Laboratory of Glycobiology, Fundación Instituto Leloir, Buenos Aires, Argentina.
PLoS One. 2011;6(11):e27025. doi: 10.1371/journal.pone.0027025. Epub 2011 Nov 2.
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.
UDP-葡萄糖:糖蛋白葡糖基转移酶(UGGT)是糖蛋白折叠质量控制中的糖蛋白构象传感器,因为它仅葡糖基化未呈现其天然构象的糖蛋白。因此形成的单糖基化糖蛋白可能与凝集素伴侣 calnexin (CNX) 和 calreticulin (CRT) 相互作用。这种相互作用可防止折叠中间体过早退出高尔基体并提高折叠效率。生物信息学分析表明,在秀丽隐杆线虫中有两个开放阅读框 (F48E3.3 和 F26H9.8,分别称为 uggt-1 和 uggt-2) 编码 UGGT 同源物。在缺乏 UGGT 活性的酿酒酵母突变体中表达这两个基因表明,uggt-1 编码一种活性 UGGT 蛋白 (CeUGGT-1)。另一方面,uggt-2 编码一种蛋白质 (CeUGGT-2),显然不具有典型的 UGGT 活性。尽管 cnx/crt 缺失的线虫仍然具有活力,但这种蛋白质对于生存是必需的。我们构建了携带 uggt-1 启动子与绿色荧光蛋白 (GFP) 编码序列相连的转基因线虫,发现 CeUGGT-1 在神经系统的细胞中表达。在未折叠蛋白反应 (UPR) 的 ire-1 臂的作用下,uggt-1 在 ER 应激下上调。实时 PCR 分析表明,uggt-1 和 uggt-2 基因在整个秀丽隐杆线虫生命周期中都有表达。RNAi 介导的 CeUGGT-1 消耗但不是 CeUGGT-2 的消耗导致寿命缩短,而 CeUGGT-1 和 CeUGGT-2 的消耗导致发育延迟。我们发现,在 ER 应激条件下,CeUGGT1 和 CeUGGT2 都发挥保护作用,因为 10µg/ml 衣霉素使 uggt-1(RNAi) 和 uggt-2(RNAi) 线虫都在 L2/L3 阶段发育停滞,但对照线虫没有。此外,我们发现 CeUGGT-2 的作用而不是 CeUGGT-1 的作用在没有 ire-1 unfolded 蛋白反应信号通路的情况下显著减轻低水平的 ER 应激。我们的结果表明,秀丽隐杆线虫的两种 UGGT 同源物具有不同的生物学功能。