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          Institute: MPI für molekulare Biomedizin     Collection: Jahrbuch 2017 (publ. 2016, arch)     Display Documents



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ID: 732091.0, MPI für molekulare Biomedizin / Jahrbuch 2017 (publ. 2016, arch)
Glycomic Characterization of Induced Pluripotent Stem Cells Derived from a Patient Suffering from Phosphomannomutase 2 Congenital Disorder of Glycosylation (PMM2-CDG)
Authors:Thiesler, C. T.; Cajic, S.; Hoffmann, D.; Thiel, C.; van Diepen, L.; Hennig, R.; Sgodda, M.; Weibetamann, R.; Reichl, U.; Steinemann, D.; Diekmann, U.; Huber, N. M.; Oberbeck, A.; Cantz, T.; Kuss, A. W.; Korner, C.; Schambach, A.; Rapp, E.; Buettner, F. F.
Date of Publication (YYYY-MM-DD):2016-04
Title of Journal:Mol Cell Proteomics
Volume:15
Issue / Number:4
Start Page:1435
End Page:1452
Review Status:Internal review
Audience:Not Specified
Abstract / Description:PMM2-CDG, formerly known as congenital disorder of glycosylation-Ia (CDG-Ia), is caused by mutations in the gene encoding phosphomannomutase 2 (PMM2). This disease is the most frequent form of inherited CDG-diseases affecting protein N-glycosylation in human. PMM2-CDG is a multisystemic disease with severe psychomotor and mental retardation. In order to study the pathophysiology of PMM2-CDG in a human cell culture model, we generated induced pluripotent stem cells (iPSCs) from fibroblasts of a PMM2-CDG-patient (PMM2-iPSCs). Expression of pluripotency factors andin vitrodifferentiation into cell types of the three germ layers was unaffected in the analyzed clone PMM2-iPSC-C3 compared with nondiseased human pluripotent stem cells (hPSCs), revealing no broader influence of the PMM2 mutation on pluripotency in cell culture. Analysis of gene expression by deep-sequencing did not show obvious differences in the transcriptome between PMM2-iPSC-C3 and nondiseased hPSCs. By multiplexed capillary gel electrophoresis coupled to laser induced fluorescence detection (xCGE-LIF) we could show that PMM2-iPSC-C3 exhibit the common hPSC N-glycosylation pattern with high-mannose-type N-glycans as the predominant species. However, phosphomannomutase activity of PMM2-iPSC-C3 was 27% compared with control hPSCs and lectin staining revealed an overall reduced protein glycosylation. In addition, quantitative assessment of N-glycosylation by xCGE-LIF showed an up to 40% reduction of high-mannose-type N-glycans in PMM2-iPSC-C3, which was in concordance to the observed reduction of the Glc3Man9GlcNAc2 lipid-linked oligosaccharide compared with control hPSCs. Thus we could model the PMM2-CDG disease phenotype of hypoglycosylation with patient derived iPSCsin vitro Knock-down ofPMM2by shRNA in PMM2-iPSC-C3 led to a residual activity of 5% and to a further reduction of the level of N-glycosylation. Taken together we have developed human stem cell-based cell culture models with stepwise reduced levels of N-glycosylation now enabling to study the role of N-glycosylation during early human development.
Free Keywords:Cells, Cultured; Congenital Disorders of Glycosylation/metabolism/*pathology; Gene Expression Profiling/methods; Glycomics/*methods; Glycosylation; High-Throughput Nucleotide Sequencing/methods; Humans; Induced Pluripotent Stem Cells/*metabolism/pathology; *Models, Biological; Phosphotransferases (Phosphomutases)/*deficiency/metabolism; Polysaccharides/metabolism
External Publication Status:published
Document Type:Article
Communicated by:Jeanine Müller-Keuker
Affiliations:MPI für molekulare Biomedizin
External Affiliations:paragraph signMax Planck Institute for Dynamics of Complex Technical Systems, 39106 Magdeburg, Germany; From the double daggerREBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany; ||Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany; **Center for Child and Adolescent Medicine, Department Kinderheilkunde I, 69120 Heidelberg, Germany; double daggerdouble daggerDepartment of Human Genetics, University Medicine Greifswald and Interfaculty Institute for Genetics and Functional Genomics, Ernst-Moritz-Arndt University, 17475 Greifswald, Germany; paragraph signMax Planck Institute for Dynamics of Complex Technical Systems, 39106 Magdeburg, Germany; section sign section signglyXera GmbH, 39120 Magdeburg, Germany; From the double daggerREBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany; paragraph sign paragraph signTranslational Hepatology and Stem Cell Biology, Dept. of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, 30625 Hannover, Germany; From the double daggerREBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany; || ||Institute of Human Genetics, Hannover Medical School, 30625 Hannover, Germany; Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany. From the double daggerREBIRTH-Cluster of Excellence, Hannover Medical School, 30625 Hannover, Germany; section signInstitute for Cellular Chemistry, Hannover Medical School, 30625 Hannover, Germany; buettner.falk@mh-hannover.de.
Identifiers:ISSN:1535-9484 (Electronic) 1535-9476 (Linking) %R 10.1... [ID No:1]
URL:https://www.ncbi.nlm.nih.gov/pubmed/26785728 [ID No:2]
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