Characterization of the human lysosomal alpha-glucosidase gene.
Ontology highlight
ABSTRACT:
SUBMITTER: Hoefsloot LH
PROVIDER: S-EPMC1149727 | biostudies-other | 1990 Dec
REPOSITORIES: biostudies-other
Similar Datasets
Project description:N-linked glycosylation is one of the important events in the post-translational modification of human lysosomal alpha-glucosidase. Phosphorylation of mannose residues ensures efficient transport of the enzyme to the lysosomes via the mannose 6-phosphate receptor. The primary structure of lysosomal alpha-glucosidase, as deduced from the cDNA sequence, indicates that there are seven potential glycosylation sites. We have eliminated these sites individually by site-directed mutagenesis and thereby demonstrated that all seven sites are glycosylated. The sites at Asn-882 and Asn-925 were found to be located in a C-terminal propeptide which is cleaved off during maturation. Evidence is presented that at least two of the oligosaccharide side chains of human lysosomal alpha-glucosidase are phosphorylated. Elimination of six of the seven sites does not disturb enzyme synthesis or function. However, removal of the second glycosylation site at Asn-233 interferes dramatically with the formation of mature enzyme. The mutant precursor is synthesized normally and assembles in the endoplasmic reticulum, but immunoelectron microscopy reveals a deficiency of alpha-glucosidase in the Golgi complex and in the more distal compartments of the lysosomal transport pathway.
| S-EPMC1132229 | biostudies-other
Project description:Pompe disease is an inherited metabolic disorder due to the deficiency of the lysosomal acid α-glucosidase (GAA). The only approved treatment is enzyme replacement therapy with the recombinant enzyme (rhGAA). Further approaches like pharmacological chaperone therapy, based on the stabilising effect induced by small molecules on the target enzyme, could be a promising strategy. However, most known chaperones could be limited by their potential inhibitory effects on patient's enzymes. Here we report on the discovery of novel chaperones for rhGAA, L- and D-carnitine, and the related compound acetyl-D-carnitine. These drugs stabilise the enzyme at pH and temperature without inhibiting the activity and acted synergistically with active-site directed pharmacological chaperones. Remarkably, they enhanced by 4-fold the acid α-glucosidase activity in fibroblasts from three Pompe patients with added rhGAA. This synergistic effect of L-carnitine and rhGAA has the potential to be translated into improved therapeutic efficacy of ERT in Pompe disease.
| S-EPMC8477953 | biostudies-literature
Project description:Previously isolated lysosomal alpha-glucosidase cDNA clones were ligated to full-length constructs for expression in vitro and in mammalian cells. One of these constructs (pSHAG1) did not code for functional enzyme, due to an arginine residue instead of a tryptophan residue at amino acid position 402. The mutation does not affect the rate of enzyme synthesis, but interferes with post-translational modification and intracellular transport of the acid alpha-glucosidase precursor. Using immunocytochemistry it is demonstrated that the mutant precursor traverses the endoplasmic reticulum and the Golgi complex, but does not reach the lysosomes. Pulse-chase experiments suggest premature degradation. The Trp-402-containing enzyme (encoded by construct pSHAG2) is processed properly, and has catalytic activity. A fraction of the enzyme is localized at the plasma membrane. It is hypothesized that membrane association of the acid alpha-glucosidase precursor, as demonstrated by Triton X-114 phase separation, is responsible for transport to this location. Transiently expressed acid alpha-glucosidase also enters the secretory pathway, since a catalytically active precursor is found in the culture medium. This precursor has the appropriate characteristics for use in enzyme replacement therapy. Efficient uptake via the mannose 6-phosphate receptor results in degradation of lysosomal glycogen in cultured fibroblasts and muscle cells from patients with glycogenosis type II.
| S-EPMC1149726 | biostudies-other
Project description:Acidic hot springs are colonized by a diversity of hyperthermophilic organisms requiring extremes of temperature and pH for growth. To clarify how carbohydrates are consumed in such locations, the structural gene (malA) encoding the major soluble alpha-glucosidase (maltase) and flanking sequences from Sulfolobus solfataricus were cloned and characterized. This is the first report of an alpha-glucosidase gene from the archaeal domain. malA is 2,083 bp and encodes a protein of 693 amino acids with a calculated mass of 80.5 kDa. It is flanked on the 5' side by an unusual 1-kb intergenic region. Northern blot analysis of the malA region identified transcripts for malA and an upstream open reading frame located 5' to the 1-kb intergenic region. The malA transcription start site was located by primer extension analysis to a guanine residue 8 bp 5' of the malA start codon. Gel mobility shift analysis of the malA promoter region suggests that sequences 3' to position -33, including a consensus archaeal TATA box, play an essential role in malA expression. malA homologs were detected by Southern blot analysis in other S. solfataricus strains and in Sulfolobus shibatae, while no homologs were evident in Sulfolobus acidocaldarius, lending further support to the proposed revision of the genus Sulfolobus. Phylogenetic analyses indicate that the closest S. solfataricus alpha-glucosidase homologs are of mammalian origin. Characterization of the recombinant enzyme purified from Escherichia coli revealed differences from the natural enzyme in thermostability and electrophoretic behavior. Glycogen is a substrate for the recombinant enzyme. Unlike maltose hydrolysis, glycogen hydrolysis is optimal at the intracellular pH of the organism. These results indicate a unique role for the S. solfataricus alpha-glucosidase in carbohydrate metabolism.
| S-EPMC107019 | biostudies-literature
Project description:This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.
| S-EPMC3687018 | biostudies-literature
Project description:Deoxynojirimycin (DNJ) is the archetypal iminosugar, in which the configuration of the hydroxyl groups in the piperidine ring truly mimic those of d-glucopyranose; DNJ and derivatives have beneficial effects as therapeutic agents, such as anti-diabetic and antiviral agents, and pharmacological chaperones for genetic disorders, because they have been shown to inhibit α-glucosidases from various sources. However, attempts to design a better molecule based solely on structural similarity cannot produce selectivity between α-glucosidases that are localized in multiple organs and tissues, because the differences of each sugar-recognition site are very subtle. In this study, we provide the first example of a design strategy for selective lysosomal acid α-glucosidase (GAA) inhibitors focusing on the alkyl chain storage site. Our design of α-1-C-heptyl-1,4-dideoxy-1,4-imino-l-arabinitol (LAB) produced a potent inhibitor of the GAA, with an IC50 value of 0.44 µM. It displayed a remarkable selectivity toward GAA (selectivity index value of 168.2). A molecular dynamic simulation study revealed that the ligand-binding conformation stability gradually improved with increasing length of the α-1-C-alkyl chain. It is noteworthy that α-1-C-heptyl-LAB formed clearly different interactions from DNJ and had favored hydrophobic interactions with Trp481, Phe525, and Met519 at the alkyl chain storage pocket of GAA. Moreover, a molecular docking study revealed that endoplasmic reticulum (ER) α-glucosidase II does not have enough space to accommodate these alkyl chains. Therefore, the design strategy focusing on the shape and acceptability of long alkyl chain at each α-glucosidase may lead to the creation of more selective and practically useful inhibitors.
| S-EPMC7357040 | biostudies-literature
Project description:The 1,4-alpha-glucosidase inhibitor. Acarbose, when injected intraperitoneally disturbs liver lysosome metabolism, causing distinct and persistent inhibition of the enzymes and acute disturbances of lysosomal glycogen metabolism. A feedback control mechanism appears to operate, affecting cytosolic carbohydrate metabolism. A model is suggested for the adult form of lysosomal storage disease. The biochemical effects closely resemble those occurring in glycogenosis type II (Pompe's disease), and these have been confirmed by electron microscopy.
| S-EPMC1144989 | biostudies-other
Project description:Lysosomal degradation plays a crucial role in the metabolism of biological macromolecules supplied by autophagy. The regulation of the autophagy-lysosome system, which contributes to intracellular homeostasis, chemoresistance, and tumor progression, has recently been revealed as a promising therapeutic approach for pancreatic cancer (PC). However, the details of lysosomal catabolic function in PC cells have not been fully elucidated. In this study, we show evidence that suppression of acid alpha-glucosidase (GAA), one of the lysosomal enzymes, improves chemosensitivity and exerts apoptotic effects on PC cells through the disturbance of expression of the transcription factor EB. The levels of lysosomal enzyme were elevated by gemcitabine in PC cells. In particular, the levels of GAA were responsive to gemcitabine in a dose-dependent and time-dependent manner. Small interfering RNA against the GAA gene (siGAA) suppressed cell proliferation and promoted apoptosis in gemcitabine-treated PC cells. In untreated PC cells, we observed accumulation of depolarized mitochondria. Gene therapy using adenoviral vectors carrying shRNA against the GAA gene increased the number of apoptotic cells and decreased the tumor growth in xenograft model mice. These results indicate that GAA is one of the key targets to improve the efficacy of gemcitabine and develop novel therapies for PC.
| S-EPMC8177769 | biostudies-literature
Project description:In humans and rodents, the lysosomal catabolism of core Man(3)GlcNAc(2) N-glycan structures is catalyzed by the concerted action of several exoglycosidases, including a broad specificity lysosomal alpha-mannosidase (LysMan), core-specific alpha1,6-mannosidase, beta-mannosidase, and cleavage at the reducing terminus by a di-N-acetylchitobiase. We describe here the first cloning, expression, purification, and characterization of a novel human glycosylhydrolase family 38 alpha-mannosidase with catalytic characteristics similar to those established previously for the core-specific alpha1,6-mannosidase (acidic pH optimum, inhibition by swainsonine and 1,4-dideoxy-1,4-imino-d-mannitol, and stimulation by Co(2+) and Zn(2+)). Substrate specificity studies comparing the novel human alpha-mannosidase with human LysMan revealed that the former enzyme efficiently cleaved only the alpha1-6mannose residue from Man(3)GlcNAc but not Man(3)GlcNAc(2) or other larger high mannose oligosaccharides, indicating a requirement for chitobiase action before alpha1,6-mannosidase activity. In contrast, LysMan cleaved all of the alpha-linked mannose residues from high mannose oligosaccharides except the core alpha1-6mannose residue. alpha1,6-Mannosidase transcripts were ubiquitously expressed in human tissues, and expressed sequence tag searches identified homologous sequences in murine, porcine, and canine databases. No expressed sequence tags were identified for bovine alpha1,6-mannosidase, despite the identification of two sequence homologs in the bovine genome. The lack of conservation in 5'-flanking sequences for the bovine alpha1,6-mannosidase genes may lead to defective transcription similar to transcription defects in the bovine chitobiase gene. These results suggest that the chitobiase and alpha1,6-mannosidase function in tandem for mammalian lysosomal N-glycan catabolism.
| S-EPMC1351102 | biostudies-literature
Project description:Lysosomal degradation plays a crucial role in the metabolism of biological macromolecules supplied by autophagy. Recent evidence demonstrated the regulation of the autophagy-lysosome system, which contributes to intracellular homeostasis, chemoresistance, and tumor progression, as a promising therapeutic approach for pancreatic cancer (PC). However, the details of lysosomal catabolic function in PC cells is not fully elucidated. In this study, we show evidence that suppression of acid alpha-glucosidase (GAA), one of the lysosomal enzymes, improves chemosensitivity and exerts apoptotic effects on PC cells through the disturbance of mitophagy via migration of the transcription factor EB. The levels of lysosomal enzyme were elevated by gemcitabine in PC cells. In particular, the levels of GAA were responsive to gemcitabine in a dose- and time-dependent manner. Small interfering RNA against the GAA gene (siGAA) suppressed cell proliferation and promoted apoptosis in gemcitabine-treated PC cells. In untreated PC cells, we observed accumulation of depolarized mitochondria. Gene therapy using adenoviral vector carrying short hairpin RNA against the GAA gene increased the number of apoptotic cells and decreased the tumor growth in xenograft model mice. These results indicate that GAA is one of the key targets to improve the efficacy of gemcitabine and develop novel therapies for PC.
2021-06-28 | GSE153460 | GEO