LACK OF GLUCOSIDASE I RESULTS IN CELL TOXICITY THAT IS RELIEVED BY AVOIDING THE ACCUMULATION OF TRIGLUCOSYLATED GLYCOPROTEINS

VALKO, AYELÉN; PARODI, ARMANDO J.; D'ALESSIO, CECILIA (CORRESPONDING AUTHOR); ETCHEGARAY, EMILIANA; ARAMBURU, SOFÍA I.; GALLO, GIOVANNA L

New Orleans

Congreso; Annual Meeting of the Society for Glycobiology; 2018

Society for Glycobiology

Glucosidase I (GI) is an endoplasmic reticulum (ER) membrane protein that removes the outermost glucose from the N-glycan Glc3Man9GlcNAc2 (G3M9) transferred to proteins. Mutations in the GI-encoding gene (gls1) result in congenital disorders of glycosylation (CDG) IIb. Even though it has been previously reported that GI absence is lethal in the fission yeast Schizosaccharomyces pombe, we were able to obtain two viable Δgls1 mutants: one with a very sick but non-lethal phenotype (Δgls1-S) and the other with a much healthier one (Δgls1-H). While the sick strain only displayed G3M9 as ER protein-linked glycan, the healthier strain had not only G3M9 but also Man9GlcNAc2. The most abundant lipid-linked glycans formed were G3M9 and Glc2Man9GlcNAc2 in the sick and healthy mutants, respectively. This result suggested a reduced alg10+ gene product activity in the Δgls1-H strain (Alg10p is responsible for the addition of the last Glc during the step-wise synthesis of lipid-linked G3M9). A mutation in the alg10+ gene was indeed observed upon sequencing the alg10 gene in the Δgls1-H strain. We constructed a Δgls1/Δalg10 double knock out mutant and, as expected, this strain showed an almost normal phenotype. Our findings indicate that abrogating G3M9 deglucosylation was responsible for the severe defects observed in Δgls1-S cells. Further results demonstrated that such defects could not be ascribed to hindrance of glycoprotein entrance into calnexin quality control of glycoprotein folding cycles, to inhibition of the oligosaccharyltransferase by the transfer reaction products, or to a reduced degradation of misfolded glycoproteins. Moreover, lack of triglucosylated glycoprotein deglucosylation did not significantly prevent Golgi glycan elongation or modify the overall cell wall monosaccharide composition. Nonetheless, GI absence produced a distorted cell wall and absence of the ER underlying structures that occur in S. pombe WT cells. We propose that accumulation of G3M9-bearing glycoproteins is at least partially responsible for the defects observed in the CDG IIb disorder.