The transmembrane domain of Endoplasmic Reticulum α-Glucosidase I influences the survival of a MOGS-CDG fission yeast model

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

Congreso; 7th Latin American Glycobiology Congress; 2023

Congenital Disorders of Glycosylation (CDG) are a group of rare genetic disorders that affect a person´s ability to properly glycosylate proteins and lipids in their cells. These disorders can manifest in a wide range of symptoms, including developmental delays, neurological problems, and several organ dysfunctions. N-glycosylation of proteins is a highly conserved process and one of the most significant post-translational modifications in eukaryotic organisms. During N-glycosylation the pre-assembled oligosaccharide Glc3Man9GlcNAc2 (G3M9) is transferred by the oligosacharyltransferase complex to asparagine residues of proteins translocating into the ER. Then, ER-glucosidase I (GI) hydrolyzes the outermost glucose from G3M9, producing G2M9 glycoproteins enabling glucosidase II (GII) to convert G2M9 to G1M9, recognized by the ER Quality Control of glycoprotein folding (ERQC) mechanism. ERQC facilitates the folding of secreted and membrane proteins, and ensures that only proper folded ones continue to the Golgi. GI related defects lead to type IIb CDG (CDG-IIb or MOGS-CDG), a disease linked to an inactive GI. Our earlier findings indicated that the accumulation of G3M9 glycoproteins due to a GI deficiency in Schizosaccharomyces pombe is highly toxic to the cell, resulting in a sick phenotype in GI yeast mutants (GI cells). Knocking alg10p alpha-1,2-glucosyltransferase, responsible for adding the outermost glucose residue during glycan biosynthesis, does partially –but not completely- suppress this adverse phenotype, indicating that bypassing GI requirement in glycoprotein processing is not enough to relieve the growth and morphological cell defects. On the other hand, most mutations observed in patients are not located in the catalytic pair residues of GI. These results suggest that the enzyme´s catalytic domain may not be the only explanation for the defects observed in GI cells, and that other domains of GI may also play a significant role. While GII is a soluble resident ER protein, GI is an ER membrane-bound one, comprising the globular catalytic domain within the ER, a transmembrane domain, and a cytosolic tail. In this work, we examined the phenotypic impact of expressing a catalytic-domain-only GI (cGI) in ΔGI mutant. We confirmed the proper expression and localization of the GI variant through fluorescence microscopy and western blotting. Its activity was assessed in vitro by using a fluorometric assay that detects hydrolyzed glucose from a synthetic oligosaccharide substrate specific of GI. We evaluated the cell viability and the growth rate of cGI expressing cells and measure cell lengths comparing with the expression of a full-length GI. Our findings demonstrate that cGI in ΔGI mutants is not able to rescue the sick phenotype observed in fission yeasts lacking the full-length GI, supporting the hypothesis that GI possesses an additional undiscovered role, potentially related to its ER membrane localization.