ANNIVERSARY
COMM
25thS

pecial Lectures ➊N

GLY1 andnon-lysosomal degradation ofN-glycans, free N-glycans anddolichol-linked oligosaccharides

Tadashi Suzuki

Glycometabolome Team,Systems Glycobiology Research Group,RIKEN Global Research Cluster

Dr. Tadashi Suzuki (Team Leader, Glycometabolome Team, RIKEN Global ResearchCluster) received B.S. (1992), M.S. (1994) and D. Sc. (1997) from the Departmentof Biochemistry and Biophysics, Graduate School of Science, University of Tokyo,
Japan. He then became a postdoctoral fellow at Department of Biochemistry andCell Biology, State University of New York at Stony Brook (1997-2000). During thisperiod he identified the gene encoding the cytoplasmic PNGase in yeast. In 2000 hebecame a Research Scientist/Research Assistant Professor at the same University.
In December 2001 he returned to Japan to start an independent career as a Researcher, Precursory Research for Embryonic Science and Technology (PRESTO),
Japan Science and Technology Agency (JST)(2001-2005). In February 2002 he became an Assistant Professor, University of Tokyo, and in January 2004 he moved toOsaka to serve as a Visiting Associate Professor, Osaka University. Since October
2007, he holds a current position as a Team Leader, Glycometabolome Team, Systems Glycobiology Research Group, RIKEN. He also serves as a Visiting Professor at
Saitama University since 2010.
​Dr. Suzuki is a recipient of Young Scientist Award, FCCA (Forum: Carbohydrate
Coming of Age)(1996), Genzyme Award, Society for Glycobiology (1997), YoungInvestigator Award, the Japanese Biochemical Society (2005), Young InvestigatorAward, the Japanese Society of Carbohydrate Research (2008) and Glycobiology
Significant Achievement Award, Society for Glycobiology (2016). He serves as an
Executive Editor for Biochimica Biophysica Acta-General Subjects (2016-) and an
editorial board member for Glycobiology (2016-).
​The biosynthetic/processing pathways for N-glycans have been
well characterized in mammalian cells. There are, however, issues
that remain to be clarified concerning aspects of their degradation.
While the molecular mechanism of the lysosomal degradation for
N-glycoproteins has been well studied in relation to genetic disorders, evidence exists to suggest that there are also “non-lysosomal”
degradation processes, which are now known to occur widely ineukaryotic cells.
The cytoplasmic peptide:N-glycanase (PNGase) is the enzyme involved in the non-lysosomal degradation of misfolded N-glycopro-42

the 25th Anniversary of Mizutani Foundation for Glycoscienceteins, a process called ERAD (ER-associated degradation) 1),2). In 2012,a patient harboring mutations of PNGase gene (NGLY1) was first reported. Symptom of these patients includes developmental delay,multifocus epilepsy, involuntary movement and liver dysfunction.
This report clearly demonstrated that the cytoplasmic PNGaseplays a pivotal role in normal human development.
The past decade we intensively analyzed Ngly1-deficient miceand found that they are embryonic lethal in C57BL/6 (B6) background. Surprisingly, the additional deletion of Engase, encoding
another cytosolic deglycosylating enzyme involved in the non-MEMORATIVE SYMPOSIUM

Figure 1

Non-lysosomal catabolism of N-glycoproteins, free N-glycans and dolichol-linked oligosaccharides in mammalian cells (Harada, et al., Cell Mol Life Sci 72, 2509-2533 (2015)). Dol:dolichol; ENGase: endo-β-N-acetylglucosaminidase; Man2C1: cytosolic α-mannosidase; OST:
oligosaccharyltransferase; PNGase/NGLY1: peptide:N-glycanaselysosomal degradation of N-glycoproteins called ENGase (endoβ-N-acetylglucosaminidase), resulted in the partial rescue of thelethality of the Ngly1-deficient mice 3). Additionally, we also foundthat a change in the genetic background of B6 mice, produced bycrossing the mice with an outbred mice strain (ICR) could rescue
the embryonic lethality of Ngly1-deficient mice 3). Viable Ngly1-deficient mice in a B6 and ICR mixed background, however, showeda very severe phenotype reminiscent of the symptoms of NGLY1deficiency subjects. Again, many of those defects were strongly
suppressed by the additional deletion of Engase in the B6 and ICR
mixed background.
We also showed that in Ngly1-KO cells, ERAD process was compromised. Interestingly, not only delayed degradation but also thedeglycosylation of a model substrate was observed in this cell.
The unexpected deglycosylation was found to be mediated by ENGase. Surprisingly, the ERAD dysregulation in Ngly1-KO cells wererestored by the additional KO of Engase gene. These observations
collectively suggest that the ENGase represents one of the poten-tial therapeutic targets for this genetic disorder 4).​Besides degradation of N-glycoproteins, there are also novel pathways exist in cells for the catabolism of free N-glycans (FNGs); freeoligosaccharides related to N-glycans, as well as dolichol-linked
oligosaccharides (DLOs). The recent years we have also analyzed
the following novel catabolic pathways for FNGs and DLOs; (i) oligosaccharyltransferase-mediated release of FNGs in the ER 5),6) ; (ii)pyrophosphatase acting on dolichol-linked oligosaccharides 7),8) ;and (iii) formation and catabolism of sialyl FNGs inside and outsideof cells 9),10). Occurrence as well as biological importance of thosenovel pathways have been largely overlooked, partly because there
are several genes for enzymes/transporters that remained to beidentified. I have a firm belief that clarification of those novel pathway, which should constitute the one of the most basic biologicalprocesses, will lead to not only providing better insight into theprocess, but also understand the human health and disease. In thissymposium, I will overview our recent findings on NGLY1 and novel
degradation pathways for FNGs/DLOs.

References
1)	Suzuki T, Huang C, Fujihira H The cy toplasmic peptide:N-glycanase (NGLY1); structure, expression and cellular functions. Gene (577): 1-7, 20162)	Suzuki T The cytoplasmic peptide:N-glycanase (PNGase) – basic science encounters a human genetic disorder. J Biochem (157): 23-34, 20153)	Fujihira H, Masahara-Negishi Y, Tamura M, Huang C, Harada Y, Wakana S, Takakura D, Kawasaki N, Taniguchi N, Kondoh G, Yamashita T, Funakoshi Y, Suzuki T
Lethality of Ngly1-knockout mice is partially rescued by the additional deletion of the Engase gene. PLOS Genet (13): e1006696, 20174)	Huang C, Harada Y, Hosomi A, Masahara-Negishi Y, Seino J, Fujihira H, Funakoshi Y, Suzuki T, Dohmae T, Suzuki T Endo-β-N-acetylglucosaminidase forms NGlcNAc protein aggregates during ER-associated degradation in Ngly1-defective cells. Proc Natl Acad Sci USA (112): 1398-1403, 20155)	Harada Y, Buser R, Ngwa EM, Hirayama H, Aebi M, Suzuki T Eukaryotic oligosaccharyltransferase generates free oligosaccharides during N-glycosylation. J BiolChem (288): 32673-32684, 20136)	Harada Y, Masahara-Negishi Y, Suzuki T Cytosolic-free oligosaccharides are predominantly generated by the degradation of dolichol-linked oligosaccharides inmammalian cells. Glycobiology (25): 1196-1205, 20157)	Harada Y, Nakajima K, Masahara-Negishi Y, Freeze HH, Angata T, Taniguchi N, Suzuki T Metabolically programmed quality control system for dolichol-linkedoligosaccharides. Proc Natl Acad Sci USA (110): 19366-19371, 20138)	Harada Y, Huang C, Yamaki S, Dohmae N, Suzuki T Non-lysosomal degradation of phophorylated oligosaccharides is initiated by the cytosolic endo-β-Nacetylglucosaminidase. J Biol Chem (291): 8048-8058, 20169)	Seino J, Wang L, Harada Y, Huang C, Ishii K, Mizushima N, Suzuki T Basal autophagy is required for the efficient catabolism of sialyl oligosaccharides. J Biol Chem
(288): 26898-26907, 201310)	Seino J, Fujihira H, Nakakita S, Masahara-Negishi Y, Miyoshi E, Hirabayashi J, Suzuki T Occurrence of sialyl oligosaccharides related to N-glycans (sialyl FNGs) inanimal sera. Glycobiology (26): 1072-1085, 2016

Commemorative Symposium43