Glycomic Characterization of Induced Pluripotent Stem Cells Derived from a Patient Suffering from Phosphomannomutase 2 Congenital Disorder of Glycosylation (PMM2-CDG)

• Published in: Molecular & cellular proteomics 2016-04, Vol.15 (4), p.1435-1452
• Main Authors: Thiesler, Christina T., Cajic, Samanta, Hoffmann, Dirk, Thiel, Christian, van Diepen, Laura, Hennig, René, Sgodda, Malte, Weiβmann, Robert, Reichl, Udo, Steinemann, Doris, Diekmann, Ulf, Huber, Nicolas M.B., Oberbeck, Astrid, Cantz, Tobias, Kuss, Andreas W., Körner, Christian, Schambach, Axel, Rapp, Erdmann, Buettner, Falk F.R.
• Format: Article
• Language: English
• Subjects: Cells, Cultured; Congenital Disorders of Glycosylation - metabolism; Congenital Disorders of Glycosylation - pathology; Gene Expression Profiling - methods; Glycomics - methods; Glycosylation; High-Throughput Nucleotide Sequencing - methods; Humans; Induced Pluripotent Stem Cells - metabolism; Induced Pluripotent Stem Cells - pathology; Models, Biological; Phosphotransferases (Phosphomutases) - deficiency; Phosphotransferases (Phosphomutases) - metabolism; Polysaccharides - metabolism; Technological Innovation and Resources
• ISSN: 1535-9476; 1535-9484
• DOI: 10.1074/mcp.M115.054122

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 and in vitro differentiation 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 iPSCs in vitro. Knock-down of PMM2 by 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.