Peptide:N-glycanase (PNGase) and free N-glycans in cytosol
	Cytoplasmic Peptide:N-glycanase Peptide:N-glycanase (PNGase) releases N-glycans from glycoproteins/glycopeptides, and this enzyme from bacteria and plants has been extensively used as a powerful "tool" reagent to study the structure and biological functions of N-linked glycans on glycoproteins. The cytoplasmic PNGases, ubiquitously found throughout eukaryotes, are now widely recognized as a component implicated in the ERAD (ER-associated degradation) process, which constitute one of the quality control mechanisms for newly synthesized misfolded glycoproteins exported from the ER lumen. The enzyme is classified as a transglutaminase-superfamily that contains a putative catalytic triad of amino acids (cysteine, histidine, and aspartic acid). The mammalian orthologues of PNGase contain the N-terminal PUB domain, which serves as a protein-protein interaction domain, as well as the C-terminus domain, which has recently been found to be a novel carbohydate-binding domain. These structural features indicate the sophisticated coordination of this protein in complex formation, as well as substrate recognition. For more details on the structure and functions of this enzyme, the reader is directed to our recent reviews (1-3).
	Free N-linked glycans: formation and degradation The occurrence of PNGase in cytosol and its participation in the ERAD process is obvious, but one question arises : what will be the fate of "free" N-glycans that are released by PNGase in the cytosol? Since the discovery of the ERAD process, extensive studies have focused on the fate of misfolded proteins. Curiously enough, however, not much attention has been paid with regard to the molecular mechanism and importance of free oligosaccharide metabolism in cytosol. In mammalian cells, so far two cytosolic glycosidases have been identified to be involved in the processing of free oligosaccharides: endo-β-N-acetylglucosaminidase (ENGase) and α-mannosidase (Man2C1) (Figure 1). It should be noted here that there are still many more enzymes/transporters that remain to be identified, although this "non-lysosomal" metabolic path for N-glycans may represent one of the very basic biological phenomena in eukaryotes (Figure 1). For further reading on this topic, the reader is directed to our recent review (4).
Fig.1 Proposed fate of free oligosaccharides generated in and out of the ER in mammalian cells. (1) Free oligosaccharides, generated in the lumen of the ER by an unknown mechanism, bear N, N'-diacetylchitobiose (Gn2) at their reducing termini. (2) Free oligosaccharides can also be generated by the action of PNGase to misfolded glycoproteins, releasing Gn2 glycans. (3) Putative pyrophosphatase, the activity of which is reported in the cytosolic face of the ER membrane, can also release free oligosaccharide-phosphate from the dolichol-linked oligosaccharide into the cytosol. Probably this oligosaccharide-phosphate will be processed by ENGase to give rise to Gn1 glycan, which has only a single GlcNAc at its reducing terminus and is readily transported into lysosomes. (4) After quick deglucosylation by -glucosidase I and II (and ER -mannosidase I), Man8~9GlcNAc2 in the lumen of the ER is transported into the cytosol by a putative transporter. (5) Once in the cytosol, ENGase (or in some cases a chitobiase) acts on Gn2 sugars (lipid- or protein-derived) and forms Gn1 glycans (Man8~9GlcNAc). (6) Gn1 is now susceptible to the action of a cytosolic -mannosidase (Man2C1), giving rise to the specific Man5GlcNAc structure. The isomeric structure Man5 is identical to that of the last dolichol intermediate oriented to the cytosolic face. (7) The Man5GlcNAc is transported into lysosomes by a specific transporter. In lysosomes the Man5GlcNAc is hydrolyzed into monomers (Man and GlcNAc) by lysosomal - and -mannosidases. This non-lysosomal degradation pathway has been deduced mainly from the structures of free oligosaccharides that have been biochemically isolated.
	Tadashi Suzuki (Department of Biochemistry and 21st COE(Center of Excellence) Program, Osaka University Graduate School of Medicine; CREST (Core Research for Evolutionary Science and Technology), JST (Japan Science and Technology Agency))
	References (1) Suzuki, T., Park, H., Lennarz, W. J. (2002) Cytoplasmic peptide:N -glycanase (PNGase) in eukaryotic cells: occurrence, primary structure, and potential functions. FASEB J., 16, 635-641 (2) Suzuki, T., Lennarz, W. J. (2003) Hypothesis: a glycoprotein-degradation complex formed by protein-protein interaction involves cytoplasmic peptide:N-glycanase. Biochem. Biophys. Res. Commun., 302, 1-5. (3) Suzuki, T. (2007) Cytoplasmic peptide:N-glycanase and catabolic pathway for free N-glycans in the cytosol. Sem. Cell Dev. Biol. In press. (4) Suzuki, T., Funakoshi, Y. (2006) Free N-linked oligosaccharide chains: formation and degradation. Glycoconj. J., 23, 291-302. Links Cytoplasmic peptide:N-glycanase (Tadashi Suzuki)
Apr. 19, 2007