Peptide:N-glycanase (PNGase; also called glycoamidase
or N-glycanase) is a deglycosylating enzyme which acts upon asparagine-linked
(N-linked) glycans on glycoproteins/glycopeptides. PNGases
isolated from plants or bacteria have been used as powerful “tool”
reagents for structural and functional studies of N-linked
glycans on glycoproteins. Curiously enough, the physiological significance
of this enzyme itself has long been undocumented until recently.
|Cytoplasmic PNGase - A Player Involved in GERAD-Process|
In eukaryotes, many proteins synthesized in the cytoplasm
are translocated across, or integrated into, the membrane of the endoplasmic
reticulum (ER), a central organelle for delivery of these soluble or
membrane proteins to their respective destination. As this organelle
is also responsible for monitoring proper folding/assembly of proteins,
those with abnormal structure arising from mutations or metabolic damage
can be eliminated. These proofreading and elimination system (“quality
control system”) ensures cells to avoid the intracellular accumulation
of nonfunctional proteins. In this system, proteins that fail to fold
productively are retained in the ER by various lumenal chaperones, assisting
their folding and appropriate subunit assembly. However, if some proteins
maintain undesired structure despite the effort by chaperone system,
they are destined for destruction by a mechanism known as an “ER-associated
ERAD involves three successive steps: (I) sensing of aberrant proteins
in the ER, (II) the retrotranslocation of substrates into the cytosol,
and (III) degradation of them by ubiquitin-proteasome system 4.
It has also been suggested that there is an evolutionally conserved
system specifically dedicated for N-linked glycoproteins, named
glycoprotein ERAD (GERAD) 5.
Understanding the detail of ERAD/GERAD system is significant from not
only for our understanding of this basic cellular mechanism but also
for the medical application regarding the treatment of various human
inherited or acquired disorders caused by the abnormality of this surveillance
|Reaction Mechanism of Cytoplasmic PNGase - Reverse Reaction with the Similar Catalytic Triad|
|The gene encoding the cytoplasmic PNGase (PNG1 in yeast) was first identified in S. cerevisiae 7. Extensive survey in sequence databases has revealed that this enzyme is conserved throughout eukaryotes. The enzymes in budding and fission yeasts have a highly homologous “core” sequence, while orthologues in higher eukaryotes, unlike those from yeast, have extended domains at both the N- and C-termini of the core domain 1, 7. One of the interesting features of the PNGases is the presence of a "transglutaminase" motif in the most conserved region by which they have been proposed to belong to the transglutaminase-like superfamily 9. Transglutaminase is an enzyme that establishes covalent links between proteins by formation of amide crosslinks between the side chains of glutamate and lysine residues (Fig. 1). In most cases, proteins in the transglutaminase family possess a putative catalytic triad consisting of cysteine, histidine, and aspartate residues. Since the proteins in the transglutaminase superfamily are so far known to be responsible for either formation (transglutamindase) or hydrolysis (protease) of amide bonds, it is not surprising that the cytoplasmic PNGase, an amidase, has been defined as a third type of enzyme belonging to this superfamily. Indeed, the potential catalytic triad in this domain is conserved in all PNGase orthologues. It is also important to note that reducing reagents such as dithiothreitol are required for in vitro enzyme activity of the cytoplasmic PNGases 10, 11. This observation is consistent with the assumption that they have a cysteine residue that acts as a critical nucleophile for its enzymatic activity. Furthermore, the extensive mutagenesis study using yeast PNGase has shown that these amino acid residues are indeed essential for enzyme catalysis, suggesting a common evolutionary lineage for the cytoplasmic PNGases and transglutaminases 12.|
Fig. 1 Schematic
representation of enzyme reaction by transglutaminase (upper) and peptide:N-glycanase
|Complex Formation of Cytoplasmic PNGase - Getting More Complex??|
|We have shown that Rad23 protein (Rad23p) is a PNGase-binding
protein both in budding yeast as well as in mammalian cells 13,
14. Rad23p, whose gene was originally identified
as the one related with DNA repair, has been shown to exhibit multiple
function in diverse cellular processes, while this protein is able to
bind to the 26S proteasome by the N-terminal ubiquitin-like domain. In
fact, the physical interaction of the yeast PNGase (Png1p) with the 26S
proteasome occurs in a Rad23p-dependent manner in yeast 13.
The Rad23p-Png1p complex has been found to exist as a distinct complex
from the well-established Rad23p-Rad4p complex, which is required for
DNA repair. Recent evidence also shows that the two UBA (ubiquitin-associated)
domains in Rad23p can actually bind to ubiquitin/multiubiquitin chain,
a major “tag” for degradation by the 26S proteasome. These
observations suggest that Rad23 may serve as an “escort” function
to connect the 26S proteasome with other proteins and exhibit diverse
functions by controlling the stability and/or the functions of various
Regarding the complex formation of PNGase, the recent finding of the PUB/PUG domain as a novel sequence motif within the extended N-terminal region of PNGases from vertebrates and insects is particularly of interest 15, 16. This domain is highly homologous across the species, while PNGases from fungi or plants does not possess it. Our preliminary experimental evidence shows that the PUB/PUG domain is important for the binding of the mPng1p to a wide variety of ubiquitin/proteasome pathway-related proteins 14. Interestingly, all of the interactions except that of Rad23p orthologue, which requires the core catalytic domain for interaction, utilize the N-terminal domain, including the PUB/PUG domain, of mouse PNGase orthologue.
The functional importance of the PUB/PUG domain in proteins has not yet been proven. Nonetheless It is noteworthy that some of proteins that have PUB/PUG domains were found to have either a UBA or a UBX domain, a sequence motif present in multiple enzyme classes in the ubiquitin-related pathway, though the functions of these proteins are so far unknown. Recent studies suggest that both UBA and, most likely UBX domains function as protein-protein interaction domains 17. Accordingly, together with our experimental observation, it has been hypothesized that the PUB/PUG domain, as proposed for UBA or UBX domain, may also serve as a protein-protein interaction domain. One can readily envision that, together with UBA or possibly UBX domains, the PUB/PUG domain is able to generate a number of protein-protein interaction networks in many cellular processes including the ubiquitin-proteasome pathway. In this case, the acquisition of PUB/PUG domain may result in a formation of a “glycoprotein-degradation complex” in order to facilitate a series of reactions such as retro-translocation, ubiquitination, deglycosylation, deubiquitination and degradation of proteins at one cellular site 2. Demonstration of the occurrence as well as the characterization of these complexes would be the next logical step to substantiate this hypothesis.
|Cytoplasmic PNGase-Catalyzed Deglycosylation of Glycoproteins - They Know the Difference!|
| The evidence that the cytoplasmic
PNGase is involved in GERAD has been presented by several experiments.
First, the small N-glycosylatable peptide, which follows the
similar fate as that of misfolded glycoproteins, is deglycosylated by
the cytoplasmic PNGase in the cytosol 18,
19. Moreover, the amount of free glycan, the majority
of which has been generated by PNGase in budding yeast 20,
is dramatically reduced by inhibition of proteasomal proteolysis 21.
This result indicates that the PNGase-mediated deglycosylation and proteasomal
degradation are possibly correlated with each other. Furthermore, recent
observation has proven that PNGase is capable of acting on misfolded glycoproteins
that are GERAD substrates 8.
This finding strongly supports the idea that PNGase deglycosylates glycoprotein
prior to proteasomal degradation. The cytoplasmic PNGase seems to distinguish
between native and unfolded status of glycoproteins, and only deglycosylates
glycoproteins when they are extensively denatured 8. It is important to
note that since the unfolding of proteins are not prerequisite for retrotranslocation
of proteins from the ER to the cytosol 22, it is feasible to assume, at
least in some cases, that there is an “unfolding” step of
proteins prior to the action of PNGase to proteins in the cytosol.
The latest study has identified the lectin-ubiquitin ligase, Fbx2, to be involved in GERAD process 23. The lectin forms a SCF (Skp1-Cullin1-F-box) complex, which serves as an E3-ligase. It was revealed that the SCFFbx2 complex was able to recognize the misfolded glycoproteins for ubiquitination in N-glycan-dependent manner. Considering its reaction mechanism, the recognition of glycoprotein substrates by SCFFbx2 complex should presumably precede their deglycosylation by PNGase (Fig. 2).
Fig. 2 Hypothetical
model for degradation of a misfolded glycoprotein; cytosolic events
|When the occurrence of cytoplasmic peptide:N-glycanase (PNGase) was first reported in 1993 24, there has always been a difficult yet fundamental question regarding this enzyme; “Why in the cytosol?”. At that time few people believed that N-glycosylated proteins, the supposedly the substrates for the PNGase, can exist in the cytosol. However, after 10 years, it is now taken for granted that the “retrotranslocation” phenomena of (glyco)proteins from the ER to the cytosol does occur ubiquitously and today few people cast a doubt upon the implication of this enzyme in GERAD process, not to mention the presence of it. Now that the gene encoding the cytoplasmic PNGases has been identified and that structural features of this enzyme has been studied, the next focus will be on its functional aspect, especially in terms of phenotypic consequences due to its defect, in cells and individuals in higher eukaryotes.|
|July 25, 2003 / Copyright (c) Glycoforum, All Rights Reserved.|