Hypoglycosylation in fission yeast mutants that reproduce the genetic defects of CDG Type I

GALLO GIOVANNA LUCRECIA; ORSI RAMIRO; PARODI ARMANDO; D'ALESSIO CECILIA

Villa General Belgrano, Córdoba

Simposio; 2nd Argentinean Glycobiology Symposium "GlycoAr 2016"; 2016

Fundación Instituto Leloir, IBYME, CIQUIBIC

N-linked glycosylation is the most frequent protein postraslational modification occurring in the endoplasmic reticulum (ER). It is catalyzed by the oligosaccharyltransferase complex (OST) which transfers a preassembled glycan (Glc3Man9GlcNAc2) from the dolichol-PP donor to the NXS/T sequon of proteins. Mutations affecting the biosynthesis of the glycan or the OST itself may result in protein hypoglycosylation (sub-ocupation of N-glycosylation sites), thus causing multisystemic human defects known as congenital disorders of glycosylation type I. We measured hypoglycosylation in S. pombe mutants which transfer truncated glycans (Glc0-3Man0-9GlcNAc2) to proteins using a GFP reporter bearing an N-glycosylation site that when it is occupied by a glycan causes a fluorescence loss (Gly-GFP). We integrated GFP and Gly-GFP variants in S. pombe genomes of wild type and mutants and expressed them in the ER by fusing it to a N-terminal signal peptide and to a C-terminal ER retention signal. We quantified fluorescence in each strain by flow cytometry. Our results showed that while GFP fluoresces in the ER of all strains, Gly-GFP fluorescence is dependent on the hypoglycosylation degree of each strain. The mutant which transfers the glycan Man9 shows the highest fluorescence level, indicating that the absence of glucoses in the glycan during N-glycosylation affects its recognition by OST and hence its transfer efficiency from the dolichol-PP to the protein.