| dc.contributor.advisor | Großhans, Jörg Prof. Dr. | |
| dc.contributor.author | Zhang, Na | |
| dc.date.accessioned | 2022-12-14T16:54:22Z | |
| dc.date.available | 2022-12-22T00:50:09Z | |
| dc.date.issued | 2022-12-14 | |
| dc.identifier.uri | http://resolver.sub.uni-goettingen.de/purl?ediss-11858/14414 | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-9613 | |
| dc.format.extent | 143 Seiten | de |
| dc.language.iso | eng | de |
| dc.rights.uri | http://creativecommons.org/licenses/by/4.0/ | |
| dc.subject.ddc | 570 | de |
| dc.title | Role of N-glycosylation for the function of E-cadherin in tissue morphogenesis | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Bastians, Holger Prof. Dr. | |
| dc.date.examination | 2022-09-27 | de |
| dc.description.abstracteng | E-cadherin is central to the formation, stabilization, and maturation of adherens junctions, which serve to physically link epithelial cells and transmit mechanical pulling forces from the outside to the actomyosin cortex inside of cells. Besides this structural function, E-cadherin complexes mediate mechanical signaling. Mechanical pulling forces are translated into biochemical signals by proteins associated with E-cadherin with multiple mechanisms. E-cadherin complexes and clusters’ activities and biochemical constitution are regulated on multiple levels, including post-translational modifications, interactions with the cortical actomyosin network, membrane trafficking, and cis/trans clustering. Here I will focus on the role of N-glycans in the function of E-cadherin, which has received little attention so far.
I utilized two experimental model systems. (1) The gene xit (xiantuan, Alg8 in Saccharomyces cerevisiae) encodes the glucosyl transferase, which adds the second but last glucose to the dolichol-oligosaccharide precursor during N-glycan synthesis in the endoplasmic reticulum. (2) At the endogenous genetic locus in Drosophila, site-specific Asn->Gln mutations in E-cadherin at N-glycosylation sites are introduced, preventing N-glycan transfer to these sites.
Besides the phenotypes in visible morphology during gastrulation, I analyzed the mobility of E-cadherin by fluorescence recovery after photobleaching (FRAP) and the mechanical properties of junctions by laser ablation and measurement of recoil. The visible spatial distribution and size of E-cadherin clusters were analyzed by high-resolution confocal microscopy. I detected consistent phenotypes in xit and hypoglycosylation mutants of E-cadherin: increased mobility, increased initial recoil velocity, and reduced clustering at junctions, which indicated that the xit phenotype is largely due to hypoglycosylation of E-cadherin in early embryos.
On the molecular level, analysis of the cadherin-catenin complex by coimmunoprecipitation (Co-IP) experiments and an E-cadherin-α-catenin fusion protein revealed an increased stoichiometry of α-catenin to E-cadherin in xit mutant,suggesting that xit and hypoglycosylation impinge on interactions with cortical actomyosin.
To experimentally confirm and identify new sites of N-glycosylation, I conducted a mass spectrometric analysis of E-cadherin isolated from wild-type and xit mutants. To my surprise, I identified sites not predicted yet, sites whose N-glycosylation is dependent, and others not dependent on xit. These findings indicate that lacking terminal glucosyl residues on the dolichol-oligosaccharide precursor alters the specificity and not simply the activity of the oligosaccharide transferase.
In summary, I present evidence that N-glycans specifically modulate E-cadherin activity and function, and constitute a so far neglected molecular mechanism for modulation of E-cadherin function. | de |
| dc.contributor.coReferee | Vorbrüggen, Gerd Dr. | |
| dc.contributor.thirdReferee | Krebber, Heike Prof. Dr. | |
| dc.contributor.thirdReferee | Wolf, Fred | |
| dc.contributor.thirdReferee | Betz, Timo Prof. Dr. | |
| dc.subject.eng | E-cadherin, N-glycosylation, cadherin-catenin complex, germband extension, tissue morphogenesis | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-ediss-14414-6 | |
| dc.affiliation.institute | Biologische Fakultät für Biologie und Psychologie | de |
| dc.subject.gokfull | Biologie (PPN619462639) | de |
| dc.description.embargoed | 2022-12-22 | de |
| dc.identifier.ppn | 1827070757 | |
| dc.notes.confirmationsent | Confirmation sent 2022-12-15T06:15:01 | de |