Altered molecular size of N-acetylglucosamine 1-phosphotransferase in I-cell disease and pseudo-Hurler polydystrophy.
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ABSTRACT:
SUBMITTER: Ben-Yoseph Y
PROVIDER: S-EPMC1148605 | biostudies-other | 1987 Dec
REPOSITORIES: biostudies-other
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Project description:The solventogenic clostridia have a considerable capacity to ferment carbohydrate substrates with the production of acetone and butanol, making them attractive organisms for the conversion of waste materials to valuable products. In common with other anaerobes, the clostridia show a marked dependence on the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) to accumulate sugars and sugar derivatives. In this study, we demonstrate that extracts of Clostridium beijerinckii grown on N-acetylglucosamine (GlcNAc) exhibit PTS activity for the amino sugar. The PTS encoded by the divergent genes cbe4532 (encoding the IIC and IIB domains) and cbe4533 (encoding a IIA domain) was shown to transport and phosphorylate GlcNAc and also glucose. When the genes were recombined in series under the control of the lac promoter in pUC18 and transformed into a phosphotransferase mutant (nagE) of Escherichia coli lacking GlcNAc PTS activity, the ability to take up and ferment GlcNAc was restored, and extracts of the transformant showed PEP-dependent phosphorylation of GlcNAc. The gene products also complemented an E. coli mutant lacking glucose PTS activity but were unable to complement the same strain for PTS-dependent mannose utilization. Both GlcNAc and glucose induced the expression of cbe4532 and cbe4533 in C. beijerinckii, and consistent with this observation, extracts of cells grown on glucose exhibited PTS activity for GlcNAc, and glucose did not strongly repress utilization of GlcNAc by growing cells. On the basis of the phylogeny and function of the encoded PTS, we propose that the genes cbe4532 and cbe4533 should be designated nagE and nagF, respectively.
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Project description:Human lymphoblast and fibroblast cell lines from a patient with I-cell disease and normal individuals were characterized with respect to certain properties of UDP-N-acetylglucosamine:lysosomal enzyme precursor N-acetylglucosamine phosphotransferase. The enzyme isolated from normal lymphoblast and fibroblast cell lines expressed similar kinetic properties, substrate specificities and subcellular localizations. Coincident with the severe reduction of N-acetylglucosamine phosphotransferase activity in both I-cell fibroblast and lymphoblast cell lines, there was an increased secretion of several lysosomal enzymes compared to normal controls. Subsequent examination of N-acetyl-beta-D-hexosaminidase secreted by the I-cell lymphoblasts demonstrated a significant increase in adsorption of the I-cell enzyme to Ricinus communis agglutinin, a galactose-specific lectin. However, the I-cell lymphoblasts did not exhibit the significant decrease in intracellular lysosomal activities seen in I-cell fibroblasts. Our results suggest that lymphoblasts not only represent an excellent source for the purification of N-acetylglucosamine phosphotransferase, but in addition, represent a unique system for studying alternate mechanisms involved in the targeting of lysosomal enzymes.
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Project description:Mucolipidosis II (MLII; I-cell disease) and mucolipidosis IIIA (MLIIIA; classical pseudo-Hurler polydystrophy) are diseases in which the activity of the uridine diphosphate (UDP)-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) is absent or reduced, respectively. In the absence of mannose phosphorylation, trafficking of lysosomal hydrolases to the lysosome is impaired. In these diseases, mistargeted lysosomal hydrolases are secreted into the blood, resulting in lysosomal deficiency of many hydrolases and a storage-disease phenotype. To determine whether these diseases are caused by mutations in the GlcNAc-phosphotransferase alpha / beta -subunits precursor gene (GNPTAB), we sequenced GNPTAB exons and flanking intronic sequences and measured GlcNAc-phosphotransferase activity in patient fibroblasts. We identified 15 different mutations in GNPTAB from 18 pedigrees with MLII or MLIIIA and demonstrated that these two diseases are allelic. Mutations in both alleles were identified in each case, which demonstrated that GNPTAB mutations are the cause of both diseases. Some pedigrees had identical mutations. One frameshift mutation (truncation at amino acid 1171) predominated and was found in both MLII and MLIIIA. This mutation was found in combination with severe mutations (i.e., mutations preventing the generation of active enzyme) in MLII and with mild mutations (i.e., mutations allowing the generation of active enzyme) in MLIIIA. Some cases of MLII and MLIIIA were the result of mutations that cause aberrant splicing. Substitutions were inside the invariant splice-site sequence in MLII and were outside it in MLIIIA. When the mutations were analyzed along with GlcNAc-phosphotransferase activity, it was possible to confidently distinguish these two clinically related but distinct diseases. We propose criteria for distinguishing these two disorders by a combination of mutation detection and GlcNAc-phosphotransferase activity determination.
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Project description:UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase is an alpha(2)beta(2)gamma(2) hexamer that mediates the first step in the synthesis of the mannose 6-phosphate recognition marker on lysosomal acid hydrolases. Using a multifaceted approach, including analysis of acid hydrolase phosphorylation in mice and fibroblasts lacking the gamma subunit along with kinetic studies of recombinant alpha(2)beta(2)gamma(2) and alpha(2)beta(2) forms of the transferase, we have explored the function of the alpha/beta and gamma subunits. The findings demonstrate that the alpha/beta subunits recognize the protein determinant of acid hydrolases in addition to mediating the catalytic function of the transferase. In mouse brain, the alpha/beta subunits phosphorylate about one-third of the acid hydrolases at close to wild-type levels but require the gamma subunit for optimal phosphorylation of the rest of the acid hydrolases. In addition to enhancing the activity of the alpha/beta subunits toward a subset of the acid hydrolases, the gamma subunit facilitates the addition of the second GlcNAc-P to high mannose oligosaccharides of these substrates. We postulate that the mannose 6-phosphate receptor homology domain of the gamma subunit binds and presents the high mannose glycans of the acceptor to the alpha/beta catalytic site in a favorable manner.
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Project description:Despite decades of research into the aetiology and pathophysiology of schizophrenia, our understanding of this devastating disorder remains incomplete, with adverse consequences for both diagnosis and treatment. Here we investigate whether differences between patients and controls can be observed in peripheral patient tissue, with a view of establishing a means for dynamic investigations into cell function. In vitro stimulation of peripheral blood CD3+ pan T cells with anti-CD3 (clone OKT3) was used to investigate disease-associated cell responses. T cells from both medicated (n = 39), unmedicated (n = 6) and minimally medicated (n = 5) schizophrenia patients were found to have significantly lower proliferative responses to stimulation, compared to well-matched controls (n = 32). Expression of CD3 and TCR (T cell receptor) alphabeta chains was equivalent between patients and controls, ensuring equal stimulation with anti-CD3, and there was no significant difference in the proportions of CD4+ and CD8+ T cells between samples (n = 12). Lower T cell proliferation in schizophrenia patients was not found to result from deficient early tyrosine phosphorylation signalling or lower IL-2 (interleukin-2) production, as these parameters were similar between patients and controls, as was the expression of CD25, the IL-2 receptor alpha chain. Analysis of CD45 isoforms, however, revealed that patients had a significantly greater percentage of CD8+ and CD4+ CD45RA+ cells before stimulation and significantly higher fluorescence intensity of CD45RA on CD4+ and CD8+ cells before and after stimulation. There was significantly higher expression of CD45 RB on both CD4+ and CD8+ unstimulated cells, with a trend towards lower numbers of CD45RO+ T cells in patient blood. Gene expression analysis in freshly isolated T cells from six minimally treated or first onset patients and six controls was carried out using human whole-genome CodeLink microarrays to identify functional pathways that may affect the ability of patient cells to respond to stimulation. Functional profiling showed prominent transcript changes in categories pertaining to cell cycle machinery, intracellular signalling, oxidative stress and metabolism. Intriguingly, chromosomal location analysis of genes significantly altered between schizophrenia and controls revealed clusters at 1p36, 1q42 and 6p22, which have previously been identified as strong susceptibility loci for schizophrenia.
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Project description:Huntington disease is a fatal neurodegenerative disorder caused by a CAG repeat expansion in the gene encoding the huntingtin protein. Expression of the mutant protein disrupts various intracellular pathways and impairs overall cell function. In particular striatal neurons seem to be most vulnerable to mutant huntingtin-related changes. A well-known and commonly used model to study molecular aspects of Huntington disease are the striatum-derived STHdh cell lines generated from wild type and huntingtin knock-in mouse embryos. However, obvious morphological differences between wild type and mutant cell lines exist, which have rarely been described and might not have always been considered when designing experiments or interpreting results. Here, we demonstrate that STHdh cell lines display differences in cell size, proliferation rate and chromosomal content. While the chromosomal divergence is considered to be a result of the cells' tumour characteristics, differences in size and proliferation, however, were confirmed in a second non-immortalized Huntington disease cell model. Importantly, our results further suggest that the reported phenotypes can confound other study outcomes and lead to false conclusions. Thus, careful experimental design and data analysis are advised when using these cell models.
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2023-03-05 | GSE148434 | GEO