Abstract
The UDP-Glc:glycoprotein glucosyltransferase (UGGT) is the sensor of glycoprotein conformations in the glycoprotein folding quality control as it exclusively glucosylates glycoproteins not displaying their native conformations. Monoglucosylated glycoproteins thus formed may interact with the lectin-chaperones calnexin (CNX) and calreticulin (CRT). This interaction prevents premature exit of folding intermediates to the Golgi and enhances folding efficiency. Bioinformatic analysis showed that in C. elegans there are two open reading frames (F48E3.3 and F26H9.8 to be referred as uggt-1 and uggt-2, respectively) coding for UGGT homologues. Expression of both genes in Schizosaccharomyces pombe mutants devoid of UGGT activity showed that uggt-1 codes for an active UGGT protein (CeUGGT-1). On the other hand, uggt-2 coded for a protein (CeUGGT-2) apparently not displaying a canonical UGGT activity. This protein was essential for viability, although cnx/crt null worms were viable. We constructed transgenic worms carrying the uggt-1 promoter linked to the green fluorescent protein (GFP) coding sequence and found that CeUGGT-1 is expressed in cells of the nervous system. uggt-1 is upregulated under ER stress through the ire-1 arm of the unfolded protein response (UPR). Real-time PCR analysis showed that both uggt-1 and uggt-2 genes are expressed during the entire C. elegans life cycle. RNAi-mediated depletion of CeUGGT-1 but not of CeUGGT-2 resulted in a reduced lifespan and that of CeUGGT-1 and CeUGGT-2 in a developmental delay. We found that both CeUGGT1 and CeUGGT2 play a protective role under ER stress conditions, since 10 µg/ml tunicamycin arrested development at the L2/L3 stage of both uggt-1(RNAi) and uggt-2(RNAi) but not of control worms. Furthermore, we found that the role of CeUGGT-2 but not CeUGGT-1 is significant in relieving low ER stress levels in the absence of the ire-1 unfolding protein response signaling pathway. Our results indicate that both C. elegans UGGT homologues have distinct biological functions.
Highlights
The endoplasmic reticulum (ER) is the subcellular compartment where glycoproteins acquire their tertiary and quaternary structures
C. elegans microsomes incubated with UDP-[14C]Glc yielded products that migrated as Glc1Man9GlcNAc, Glc1Man8GlcNAc and Glc1Man7GlcNAc standards on paper chromatography (Figure 1A)
The high level of hsp-4 induction has already been observed in C.elegans [17]. These results suggest that only CeUGGT-1 is upregulated under ER stress conditions induced by TN treatment
Summary
The endoplasmic reticulum (ER) is the subcellular compartment where glycoproteins acquire their tertiary and quaternary structures. Monoglucosylated glycoproteins bearing glycans of compositions Glc1Man7–9GlcNAc2 may interact with two ER-resident lectin chaperones, membrane bound calnexin (CNX) or its soluble homologue calreticulin (CRT). Monoglucosylated N-glycans may be formed by reglucosylation of deglucosylated glycans of structure Man7–9GlcNAc2 by the UDP-Glc:glycoproteinglucosyltransferase (UGGT). This enzyme is the key component of the folding quality control mechanism. Lectin–glycoprotein binding and unbinding as a result of the opposing activities of UGGT and GII continues until glycoproteins either acquire their native structures or, alternatively are recognized by cells as irreparably misfolded species or as complexes unable to acquire their full subunit complement. The interaction of folding intermediates, incomplete complexes and irreparably misfolded glycoproteins with the lectin-chaperones prevents Golgi exit of the former and decreases the folding rate and increases folding efficiency by preventing aggregation and facilitating correct disulfide bond formation through their interaction with ERp57, a protein disulfide isomerase loosely associated with CNX and CRT [1,2,3,4]
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