Development of a molecular tool to measure in vivo protein hypoglycosylation in fission yeasts

GALLO, GL; HERRERA AGUILAR, N; ORSI, R.; MATTERA, V; PARODI, AJ; D'ALESSIO, C

Rosario

Congreso; L reunión anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2014

SAIB

¨ 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 may result in protein hypoglycosylation (lack of glycosylation of sites normally occupied) 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 (Gly-GFP) loses fluorescence when such site is occupied by a glycan. We expressed GFP and Gly-GFP 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 an Dalg6 mutant 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 Dalg6 mutant, indicating that the fluorescence intensity of this construction may be used to test the glycoprotein hypoglycosylation in vivo in S. pombe.