Yoichiro Harada

Oligosaccharyltransferase directly binds to ribosome at a location near the translocon-binding site

Oligosaccharyltransferase (OT) transfers high mannose-type glycans to the nascent polypeptides that are translated by the membrane-bound ribosome and translocated into the lumen of the endoplasmic reticulum through the Sec61 translocon complex. In this article, we show that purified ribosomes and OT can form a binary complex with a stoichiometry of ≈1 to 1 in the presence of detergent. We present evidence that OT may bind to the large ribosomal subunit near the site where nascent polypeptides exit. We further show that OT and the Sec61 complex can simultaneously bind to ribosomes in vitro. Based on existing data and our findings, we propose that cotranslational translocation and N-glycosylation of nascent polypeptides are mediated by a ternary supramolecular complex consisting of OT, the Sec61 complex, and ribosomes.

Structural Studies and the Assembly of the Heptameric Post-translational Translocon Complex

In Saccharomyces cerevisiae, some of the nascent chains can be post-translationally translocated into the endoplasmic reticulum through the heptameric post-translational translocon complex (post-translocon). This membrane-protein complex is composed of the protein-conducting channel and the tetrameric Sec62/63 complex. The Sec62/63 complex plays crucial roles in targeting of the signal recognition particle-independent protein substrate to the protein-conducting channel and in assembly of the post-translocon. Although the molecular mechanism of the post-translational translocation process has been well established, the structure of the post-translocon and how the channel and the Sec62/63 complex form the heptameric complex are largely uncharacterized. Here, we report a 20-Å resolution cryo-electron microscopy structure of the post-translocon. The purified post-translocon was found to have a mass of 287 kDa, which is consistent with the unit stoichiometry of the seven subunits as determined by a cysteine labeling experiment. We demonstrated that Triton X-100 dissociated the heptameric complex into three subcomplexes identified as the trimeric translocon Sec61-Sbh1-Sss1, the Sec63-Sec71-Sec72 trimer, and the heterotetramer Sec62-Sec63-Sec71-Sec72, respectively. Additionally, a role of the sixth cytosolic loop of Sec61 in assembly of the post-translocon was demonstrated. Mutations of conserved, positively charged amino acid residues in the loop caused decreased formation of the post-translocon. These studies provide the first architectural description of the yeast post-translocon.

Endo-β-N-acetylglucosaminidase forms N-GlcNAc protein aggregates during ER-associated degradation in Ngly1-defective cells

Significance In the endoplasmic reticulum (ER), N-glycans on glycoproteins play important roles in dictating the folding status of proteins by a sophisticated N-glycan–dependent protein quality control machinery. In this study we identified the dysregulation of ER-associated degradation (ERAD) in cells that were defective in the cytosolic deglycosylating enzyme, Ngly1. ERAD dysregulation was caused by an unexpected deglycosylating activity of endo-β-N-acetylglucosaminidase, another cytosolic deglycosylation enzyme, and this action resulted in the intracellular formation of protein aggregates. Our results clearly point to the critical role of N-glycans even in cytosolic events of the ERAD process by controlling the conformation/solubility of proteins. This study may also provide a potential mechanism for explaining the pathology of a human genetic disorder caused by mutations in the NGLY1 gene. The cytoplasmic peptide:N-glycanase (PNGase; Ngly1 in mice) is a deglycosylating enzyme involved in the endoplasmic reticulum (ER)-associated degradation (ERAD) process. The precise role of Ngly1 in the ERAD process, however, remains unclear in mammals. The findings reported herein, using mouse embryonic fibroblast (MEF) cells, that the ablation of Ngly1 causes dysregulation of the ERAD process. Interestingly, not only delayed degradation but also the deglycosylation of a misfolded glycoprotein was observed in Ngly1−/− MEF cells. The unconventional deglycosylation reaction was found to be catalyzed by the cytosolic endo-β-N-acetylglucosaminidase (ENGase), generating aggregation-prone N-GlcNAc proteins. The ERAD dysregulation in cells lacking Ngly1 was restored by the additional knockout of ENGase gene. Thus, our study underscores the functional importance of Ngly1 in the ERAD process and provides a potential mechanism underlying the phenotypic consequences of a newly emerging genetic disorder caused by mutation of the human NGLY1 gene.

Non-lysosomal degradation pathway for N-linked glycans and dolichol-linked oligosaccharides.

There is growing evidence that asparagine (N)-linked glycans play pivotal roles in protein folding and intra- or intercellular trafficking of N-glycosylated proteins. During the N-glycosylation of proteins, significant amounts of free oligosaccharides (fOSs) and phosphorylated oligosaccharides (POSs) are generated at the endoplasmic reticulum (ER) membrane by unclarified mechanisms. fOSs are also formed in the cytosol by the enzymatic deglycosylation of misfolded glycoproteins destined for proteasomal degradation. This article summarizes the current knowledge of the molecular and regulatory mechanisms underlying the formation of fOSs and POSs in mammalian cells and Saccharomyces cerevisiae.

Eukaryotic oligosaccharyltransferase generates free oligosaccharides during N-glycosylation

Background: The enzyme generating free oligosaccharides (fOSs) in the lumen of the endoplasmic reticulum (ER) has been unidentified. Results: Oligosaccharyltransferase (OST), the N-glycosylating enzyme, hydrolyzes dolichol-linked oligosaccharides to release the fOSs. Conclusion: OST is responsible for the generation of fOSs in the ER lumen. Significance: This study provides a mechanistic insight into the formation of luminal fOSs in yeast. Asparagine (N)-linked glycosylation regulates numerous cellular activities, such as glycoprotein quality control, intracellular trafficking, and cell-cell communications. In eukaryotes, the glycosylation reaction is catalyzed by oligosaccharyltransferase (OST), a multimembrane protein complex that is localized in the endoplasmic reticulum (ER). During N-glycosylation in the ER, the protein-unbound form of oligosaccharides (free oligosaccharides; fOSs), which is structurally related to N-glycan, is released into the ER lumen. However, the enzyme responsible for this process remains unidentified. Here, we demonstrate that eukaryotic OST generates fOSs. Biochemical and genetic analyses using mutant strains of Saccharomyces cerevisiae revealed that the generation of fOSs is tightly correlated with the N-glycosylation activity of OST. Furthermore, we present evidence that the purified OST complex can generate fOSs by hydrolyzing dolichol-linked oligosaccharide, the glycan donor substrate for N-glycosylation. The heterologous expression of a single subunit of OST from the protozoan Leishmania major in S. cerevisiae demonstrated that this enzyme functions both in N-glycosylation and generation of fOSs. This study provides insight into the mechanism of PNGase-independent formation of fOSs.

Basal Autophagy Is Required for the Efficient Catabolism of Sialyloligosaccharides

Background: The role of autophagy in glycan catabolism remains to be clarified. Results: In Atg5−/− cells, defective in autophagosome formation, sialyloligosaccharides accumulate specifically in the cytosol. Conclusion: Basal autophagy is essential for lysosomal catabolism of sialyloligosaccharides. Significance: This result not only underscores the importance of autophagy in glycan catabolism but also suggests that basal autophagy is required for proper function of lysosomes. Macroautophagy is an essential, homeostatic process involving degradation of a cells own components; it plays a role in catabolizing cellular components, such as protein or lipids, and damaged or excess organelles. Here, we show that in Atg5−/− cells, sialyloligosaccharides specifically accumulated in the cytosol. Accumulation of these glycans was observed under non-starved conditions, suggesting that non-induced, basal autophagy is essential for their catabolism. Interestingly, once accumulated in the cytosol, sialylglycans cannot be efficiently catabolized by resumption of the autophagic process, suggesting that functional autophagy is important for preventing sialyloligosaccharides from accumulating in the cytosol. Moreover, knockdown of sialin, a lysosomal transporter of sialic acids, resulted in a significant reduction of sialyloligosaccharides, implying that autophagy affects the substrate specificity of this transporter. This study thus provides a surprising link between basal autophagy and catabolism of N-linked glycans.

Generation and degradation of free asparagine-linked glycans

Asparagine (N)-linked protein glycosylation, which takes place in the eukaryotic endoplasmic reticulum (ER), is important for protein folding, quality control and the intracellular trafficking of secretory and membrane proteins. It is known that, during N-glycosylation, considerable amounts of lipid-linked oligosaccharides (LLOs), the glycan donor substrates for N-glycosylation, are hydrolyzed to form free N-glycans (FNGs) by unidentified mechanisms. FNGs are also generated in the cytosol by the enzymatic deglycosylation of misfolded glycoproteins during ER-associated degradation. FNGs derived from LLOs and misfolded glycoproteins are eventually merged into one pool in the cytosol and the various glycan structures are processed to a near homogenous glycoform. This article summarizes the current state of our knowledge concerning the formation and catabolism of FNGs.

Metabolically programmed quality control system for dolichol-linked oligosaccharides

Significance In mammals, asparagine (N)-linked glycosylation of nascent polypeptides synthesized in the endoplasmic reticulum regulates folding, degradation, and intracellular trafficking of the glycoproteins. The normal N-glycosylation requires the completely-assembled dolichol-linked oligosaccharide (DLO) as the optimal glycan donor substrate; however, a low-glucose environment causes arrest of the DLO assembly, which results in the synthesis of extensively truncated premature DLOs, thereby increasing a risk of abnormal N-glycosylation. Here, we report that under low-glucose conditions, the premature DLOs are efficiently degraded by unidentified pyrophosphatase, catabolizing them to singly phosphorylated oligosaccharides. Our results suggest that the pyrophosphatase-mediated degradation of premature DLOs functions as a quality control system to avoid abnormal N-glycosylation under conditions that impair efficient DLO biosynthesis. The glycolipid Glc3Man9GlcNAc2-pyrophosphate-dolichol serves as the precursor for asparagine (N)-linked protein glycosylation in mammals. The biosynthesis of dolichol-linked oligosaccharides (DLOs) is arrested in low-glucose environments via unknown mechanisms, resulting in abnormal N-glycosylation. Here, we show that under glucose deprivation, DLOs are prematurely degraded during the early stages of DLO biosynthesis by pyrophosphatase, leading to the release of singly phosphorylated oligosaccharides into the cytosol. We identified that the level of GDP-mannose (Man), which serves as a donor substrate for DLO biosynthesis, is substantially reduced under glucose deprivation. We provide evidence that the selective shutdown of the GDP-Man biosynthetic pathway is sufficient to induce the release of phosphorylated oligosaccharides. These results indicate that glucose-regulated metabolic changes in the GDP-Man biosynthetic pathway cause the biosynthetic arrest of DLOs and facilitate their premature degradation by pyrophosphatase. We propose that this degradation system may avoid abnormal N-glycosylation with premature oligosaccharides under conditions that impair efficient DLO biosynthesis.

Cytosolic-free oligosaccharides are predominantly generated by the degradation of dolichol-linked oligosaccharides in mammalian cells

During asparagine (N)-linked protein glycosylation, eukaryotic cells generate considerable amounts of free oligosaccharides (fOSs) in the cytosol. It is generally assumed that such fOSs are produced by the deglycosylation of misfolded N-glycoproteins that are destined for proteasomal degradation or as the result of the degradation of dolichol-linked oligosaccharides (DLOs), which serve as glycan donor substrates in N-glycosylation reactions. The findings reported herein show that the majority of cytosolic fOSs are generated by a peptide:N-glycanase (PNGase) and an endo-β-N-acetylglucosaminidase (ENGase)-independent pathway in mammalian cells. The ablation of the cytosolic deglycosylating enzymes, PNGase and ENGase, in mouse embryonic fibroblasts had little effect on the amount of cytosolic fOSs generated. Quantitative analyses of fOSs using digitonin-permeabilized cells revealed that they are generated by the degradation of fully assembled Glc3Man9GlcNAc2-pyrophosphate-dolichol (PP-Dol) in the lumen of the endoplasmic reticulum. Because the degradation of Glc3Man9GlcNAc2-PP-Dol is greatly inhibited in the presence of an N-glycosylation acceptor peptide that is recognized by the oligosaccharyltransferase (OST), the OST-mediated hydrolysis of DLO is the most likely mechanism responsible for the production of a large fraction of the cytosolic fOSs.

Lethality of mice bearing a knockout of the Ngly1-gene is partially rescued by the additional deletion of the Engase gene

The cytoplasmic peptide:N-glycanase (Ngly1 in mammals) is a de-N-glycosylating enzyme that is highly conserved among eukaryotes. It was recently reported that subjects harboring mutations in the NGLY1 gene exhibited severe systemic symptoms (NGLY1-deficiency). While the enzyme obviously has a critical role in mammals, its precise function remains unclear. In this study, we analyzed Ngly1-deficient mice and found that they are embryonic lethal in C57BL/6 background. Surprisingly, the additional deletion of the gene encoding endo-β-N-acetylglucosaminidase (Engase), which is another de-N-glycosylating enzyme but leaves a single GlcNAc at glycosylated Asn residues, resulted in the partial rescue of the lethality of the Ngly1-deficient mice. Additionally, we also found that a change in the genetic background of C57BL/6 mice, produced by crossing the mice with an outbred mouse strain (ICR) could partially rescue the embryonic lethality of Ngly1-deficient mice. Viable Ngly1-deficient mice in a C57BL/6 and ICR mixed background, however, showed a very severe phenotype reminiscent of the symptoms of NGLY1-deficiency subjects. Again, many of those defects were strongly suppressed by the additional deletion of Engase in the C57BL/6 and ICR mixed background. The defects observed in Ngly1/Engase-deficient mice (C57BL/6 background) and Ngly1-deficient mice (C57BL/6 and ICR mixed background) closely resembled some of the symptoms of patients with an NGLY1-deficiency. These observations strongly suggest that the Ngly1- or Ngly1/Engase-deficient mice could serve as a valuable animal model for studies related to the pathogenesis of the NGLY1-deficiency, and that cytoplasmic ENGase represents one of the potential therapeutic targets for this genetic disorder.