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Biological Roles of N-glycanase in Cells |
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Anyone who has some interest in glycobiology may be familiar with N-glycanase [peptide-N4(N-acetyl-beta-D-glucosaminyl)asparagine amidase, EC 3.5.1.52, peptide:N-glycanase abbreviated as PNGase ] used as a tool to cleave N-glycan chains from glycopeptides and glycoproteins but only a limited number of individuals have some knowledge of the biological roles of N-glycanase in living cells. It is a subject that has come to be recognized by glycobiologists only recently, simply because only very few researchers have come across biological phenomena that can be logically explained by the action of N-glycanase in animal cells. The first identification of N-glycanase in animal cells was made in 1991 by Seko et al. in the embryos of medaka fish. The finding of accumulation in the embryos of certain fish species of free N-glycan chains terminating with a di-N-acetylchitobiosyl structure at their recucing termini led to the discovery of N-glycanase in fish. In studies on both identification and function of N-glycanase, the structural features of the products of enzyme reaction should be noted: the free oligosaccharide chain should retain a di-N-acetylchitobiosyl structure, and the protein (peptide) chain should have an Asp residue in place of Asn to which the glycan chain was originally linked (Fig.1). These structural features are important in discriminating N-glycanase activity from that of endo-beta-N-acetylglucosaminidase that also cleaves the N-glycan chain leaving a single residue of N-acetylglucosamine on the Asn residue of the peptide chain. Di-N-acetylchitobiosyl structure is often lost from the liberated free glycan chains because it is cleaved by endogenous enzyme(s) (chitobiase activity has been identified in mammalian cells). The transformation of the glycosylated Asn−> Asp in the deglycosylated protein (peptide) results in a large change in the properties of the molecule by the acquirement of a negative charge in addition to the loss of the glycan chain. This change is important to identify the deglycosylated protein (peptide) by electrophoretic techniques and helps in the identification of N-glycanase activity. In 1993 Suzuki et al. showed the occurrence of N-glycanase in mammalian cells including those of human origin. Studies on the properties of partially purified N-glycanase from L-929 cells revealed that the enzyme also possesses a lectin-like property. In 1995 the occurrence of N-glycanase in various organs of mouse was shown. |
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 Fig. 1 De-N-glycosylation catalyzed by N-glycanase |
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| The results obtained from studies aiming to clarifying the biological function of N-glycanase strongly suggest its involvement in a "quality control" mechanism of the newly synthesized ovalbumin in hen oviduct. Ovalbumin isolated from egg white contains a single N-glycan chain linked to Asn-292, whereas an "intermediate form" of ovalbumin containing two N-glycans at Asn-292 and Asn-311 had been isolated from hen oviduct. N-glycanase purified from hen oviduct site-specifically cleaved the glycan chain at Asn-311 from its physiological substrate, the "intermediate" ovalbumin, but did not act on hen egg ovalbumin. The results suggested that while monoglycosylated albumin was destined for secretion the diglycosylated molecule was arrested by "quality control check" and deglycosylated by N-glycanase. The problem whether deglycosylated albumin is deployed to compartments of the secretory pathway or is degraded after further proteolytic degradation is yet to be clarified. The reports described below may support the latter pathway. To clarify intracellular localization of N-glycanase is important to evaluate its physiological significance. A number of reports in the fields of cell biology and immunology have appeared since 1996 in which the observed phenomena can be best explained if possible involvement of N-glycanase is considered. One is related to the "quality control" of nascent proteins that failed to fold or to oligomerize correctly, or more directly to the mechanism for degrading these unsecreted proteins. Cases observed in the formation of MHC (major histocompatiblity antigen complex) and TCR (T cell antigen receptor) have been reported. At one time the entire processes were assumed to occur within the ER (endoplasmic reticulum) but failure to find proteolytic activity in the ER and inhibition of the process by inhibitors of the cytoplasmic proteasome led to an updated concept that major degradation processes occur in cytoplasm. The mechanism to extrude proteins back into the cytosol, i.e., a process known as reverse translocation, has been solved. Thus, the glycosylated subunits are extruded into the cytosol and subjected to deglycosylation by N-glycanase prior to proteolysis by the proteasome (Fig.2). In one report the amino acid sequence of the viral peptide found in the cytosol was compared with those predicted from viral DNA to show the involvement of N-glycanase, while some reported a change in the isoelectric point when the putative proteins found in the cytosol were compared. In other reports the evidence was poor and only the lack of N-glycans in these proteins was shown. A similar mechanism was shown in the rapid degradation of MHC class I molecules in human cytomegalovirus (HCMV)-infected cells. Viruses have evolved sophisticated mechanisms to evade host immune systems. In HCMV viral proteins are associated with newly synthesized MHC class I molecules and quickly escape from the ER to cytosol together with MHC class I molecules. It has been proposed that not only MHC class I molecule but also the virus protein (e.g.,US2) are deglycosylated by N-glycanase before they are transported to the cytosolic proteasome. Thus in the cell infected with the virus not only are the sites of antigen presentation lost but the structure of viral peptide is also changed. In these studies more detailed information on subcellular localization of N-glycanase is necessary to clarify the proposed mechanism. It may be difficult to answer the question what happens if N-glycanase is absent in the cell. Perhaps it is like the question as what happens to the cell or animal if glycosylation does not occur on some glycoproteins. In many systems living organisms may have evolved the ability to do without N-glycanase. Most recently studies aimed at solving these questions have been started using yeast, and the presence of N-glycanase in the cytosolic fraction of yeast has been reported. |
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 Fig. 2 Fate of unfolded and mistold glycoproteins |
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| It is well known that N-glycanase was first found in almond seeds. Relatively high activity of N-glycanase was detected in various plant seeds. Some preliminary studies aimed at solving functions of these plant N-glycanase have appeared recently. In plants the presence of free N-glycan chains has been reported. Involvement of N-glycanase in the accumulation of these glycan chains, and the physiological significance of free glycan chains in the budding of seeds and maturation of fruits are problems to be solved in the future. As in the case of fish embryo the pool of free glycan chains in these cells is large and may have a distinctive function for the cell, as opposed to just being waste. Studies on the embryonic development of fish manifested expression of two different types of N-glycanase in the different stages of development. Interestingly the putative endogenous substrate for each type of N-glycanase was shown to be different types of glycoproteins. Contributions from researchers working in fields other than glycobiology are desired to obtain a solution to this problem as well as for the further development of these studies. | |||||||||||||||||
| Sadako Inoue (Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan) |
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| Jun. 15, 1999 | |||||||||||||||||
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