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A J Reuser

Publications and source records attributed to A J Reuser.

At least 55 records · Page 3Linked to original sources
PubMed1994

An evaluation of the potential side-effects of alpha-glucosidase inhibitors used for the management of diabetes mellitus.

Orally taken alpha-glucosidase inhibitors are used for the management of diabetes mellitus. These drugs can prevent the postprandial rise of the blood glucose level by inhibiting the enzymatic digestion of carbohydrates in the intestinal lumen. Non-absorbable inhibitors such as acarbose are expected to function exclusively in the intestine, but absorbable inhibitors such as miglitol may exert an inhibitory effect on non-intestinal alpha-glucosidases present in the various cell types of the body. The potential side-effects of absorbable inhibitors are evaluated in this literature review. It is concluded that there is little risk of inducing unwanted side-effects when miglitol is taken in an oral dose of approximately 1 mg kg-1 body weight. The use of absorbable inhibitors is, however, not advised in case of kidney dysfunction.

1-Deoxynojirimycin↗
PubMed1994

Lysosomal storage diseases: cellular pathology, clinical and genetic heterogeneity, therapy.

Lysosomal storage diseases are genetically determined metabolic diseases characterized by dysmorphology and dysfunction of the lysosomal system. The lysosomal pathology can have different causes; these are (i) the deficiency of a lysosomal enzyme or subunit thereof, (ii) the deficiency of a protein assisting one or more lysosomal enzymes in their catalytic function by activation and/or stabilization, or by substrate presentation, (iii) the deficiency or dysfunction of a lysosomal membrane carrier protein essential for the export of degradation products from the lysosomal interior to the cytoplasm or, (iiii) defective targeting of lysosomal proteins to the lysosomes. This excerpt of an oral presentation given at Eurolab 93 starts with a general introduction on lysosomes and lysosomal storage diseases and devotes attention to current issues in this field.

Humans↗
PubMed1993

Characterization and localization of the FMR-1 gene product associated with fragile X syndrome.

The fragile X syndrome is the most frequent form of inherited mental retardation after Down's syndrome, having an incidence of one in 1,250 males. The fragile X syndrome results from amplification of the CGG repeat found in the FMR-1 gene. This CGG repeat shows length variation in normal individuals and is increased significantly in both carriers and patients; it is located 250 base pairs distal to a CpG island which is hypermethylated in fragile X patients. The methylation probably results in downregulation of FMR-1 gene expression. No information can be deduced about the function of the FMR-1 protein from its predicted sequence. Here we investigate the nature and function of the protein encoded by the FMR-1 gene using polyclonal antibodies raised against the predicted amino-acid sequences. Four different protein products, possibly resulting from alternative splicing, have been identified by immunoblotting in lymphoblastoid cell lines of healthy individuals. All these proteins were missing in cell lines from patients not expressing FMR-1 messenger RNA. The intracellular localization of the FMR-1 gene products was investigated by transient expression in COS-1 cells and found to be cytoplasmic. Localization was also predominantly cytoplasmic in the epithelium of the oesophagus, but in some cells was obviously nuclear.

Alternative Splicing↗
PubMed1993

Biochemical genetics of glycogenosis type II in Brahman cattle.

Glycogenosis type II is an inherited lysosomal storage disorder caused by acid alpha-glucosidase deficiency. The disorder is inbred in Brahman cattle, and the incidence of carriers in Australian herds averages 15%. Affected animals are lethargic and die typically in the eighth or ninth month after birth. A complete lack of acid alpha-glucosidase synthesis was demonstrated in cultured fibroblasts and muscle tissue of affected animals. Moreover, the tissue was found to be devoid of acid alpha-glucosidase mRNA. Gross abnormalities of the acid alpha-glucosidase gene itself were not detected by Southern blot analysis. These results suggest Brahman glycogenosis type II to be caused by a point mutation or a micro deletion/insertion in the acid alpha-glucosidase gene.

Animals↗
PubMed1993

Human lysosomal alpha-glucosidase: functional characterization of the glycosylation sites.

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.

Animals↗
PubMed1993

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.

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.

Adult↗
PubMed1993

Structural and functional changes of lysosomal acid alpha-glucosidase during intracellular transport and maturation.

The synthesis and posttranslational modification of lysosomal acid alpha-glucosidase were studied in a cell-free translation system and in mammalian cells transfected with acid alpha-glucosidase cDNA constructs. The newly synthesized precursor, sequestered in the endoplasmic reticulum, was demonstrated to be membrane-bound by lack of signal peptide cleavage, and to be catalytically inactive. Sugar chain modification was shown to occur in the Golgi complex and to be dependent on the rate of transport. From the trans-Golgi network different routes were found to be followed by acid alpha-glucosidase. A fraction of precursor molecules, proteolytically released from the membrane anchor, appeared to enter the secretory pathway and was recovered from the cell culture medium in a catalytically active form. A second fraction was transported to the lysosomes and was trimmed in a stepwise process at both the amino- and carboxyl-terminal ends. The intramolecular cleavage sites were determined. Involvement of thiol proteinases was demonstrated. Specificity for the natural substrate glycogen was gained during the maturation process. The phosphomannosyl receptor is assumed to be instrumental in the lysosomal targeting of acid alpha-glucosidase, but a phosphomannosyl receptor-independent transport of membrane-bound precursor molecules to the lysosomes, either directly or via the plasma membrane, cannot be excluded.

Amino Acid Sequence↗
PubMed1993

Two mutations affecting the transport and maturation of lysosomal alpha-glucosidase in an adult case of glycogen storage disease type II.

The autosomal recessive glycogen storage disease type II is associated with a deficiency of lysosomal alpha-glucosidase (acid maltase). This paper reports on the mutations in the lysosomal alpha-glucosidase alleles of an adult patient. A G-1927 to A transition was discovered in exon 14 causing the substitution of Gly-643 by Arg and a second C-2173 to T transition in exon 15 resulting in the substitution of Arg-725 by Trp. Each of the mutations was located in a different allele. The mutations were introduced in the wild-type lysosomal alpha-glucosidase cDNA and expressed in COS cells. Both mutations had a similar effect. The synthesis of the mutant enzyme precursors was not disturbed but the intracellular transport and maturation were impaired. As a result there was an overall deficiency of catalytic activity.

Adult↗
PubMed1991

Identification of a point mutation in the human lysosomal alpha-glucosidase gene causing infantile glycogenosis type II.

Two patients in a consanguineous Indian family with infantile glycogenosis type II were found to have a G to A transition in exon 11 of the human lysosomal alpha-glucosidase gene. Both patients were homozygous and both parents were heterozygous for the mutant allele. The mutation causes a Glu to Lys substitution at amino acid position 521, just three amino acids downstream from the catalytic site at Asp-518. The mutation was introduced in wild type lysosomal alpha-glucosidase cDNA and the mutant construct was expressed in vitro and in vivo. The Glu to Lys substitution is proven to account for the abnormal physical properties of the patients lysosomal alpha-glucosidase precursor and to prevent the formation of catalytically active enzyme. In homozygous form it leads to the severe infantile phenotype of glycogenosis type II.

Alleles↗
PubMed1991

Human lysosomal alpha-glucosidase. Characterization of the catalytic site.

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.

Amino Acid Sequence↗
PubMed1991

Biosynthesis of human alpha-N-acetylgalactosaminidase: defective phosphorylation and maturation in infantile alpha-NAGA deficiency.

The biosynthesis of human alpha-N-acetylgalactosaminidase (alpha-NAGA) was studied in normal fibroblasts and in cells from patients with infantile alpha-NAGA deficiency. Normal alpha-NAGA is synthesized as a 52 kDa precursor which matures to a 49 kDa species through phosphorylation and carbohydrate triming. Fibroblasts from the patients synthesize normal amounts of a 52 kDa precursor, however phosphorylation does not occur and this precursor is subsequently degraded intracellularly.

Carbohydrate Metabolism, Inborn Errors↗
PubMed1991

A quantitative immunoelectronmicroscopic study on soluble, membrane-associated and membrane-bound lysosomal enzymes in human intestinal epithelial cells.

We have used quantitative immunoelectronmicroscopy to compare the in situ localization of acid alpha-glucosidase, lysosomal acid phosphatase, beta-hexosaminidase and glucocerebrosidase in intestinal epithelial cells of the human duodenum. Differences between these four lysosomal enzymes were observed with respect to their presence at the apical cell surface. Transport to the apical membrane seems to be a more important intracellular route for lysosomal acid phosphatase and acid alpha-glucosidase than it is for beta-hexosaminidase. The membrane associated lysosomal enzyme glucocerebrosidase is not transported to the microvilli. The studies emphasize that lysosomal enzyme transport pathways are enzyme and cell type specific.

Cell Membrane↗
PubMed1991

Intravenous administration of phosphorylated acid alpha-glucosidase leads to uptake of enzyme in heart and skeletal muscle of mice.

The lysosomal storage disorder glycogenosis type II is caused by acid alpha-glucosidase deficiency. In this study we have investigated the possible applicability of mannose 6-phosphate receptor-mediated enzyme replacement therapy to correct the enzyme deficiency in the most affected tissues. Bovine testes acid alpha-glucosidase containing phosphorylated mannose residues was intravenously administered to mice and found to be taken up by heart (70% increase of activity) and skeletal muscle (43% increase); the major target organs. The uptake of nonphosphorylated human placenta acid alpha-glucosidase by heart and skeletal muscle appeared to be significantly less efficient, whereas uptake of dephosphorylated bovine testes enzyme was not detectable. The phosphorylated bovine testes acid alpha-glucosidase remained present in mouse skeletal muscle up to 9-15 d after administration, with a half-life of 2-4 d. Besides being measured in skeletal muscle and heart, uptake of phosphorylated bovine testes and nonphosphorylated human placenta acid alpha-glucosidase was measured in several other organs, but not in brain. The increase of acid alpha-glucosidase activity was highest in liver and spleen. We concluded that application of mannose 6-phosphate receptor-mediated enzyme replacement therapy may offer new perspectives for treatment of glycogenesis type II.

Animals↗
PubMed1990

Expression and routeing of human lysosomal alpha-glucosidase in transiently transfected mammalian cells.

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.

Animals↗
PubMed1990

Characterization of the human lysosomal alpha-glucosidase gene.

The gene coding for human lysosomal alpha-glucosidase was cloned and its structure was determined. The gene is approx. 20 kb long, and contains 20 exons. The first exon is non-coding. The coding sequence of the putative catalytic site domain is interrupted in the middle by an intron of 101 bp. This intron is not conserved in the highly similar region of the human and rabbit isomaltase genes. The promoter region was defined by a CAT assay and the start of the mRNA was determined by primer extension. The promoter has features characteristic of a 'housekeeping' gene. The GC content is high (80%) and distinct TATA and CCAAT motifs are lacking. Two potential binding sites for the AP-2 transcription factor are present. Four potential Sp-1 binding sites are located downstream of the 5' end of the mRNA.

Animals↗