earticle

영어

To guarantee proper glycoprotein folding, eukaryotes evolved a highly conserved N-glycan-dependent endoplasmic reticulum quality control mechanism (ERQC). In the ER, glycoprotein conformations are sensed by UDP-glucose: glycoprotein glucosyltransferase (UGGT), which selectively re-glycosylates misfolded glycoproteins for re-binding to the folding chaperones. ER-resident α-1,2-mannosidase I (MNS1) trims one mannose residue from the B branch of core N-oligosaccharides, initiating the formation of the molecular signals that either lead folded proteins towards the Golgi apparatus, or direct misfolded glycoproteins towards degradation. In this study, we investigated the molecular assembly and function of N-glycan-dependent ERQC in the human fungal pathogen Cryptococcus neoformans, which has a unique N-glycosylation pathway lacking glucosyltransferases but carrying multiple mannosidases. We constructed and functionally analyzed the C. neoformans mutant strains lacking UGGT (uggtΔ), MNS1A (mns1AΔ) and MNS1B (mns1BΔ). The uggtΔ mutant displayed reduced growth rates with aggregated forms, increased stress sensitivity, and induced the unfolded protein response even in the absence of exogenous ER stress. The mns1AΔ and mns1BΔ showed altered N-glycan profiles, indicating that whereas MNS1A acts as the widely known ER-α1,2-mannosidase I, MNS1B appears to be a novel mannosidase involved in mannose processing in the Golgi. Simultaneous deletion of MNS1A and MNS1B (mns1AΔ1BΔ) displayed mild growth retardation under several stress conditions. Moreover, qRT-PCR analysis of UGGT, MNS1A and MNS1B mRNA levels revealed that these genes were up-regulated upon DTT treatment, indicating these genes are essential upon ER stress induced by the presence of misfolded proteins. Notably, the uggtΔ mutant and the mns1AΔ1BΔ double mutant strains commonly displayed defects in capsule formation, which might contribute to a decrease in survival inside phagocytic cells and to avirulence in a murine model. Altogether, our data demonstrates the evolutionary unique C. neoformans N-glycan-dependent ERQC plays critical roles in cellular fitness under adverse conditions of the host environment, required for full fungal pathogenicity.