an autosomal recessive inheritance of lysosomal alpha-glucosidase deficiency. By sequence analysis we have identified the mutations in the lysosomal alpha-glucosidase gene (GAA) of two unrelated patients, who have one and two copies, respectively, of the same missense mutation. The milder affected adult patient was found to be homozygous for a C1634T transition resulting in the substitution of pro545 by leu. The more severely affected adolescent patient had this same mutant allele combined with a 1 base pair deletion (delta T525) in the second allele causing premature termination at nucleotide positions 658-660. Both these mutations were introduced in wild-type alpha-glucosidase cDNA and expressed in COS-1 cells to analyse their effect. The delta T525 mutation prohibits the formation of lysosomal alpha-glucosidase completely. The pro545-->leu substitution is compatible with normal synthesis but hampers enzyme maturation and results in a 92% net loss of lysosomal alpha-glucosidase activity. The patient with adult GSDII has, in accordance with the allelic constitution, a 2-fold higher residual activity than the patient with juvenile GSDII. The delta T525 deletion was detected in two other unrelated patients, and also the C1634T transition was encountered in two more Caucasian patients with GSDII.
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PMID:The effect of a single base pair deletion (delta T525) and a C1634T missense mutation (pro545leu) on the expression of lysosomal alpha-glucosidase in patients with glycogen storage disease type II. 788 22

Glycogen-storage disease type II (GSDII) is caused by the deficiency of lysosomal alpha-glucosidase (acid maltase). This paper reports on the analysis of the mutant alleles in an American black patient with an adult form of GSDII (GM1935). The lysosomal alpha-glucosidase precursor of this patient has abnormal molecular features: (i) the molecular mass is decreased, (ii) the phosphorylation is deficient and (iii) the proteolytic processing is impaired. Sequence analysis revealed four mutations leading to amino acid alterations: Asp-645-->Glu, Val-816-->Ile, Arg-854-->Stop and Thr-927-->Ile. By using allele-specific oligonucleotide hybridization on PCR-amplified cDNA we have demonstrated that the Arg-854-->Stop mutation is located in one allele that is not expressed, and that the other allele contains the remaining three mutations. Each of the mutations was introduced in wild-type cDNA and expressed in COS cells to analyse the effect on biosynthesis, transport and phosphorylation of lysosomal alpha-glucosidase. The Val-816-->Ile substitution appeared to have no significant effect in contrast with results [Martiniuk, Mehler, Bodkin, Tzall, Hirshhorn, Zhong and Hirschhorn (1991) DNA Cell Biol. 10, 681-687] and was therefore defined as a polymorphism. The Thr-927-->Ile substitution deleting one of the seven glycosylation sites was found to be responsible for the decrease in molecular-mass, but not for the deficient proteolytic processing and phosphorylation. It did not cause the enzyme deficiency either. The third mutation leading to the Asp-645-->Glu substitution was proven to account in full for the observed defects in transport, phosphorylation and proteolytic processing of the newly synthesized alpha-glucosidase precursor of the patient.
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PMID:The conservative substitution Asp-645-->Glu in lysosomal alpha-glucosidase affects transport and phosphorylation of the enzyme in an adult patient with glycogen-storage disease type II. 809 13

Glycogen-storage disease type II, Pompe disease, is caused by the deficiency of acid alpha-D-glucosidase in lysosome. Previously we found that acid alpha-D-glucosidase did exist in the skin fibroblasts and there was also no difference of mRNA in quantity and size of Chinese infantile type Pompe disease patients in Taiwan. However, functional assay of the acid alpha-D-glucosidase of these patients showed its enzyme function to be defective. In the present study, first we identified a substitution site in four Chinese infantile patients with Pompe disease which is a cytidine to adenosine (C1935-->A) transversion at 5' end of exon 14 causing substitution of glutamic acid for aspartic acid at position 645 of the acid alpha-D-glucosidase. This substitution was introduced in wild-type cDNA and expressed in COS-1 cells. The Asp-645-->Glu substitution resulted in significant reduction of acid alpha-D-glucosidase activity. Second, according to the screening data in 25 Chinese Pompe disease patients using digestion of RT-PCR amplified specific fragment with Aat II, the restriction fragment length analysis showed that patients presented the 861 bp band and the normal individuals presented the 728 bp and 133 bp polymorphic bands. We found that the frequency of mutant allele is 0.8 in infantile patients with Chinese Pompe disease and 0 in normal individuals. These results therefore indicate that Asp-645-->Glu mutation results in infantile form of Pompe disease as the major cause in Chinese patients in Taiwan.
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PMID:Molecular study on the infantile form of Pompe disease in Chinese in Taiwan. 893 10

A point mutation in the cDNA of human intestinal sucrase-isomaltase has been recently identified in phenotype II of congenital sucrase-isomaltase deficiency. The mutation results in a substitution of glutamine by proline at position 1098 (Q1098P) in the sucrase subunit. Expression of this mutant sucrase-isomaltase cDNA in COS-1 cells results in an accumulation of sucrase-isomaltase in the ER, intermediate compartment and the cis-Golgi cisternae similar to the accumulation in phenotype II intestinal cells. An interesting feature of the Q1098P substitution is its location in a region of the sucrase subunit that shares striking similarities with the isomaltase subunit and other functionally related enzymes, such as human lysosomal acid alpha-glucosidase and Schwanniomyces occidentalis glucoamylase. We speculated that the Q-->P substitution in these highly conserved regions may result in a comparable accumulation. Here we examined this hypothesis using lysosomal alpha-glucosidase as a reporter gene. Mutagenesis of the glutamine residue at position 244 in the homologous region of alpha-glucosidase to proline results in a protein that is neither transported to the lysosomes nor secreted extracellularly but accumulates in the ER, intermediate compartment and cis-Golgi as a mannose-rich polypeptide similar to mutant sucrase-isomaltase in phenotype II. We propose that the Q1098P and Q244P mutations (in sucrase-isomaltase and alpha-glucosidase, respectively) generate structural alterations that are recognized by a control mechanism, operating beyond the ER in the intermediate compartment or cis-Golgi.
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PMID:A mutation in a highly conserved region in brush-border sucrase-isomaltase and lysosomal alpha-glucosidase results in Golgi retention. 909 38

Acid alpha-glucosidase (GAA) hydrolyzes alpha-1, 4 and alpha-1, 6 glucosidic linkages of oligosaccharides and degrades glycogen in the lysosomes. The full-length GAA I cDNA, pQAM8, was isolated from a cDNA library derived from Japanese quail liver. The cDNA is 3569 base pairs long and has an open reading frame capable of coding 932 amino acids. The deduced amino acid sequence shares 52% identity with human GAA. Transfection of expression vector pETAM8 into COS-7 cells or acid maltase deficient (AMD) quail embryonic fibroblasts increased the level of GAA 20-50-fold. Compared to normal quail, the levels of GAA I mRNA were significantly reduced in the muscle, liver, heart, and brain of AMD quails, suggesting the GAA deficiency in AMD quail is due to a lack of GAA I mRNA. A second GAA II cDNA was identified after probing the cDNA library from the ovarian large follicles of quails with a PCR product derived from cultured quail skin fibroblasts. This clone having 3.1 kb insert, has GAA activity as well (3 to 10 fold increase). This cDNA, designated GAA II, predicted an 873 amino acid polypeptide showing 63% identity to human GAA and 51% identity to the GAA I. The RT-PCR analysis demonstrated that GAA II mRNAs were barely detectable in normal tissues, while they were enhanced to higher levels in AMD tissues. These results suggest that GAA II expression is up-regulated at the transcription levels, and quail GAA gene redundancy performs the same function of satisfying GAA demand at the two different phases represented by normal and AMD.
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PMID:Molecular cloning of acid alpha-glucosidase cDNA of Japanese quail (Coturnix coturnix japonica) and the lack of its mRNA in acid maltase deficient quails. 954 Aug 58

To understand the process of expression of tyrosinase, a key enzyme of melanogenesis, we examined its maturation in the endoplasmic reticulum (ER) by using a heterogeneous expression system. When human tyrosinase cDNA was introduced into COS 7 cells, tyrosinase activity was minimally detected. Immunofluorescence study revealed that tyrosinase was immunolocalized in the nuclear rim, the reticular network, and the punctuated structures. Because a cytoplasmic tail of tyrosinase-gene family protein functions as a lysosomal targeting signal in non-melanocytic cells, and immature and/or misfolded molecules are selectively retained in the ER, the observed localization suggested the inefficient maturation in the COS 7 cells. We thus examined if supplementation of calnexin, a membrane-bound chaperone with affinity for oligosaccharide-processing intermediates containing monoglucose, could improve the process. As expected, the activity was enhanced approximately 2-fold by co-transfection of cDNA encoding calnexin. In contrast, co-transfection of the cytosolic tail-free calnexin, which inhibits calnexin function by allowing premature egress of its ligands from the ER, suppressed expression of this enhanced tyrosinase activity. When alpha-glucosidase activity, which is required for calnexin function, was inhibited by castanospermine (CST) treatment, expression of tyrosinase activity was completely abolished. To confirm the direct involvement of calnexin in tyrosinase maturation, the interaction of calnexin with tyrosinase was examined. Immunoprecipitation of calnexin from extracts of [35S]methionine labeled cells with anti-calnexin antibody revealed that the association is highest immediately after the pulse and that nascent tyrosinase is gradually dissociated upon chase. The association was completely inhibited when CST was included in the medium. Hence, we suggest that the proper folding of tyrosinase is largely dependent on its direct interaction with calnexin for the determined duration in the ER.
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PMID:Promotion of tyrosinase folding in COS 7 cells by calnexin. 988 Aug 1

A six-year-old child presented at 8 months of age with proximal muscle weakness and mild cardiac hypertrophy. Some alpha-glucosidase activity was detected in muscle but not in fibroblasts. As none of the two pathogenic mutations, [c.1933G>A]+[c.2702T>A] (Asp645Asn/Leu901Gln), led to detectable alpha-glucosidase activity upon expression in COS cells, the phenotype of the patient remained unexplained. A functionally comparable set of mutations, Asp645Asn/insGnt2243, was reported previously to cause classic infantile Pompe disease [Biochem Biophys Res Commun 244 (1998) 921]. We conclude that secondary genetic or environmental factors can be decisive for the phenotypic outcome of classic infantile versus childhood Pompe disease, when the acid alpha-glucosidase activity is extremely low.
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PMID:A case of childhood Pompe disease demonstrating phenotypic variability of p.Asp645Asn. 1514 38

Pompe disease is a lysosomal storage disorder (LSD) caused by mutations in the gene that encodes acid alpha-glucosidase (GAA). Recently, small molecule pharmacological chaperones have been shown to increase protein stability and cellular levels for mutant lysosomal enzymes and have emerged as a new therapeutic strategy for the treatment of LSDs. In this study, we characterized the pharmacological chaperone 1-deoxynojirimycin (DNJ) on 76 different mutant forms of GAA identified in Pompe disease. DNJ significantly increased enzyme activity and protein levels for 16 different GAA mutants in patient-derived fibroblasts and in transiently transfected COS-7 cells. Additionally, DNJ increased the processing of these GAA mutants to their mature lysosomal forms, suggesting facilitated trafficking through the secretory pathway. Immunofluorescence microscopy studies showed increased colocalization of GAA with the lysosomal marker LAMP2 after incubation with DNJ, confirming increased lysosomal trafficking. Lastly, a GAA structural model was constructed based on the related eukaryotic glucosidase maltase-glucoamylase. The mutated residues identified in responsive forms of GAA are located throughout most of the structural domains, with half of these residues located in two short regions within the catalytic domain. Taken together, these data support further evaluation of DNJ as a potential treatment for Pompe disease in patients that express responsive forms of GAA.
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PMID:The pharmacological chaperone 1-deoxynojirimycin increases the activity and lysosomal trafficking of multiple mutant forms of acid alpha-glucosidase. 1986 43

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