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

PARODI, AJ; ORSI, R.; GALLO, GL; D'ALESSIO, C

Villa General Belgrano, Córdoba

Congreso; 2nd Argentinean Symposyum on Glycobiology; 2016

N-linked glycosylation is the most frequent protein postraslationalmodification occurring in the endoplasmic reticulum (ER). It is catalyzed by theoligosaccharyltransferase complex (OST) which transfers a preassembled glycan (Glc3Man9GlcNAc2)from the dolichol-PP donor to the NXS/T sequon of proteins. Mutations affectingthe biosynthesis of the glycan or the OST itself may result in proteinhypoglycosylation (sub-ocupation of N-glycosylationsites), thus causing multisystemic human defects known as congenital disordersof 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 afluorescence loss (Gly-GFP). We integrated GFP and Gly-GFP variants in S. pombe genomes of wild type and mutantsand expressed them in the ER by fusing it to a N-terminal signal peptide and toa C-terminal ER retention signal. We quantified fluorescence in each strain byflow cytometry. Our results showed that while GFP fluoresces in the ER of allstrains, Gly-GFP fluorescence is dependent on the hypoglycosylation degree ofeach strain.  The mutant which transfersthe glycan Man9 shows the highest fluorescence level, indicatingthat the absence of glucoses in the glycan during N-glycosylation affects its recognition by OST and hence itstransfer efficiency from the dolichol-PP to the protein.