Debus, Jana Davina; Milting, Hendrik; Brodehl, Andreas; Kassner, Astrid; Anselmetti, Dario; Gummert, Jan; Gaertner-Rommel, Anna: In vitro analysis of arrhythmogenic cardiomyopathy associated desmoglein-2 (DSG2) mutations reveals diverse glycosylation patterns.. In: Journal of molecular and cellular cardiology. Jg.129. 2019, S. 303-333
Inhalt
- Cell culture
- Differentiation of human induced pluripotent stem cells into cardiomyocytes
- Construction of plasmids
- Purification of recombinant DSG2
- Peptide N-glycosidase F (PNGase F) digest
- Sugar staining with Pro-Q® Emerald 300 glycoprotein gel and blot stain
- Lectin blotting
- Transfection of HT1080 cells
- Transfection of hiPSC derived cardiomyocytes
- Detection of secreted DSG2-ECD N-glycosylation mutants
- Tunicamycin treatment
- Immunoblotting
- Confocal microscopy
- Dispase based dissociation assay
- Molecular modeling
- Statistical analyses
- Results
- The recombinantly expressed DSG2-ECD of WT and ARVC associated mutants are differentially modified
- The mutant DSG2-ECD p.D154E is differently N-glycosylated
- Mutant DSG2-ECDs p.K294E and p.V392I display differential lectin binding
- Mutant DSG2-ECD p.D154E is differentially glycosylated at p.N182
- Removal of multiple N-glycosylation sites abolishes the transport of DSG2 to the cell membrane
- Cell-cell adhesion strength is affected by DSG2 mutation
- Discussion
- Conclusions
- Acknowledgements
- Funding
- Supplementary data
- References