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

Abstract

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.

• Authors: Christina T Thiesler, Samanta Cajic, Dirk Hoffmann, Christian Thiel, Laura van Diepen, René Hennig, Malte Sgodda, Robert Weiβmann, Udo Reichl, Doris Steinemann, Ulf Diekmann, Nicolas M B Huber, Astrid Oberbeck, Tobias Cantz, Andreas W Kuss, Christian Körner, Axel Schambach, Erdmann Rapp, Falk F R Buettner
• Journal: Molecular & cellular proteomics : MCP (Mol Cell Proteomics) Vol. 15 Issue 4 Pg. 1435-52 (Apr 2016) ISSN: 1535-9484 [Electronic] United States
• PMID: 26785728 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Chemical References

• Polysaccharides
• Phosphotransferases (Phosphomutases)

Topics

• 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)