Project Details
Projekt
Characterization of induced pluripotent stem cell-based disease models for congenital disorders of glycosylation
Applicant
Professor Dr. Falk Büttner
Subject Area
Developmental Biology
Biochemistry
Biochemistry
Term
since 2017
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 289991887
Three ER protein glycosylation pathways (N-glycosylation, C-mannosylation, and O-mannosylation) use the donor sugar dolicholphosphate-mannose (Dol-P-Man). Dol-P-Man is synthesized from mannose via Man-6-P, Man-1-P and GDP-Man. Isomerization of Man-6-P to Man-1-P is catalyzed by phosphomannomutase 2 (PMM2). Hypomorphic mutations in PMM2 cause the most frequent form of congenital disorders of glycosylation, PMM2-CDG, a severe multisystemic disorder affecting with high penetrance nervous system and liver. So far defects in N-glycosylation are regarded as causative for PMM2-CDG. Considering, however, that the three pathways merge in their need for Dol-P-Man and also C-mannosylation and O-mannosylation are known to impact folding and functionality of proteins with major roles in development (e.g. cadherins and cytokine receptors are O-mannosylated and C-mannosylated, respectively) it is likely that the PMM2-CDG syndrome combines alterations in all these pathways. We recently established induced pluripotent stem cells from PMM2-CDG fibroblasts (PMM2-iPSCs) and demonstrated alterations in N-glycosylation. Here we suggest using these cells together with controls to comparatively investigate the three Dol-P-Man dependent pathways by applying established as well as specifically adapted protocols in glycoproteomics. With a focus at the organs chiefly affected in PMM2-CDG patients, nervous system and liver, we will perform time laps analyses starting from human pluripotent stem cells (hPSCs) and continuing over different stages during neural and hepatic differentiation. Lacking information on C-mannosyltransferases has long prevented the study of this pathway. We recently cloned the first C-mannosyltransferase from Caenorhabditis elegans (DPY-19) and identified four homologues in mammals (DPY19L1 - DPY19L4), with DPY19L1 showing highest evolutionary conservation. To identify DPY19L1 targets and analyse its functional role in early steps of human development, we plan to establish hPSCs with DPY19L1 disruption. As preliminary data indicate that CRISP-Cas-mediated knock-out of DPY19L1 is lethal in hPSCs, we will follow a strategy that allows inducible knock-down or knock-out of DPY19L1 in hPSCs and after differentiation into neuronal or hepatic linages. Obtained cell models will in particular be used to reenact defects described in literature to be caused by aberrant C-mannosylation e.g. defective migration of glutamatergic neurons as observed upon knock-down of Dpy19L1 in mice. Using both, the available PMM2-CDG and DPY19L1 stem cell models, we will finally address the question if alterations in these pathways reflect at transcriptomic level. The rationale behind this approach bases on the fact that we expect that aberrant glycosylation of cytokines or membrane receptors causes perturbations on gene expression that can be deciphered by bioinformatics analyses of quantitative gene expression data.
DFG Programme
Research Units
