A hidden player: the influence of transmembrane domain of α-Glucosidase I in a fission yeast MOGS-CDG model

IDROVO-HIDALGO, TOMMY; ORIOLI, SOFÍA; D'ALESSIO, CECILIA

Hawaii

Congreso; Glycobiology 2023 Transformative Advances in the Biological Functions of Glycans; 2023

Society for Glycobiology

N-glycosylation is a highly conserved process among eukaryotes and represents one of the most relevant posttranslational modifications in the secretory pathway. It starts with the transfer of a pre-assembled oligosaccharide Glc3Man9GlcNAc2 (G3M9) in the endoplasmic reticulum (ER) membrane to asparagine residues of a consensus sequence of proteins entering the ER. Immediately, the ER- glucosidase I (GI) trims the outermost glucose of glycoproteins converting G3M9 into G2M9 and glucosidase II (GII) generates G1M9-bearing glycoproteins. These are recognized by calnexin/calreticulin lectins involved in the endoplasmic reticulum quality control of glycoprotein folding mechanism (ERQC), which ensures that only proper folded glycoproteins continue the secretory pathway. GI associated defects produce congenital disorders of glycosylation (CDG) type IIb (or MOGS-CDG). This disease manifests with multisystemic failures at different ages in human patients. It has been stated that unactive GI fails to trim G3M9 into G2M9 and our previous findings demonstrated that the accumulation of G3M9 glycoproteins due to the lack of GI in Schizosaccharomyces pombe (GI mutants), is extremely toxic for the cell. GI yeast mutants are shortened and clumped, with a slow cell growth and a low viability. This sick phenotype could be partially -but not fully- suppressed by knocking out an alpha-1,2-glucosyltransferase (alg10p) that adds the outermost residue of glucose during glycan biosynthesis, the same glucose that is a substrate for GI. On the other hand, most mutations found in patients are not within the proposed catalytic pair residues of GI. Both results could imply that not only the catalytic function of GI is involved in the sick phenotype of cells lacking GI, but that other domain/s could also be important. Interestingly, while GII is a soluble protein of the ER lumen, GI is an ER membrane bound protein that contains three domains: a globular catalytic domain inside the ER lumen, a transmembrane domain, and a short cytosolic tail. In this study, we examined the impact of expressing a soluble catalytic-domain-only GI (cGI) in fission yeast ΔGI ER lumen. We verified the proper expression and localization of the cGI variant by western blot and confocal fluorescence microscopy. We also confirmed the presence of GI activity by an in vitro fluorometric assay that detects glucose hydrolysis from a GI-specific synthetic substrate. Even though the cells express GI activity in the ER lumen the cell viability, growth rates and cell lengths are similar to those in ΔGI cells. Our results indicate that the expression of an active cGI in the ER lumen is not able to rescue the sick phenotype of cells lacking the full gene coding GI, supporting the hypothesis that GI may have an undiscovered additional role, possibly related to its ER transmembrane domain