Desarrollo de herramientas moleculares para determinar hipoglicosilación de proteínas in vivo en levaduras
During N-glycosylation the endoplasmic reticulum (ER) membrane oligosaccharytransferase complex (OST) transfers Glc3Man9GlcNAc2 from a dolichol-PP donor to the sequence Asn-X-Ser/Thr of proteins that are entering into the ER. The addition of a bulky hydrophilic glycan enhances protein folding effici...
Guardado en:
| Autor principal: | |
|---|---|
| Otros Autores: | |
| Formato: | Tesis de maestría acceptedVersion |
| Lenguaje: | Español |
| Publicado: |
Facultad de Farmacia y Bioquímica
2014
|
| Materias: | |
| Acceso en línea: | http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_1228 http://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_1228.dir/1228.PDF |
| Aporte de: |
| Sumario: | During N-glycosylation the endoplasmic reticulum (ER) membrane oligosaccharytransferase complex (OST) transfers Glc3Man9GlcNAc2 from a dolichol-PP donor to the sequence Asn-X-Ser/Thr of proteins that are entering into the ER. The addition of a bulky hydrophilic glycan enhances protein folding efficiency as it reduces aggregation of folding intermediates. Defects in the glycan transfer reaction due either to OST mutations or to truncated glycan structures built in the Dol-PP may result in protein hypoglycosylation (the N-glycosylation sites that are normally occupied with a glycan are empty) thus causing ubiquitous defects and in human diseases known as congenital disorders of glycosylation type I. It has been recently shown that a GFP variant in which an N-glycosylation site was introduced (GlyGFP) loses its fluorescence when such site is occupied by a glycan. We expressed GFP and GlyGFP variants fused to an N-terminal S. pombe signal peptide and a C-terminal ER retention signal (ER-GFP and ER-GlyGFP), both in a wild type and in a ?alg6 mutant strain in which hypoglycosylation occurs. Our results showed that while expressed ER-GFP fluoresces in the ER of both strains, ER-GlyGFP only fluoresces in the ER of ?alg6 mutant, indicating that the fluorescence intensity of this construction may be used to test the glycoprotein hypoglycosylation in vivo in S. pombe. |
|---|