A journey in the early steps of N-glycosylation and glycoprotein folding in the fission yeast secretory pathway

CECILIA D'ALESSIO

Congreso; Congreso conjunto SAIB-SAMIGE; 2021

SAIB-SAMIGE

Congenital Disorders of Glycosylation (CDG) are multisysteminherited human diseases produced by defects in cell glycosylation processes.Most of them are caused by deficiencies in protein N-glycosylation, which consist in the transfer by theoligosaccharyltransferase (OST) of Glc3Man9GlcNAc2pre-assembled as a lipid-linked oligosaccharide (LLO) to Asn residues ofproteins that are entering the endoplasmic reticulum (ER), and the following remodelingof the N-glycan that occurs in thesecretory pathway. We used the fission yeast Schizosaccharomyces pombe as a model system to study the earlysteps of N-glycosylation and the molecular bases of CDG. To study defectsduring LLO synthesis and the impact ofthe structures produced in the transfer efficiency to proteins by OST weconstructed a collection of 16 strains which synthesize all possiblecombinations of LLOs containing three to zero Glc and nine to five Man. We used the set of mutants as a platform to quantifyprotein hypoglycosylation produced ina fluorescent biosensor. Our results showed that in S. pombe, the presence of Glc in the LLO is more relevant in thetransfer efficiency than the amount of Man residues, although surprisingly adecrease in the number of Man in glycans somehow improves their transfer toproteins. The most severe hypoglycosylation was produced in cells completelylacking Glc and having a high number of Man, a deficiency that could bereverted by expressing a single subunit OST with a broad range of substratespecificity. We then move to the following steps of the N-glycosylation and analyze the effect produced by mutations inGlucosidases I and II (GI and GII), the enzymes that allow glycoproteinentrance in the quality control of protein folding in the ER. We demonstratedthat the inability to deglucosylate protein-linked G3M9 but not G2M9 in the ERis extremely toxic to the cell and showed the occurrence of alterations in thesecretory/endocytic pathway in cells lacking GI, suggesting an interplaybetween N-glycosylation and the cell endomembrane system. Finally, we analyzedstructural features of GII and showed that its MRH domain, which is present inother proteins of the secretory pathway, is responsible of glycoprotein fatewithin the cell.