The two Caenorhabditis elegans UDP-glucose:glycoprotein glucosyltransferase homologues have distinct biological functions

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.

Figure 1. UGGT activity in C. elegans.

C. elegans (A) and rat liver (B) microsomal proteins were incubated in a mixture that contained 5 mM Tris-maleate buffer, pH 7.5, 10 mM CaCI₂, 0.6% Triton X-100, 5 mM NMDNJ and 3 µCi UDP-[¹⁴C]Glc, at 24°C (A) and 37°C (B) for 60 min. Glycans obtained by Endo H treatment were subjected to paper chromatography with solvent A. Standards G1M9: Glc₁Man₉GlcNAc; G1M8: Glc₁Man₈GlcNAc and G1M7: Glc₁Man₇GlcNAc.

Figure 2. uggt-1 codes for an active UGGT activity.

In vitro assays. S. pombe (gpt1/alg6) microsomal proteins prepared from cells carrying pREP3X-uggt-1 (A) or pREP3X-gpt1 (B) were incubated in a mixture containing 5 mM Tris-maleate buffer, pH 7.5, 10 mM CaCI₂, 0.6% Triton X-100, 5 mM NMDNJ and 3 µCi UDP-[¹⁴C]Glc (300 Ci/mol), at 24°C for 30 min. Glycans obtained by Endo H treatment were subjected to paper chromatography with solvent A. (C) Total RNA was isolated from S. pombe gpt1/alg6 cells carrying pREP3X-gpt1+, pREP3X-uggt-1, pREP3X-uggt-2 or pREP3X and used to generate cDNA. RT-PCR analysis was performed with specific primers for gpt-1, uggt-1, uggt-2 and β-actin mRNAs.

Figure 3. uggt-1 codes for an active UGGT activity.

In vivo assays. Indicated cells were preincubated for 60 min with NMDNJ, the final 5 min in presence of 5 mM DTT and pulsed for 30 min in 5 mM Glc with 150 µCi of [¹⁴C]Glc at 24°C. Glycans liberated by Endo H treatment were subjected to paper chromatography with solvent A (Figures 3A–C). Glycans migrating between 24 and 35 cm in panels A–C were submitted to strong acid hydrolysis and run on paper chromatography with solvent B (Figures 3D–F). Standards: G1M9, Glc₁Man₉GlcNAc; M9, Man₉GlcNAc; G1M8, Glc₁Man₈GlcNAc and M8 Man₈GlcNAc.

Figure 4. UGGT activity decreases in uggt-1(RNAi) worms but no decrease was observed in uggt-2 (RNAi) worms.

F2 uggt-1(RNAi), uggt-2(RNAi) and control gfp(RNAi) worm microsomal proteins were incubated in a mixture that contained 5 mM Tris-HCl buffer, pH 7.5, 10 mM CaCI₂, 0.6% Triton X-100, 5 mM NMDNJ and 3 µCi UDP-[¹⁴C]Glc, at 20°C for 30 min. Reactions were stopped with 1 mL of 10% of trichloroacetic acid. After centrifugation, the pellets were twice washed with 1 mL of 10% trichloroacetic acid and counted. The values shown are the mean of two independent experiments. Error bars represent standard deviations * indicates significant differences.

Figure 5. uggt-1 and uggt-2 expression pattern during development.

Expression levels of uggt-1 mRNA (A) and uggt-2 mRNA (B) relative to those of ama-1 mRNA during development as measured by Real-time PCR. The values shown are the mean of three independent experiments. Error bars represent standard deviations.

Figure 6. uggt-1 but not uggt-2 is upregulated under stress conditions.

Total RNAs from untreated and 5 µg/ml TN-treated animals were isolated and the levels of uggt-1, uggt-2 and hsp4 expression were quantified by real-time PCR using ama-1 as reference gene. Relative expression levels represents RNA expression in TN treated worms/RNA expression in untreated worms. The value obtained for untreated samples was considered as one. The values shown are the mean of three independent experiments. Error bars represent standard deviations * indicates significant differences.

Figure 7. uggt-1 expression is regulated by the ire-1 arm of the unfolded response pathway.

Total RNA from young adults, wild type, ire-1, atf-6 and pek-1 worms, treated and not treated with 5 µg/ml TN was extracted and the levels of mRNA coding for uggt-1, uggt-2 and hsp-4 were analyzed by real time PCR using ama-1 as reference gene. Gene expression level represents RNA expression in TN treated worms/RNA expression in untreated worms. The value obtained for untreated samples was considered as one. The values shown are the mean of three independent experiments. Error bars represent standard deviations * indicates significant differences.

Figure 8. UGGT-1 is expressed in cells of the nervous system.

N2 transgenic worms expressing GFP under the control of uggt-1 promoter were placed in agarose pads and visualized by fluorescence confocal microscopy. A) Amphid neurons of the head and nerve ring, B and C) Neurons in the dorsal and ventral nerve cord and neurons along the body, D) phasmid neurons located at the lateral side of the tail.

Figure 9. CeUGGT-1 depletion causes a reduced lifespan and that of CeUGGT-1 and CeUGGT-2 a delayed development.

F2 uggt-1(RNAi) uggt-2(RNAi) and control gfp (RNAi) worms were seeded on RNAi plates and monitored for survival at 24 h intervals for the next 16 days (A). The survival analysis was performed using the Kaplan-Meier method and the survival curves were compared using the logarithmic rank test. Development to progressive larval stages (B). Gravid hermaphrodites were transferred to RNAi plates for 6 h to lay eggs and the developmental stages of the worms were analyzed for 5 days at intervals of 24 h and the percentages of L1, L2/L3, L4 and adults were determined. These studies were performed for three independent cohorts (n>60) and the results are representative of triplicate experiments (A) and the mean of the three experiments (B).

Figure 10. uggt-2 is an essential gene.

Heterozygous uggt-2 (ok2510)/+ (VC1961) and N2 wild type gravid hermaphrodites were allowed to lay eggs for 6 h and then removed from plates. Developmental stages were analyzed for 5 d at intervals of 24 h and the percentages of arrested eggs, of L1, L2/L3, L4, adults and gravid hermaphrodites were determined A) Developmental stages were analyzed as in (A) and the percentages of L1, L2/L3, L4, adults and gravid hermaphrodites were determined from hatched eggs. Studies were performed for three independent cohorts (n>200) and the values shown are the mean of the three experiments.

Figure 11. TN sensitivity assay.

F2 Control gfp(RNAi) (A), uggt-1(RNAi) (B) and uggt-2(RNAi) (C) worms were tested under several TN concentrations (0, 2.5, 5 or 10 µg/ml). Gravid hermaphrodites were allowed to lay eggs for 6 h and the developmental stages were analyzed for 5 days at intervals of 24 h and the percentages of L1, L2/L3, L4 and adults were determined. Studies were performed for three independent cohorts (n>100) and the values shown are the mean of the three experiments.

Figure 12. CeUGGT-2 plays a role in alleviating endogenous ER stress during development.

F2 Control gfp(RNAi), uggt-1(RNAi) and uggt-2(RNAi) worms of ire-1 (A), atf-6 (B) and pek-1 (C) strains were tested under 0 and 2.5 µg/ml TN concentrations. Gravid hermaphrodites were allowed to lay eggs for 6 h and the developmental stages were analyzed for 5 days at intervals of 24 h. The percentages of L1, L2/L3, L4 and adults were determined. Studies were performed for three independent cohorts (n>100) and the values are the mean of the three experiments.