Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.5.1.52 (PNGase F)
1,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The human cytomegalovirus-encoded glycoprotein US2 catalyzes proteasomal degradation of Class I major histocompatibility complex (MHC) heavy chains (HCs) through dislocation of the latter from the endoplasmic reticulum (ER) to the cytosol. During this process, the Class I MHC HCs are deglycosylated by an N-glycanase-type activity. siRNA molecules designed to inhibit the expression of the light chain, beta(2)-microglobulin, block the dislocation of Class I MHC molecules, which implies that US2-dependent dislocation utilizes correctly folded Class I MHC molecules as a substrate. Here we demonstrate it is peptide: N-glycanase (PNGase or PNG1) that deglycosylates dislocated Class I MHC HCs. Reduction of PNGase activity by siRNA expression in US2-expressing cells inhibits deglycosylation of Class I MHC HC molecules. In PNGase siRNA-treated cells, glycosylated HCs appear in the cytosol, providing the first evidence for the presence of an intact N-linked type I membrane glycoprotein in the cytosol. N-glycanase activity is therefore not required for dislocation of glycosylated Class I MHC molecules from the ER.
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PMID:A glycosylated type I membrane protein becomes cytosolic when peptide: N-glycanase is compromised. 1474 36

The bovine herpesvirus 5 (BHV-5) gE ectodomain contains a glycine-rich epitope coding region (gE5 epitope), residues 204 to 218, that is significantly different from the corresponding gE region of BHV-1. Deletion of the gE epitope significantly reduced the neurovirulence of BHV-5 in rabbits. Pulse-chase analyses revealed that the epitope-deleted and wild-type gE were synthesized as N-glycosylated endoglycosidase H-sensitive precursors with approximate molecular masses of 85 kDa and 86 kDa, respectively. Like the wild-type gE, epitope-deleted gE complexed with gI and was readily transported from the endoplasmic reticulum. Concomitantly, the epitope-deleted and wild-type gE acquired posttranslational modifications in the Golgi leading to an increased apparent molecular mass of 93-kDa (epitope-deleted gE) and 94-kDa (wild-type gE). The kinetics of mutant and wild-type gE processing were similar, and both mature proteins were resistant to endoglycosidase H but sensitive to glycopeptidase F. The gE epitope-deleted BHV-5 formed wild-type-sized plaques in MDBK cells, and the epitope-deleted gE was expressed on the cell surface. However, rabbits infected intranasally with gE epitope-deleted BHV-5 did not develop seizures, and only 20% of the infected rabbits showed mild neurological signs. The epitope-deleted virus replicated efficiently in the olfactory epithelium. However, within the brains of these rabbits there was a 10- to 20-fold reduction in infected neurons compared with the number of infected neurons within the brains of rabbits infected with the gE5 epitope-reverted and wild-type BHV-5. In comparison, 70 to 80% of the rabbits exhibited severe neurological signs when infected with the gE5 epitope-reverted and wild-type BHV-5. These results indicated that anterograde transport of the gE epitope-deleted virus from the olfactory receptor neurons to the olfactory bulb is defective and that, within the central nervous system, the gE5 epitope-coding region was required for expression of the full virulence potential of BHV-5.
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PMID:A glycine-rich bovine herpesvirus 5 (BHV-5) gE-specific epitope within the ectodomain is important for BHV-5 neurovirulence. 1507 62

The proton-pumping H+,K+-adenosinetriphosphatase (H,K-ATPase), responsible for acid secretion by the gastric parietal cell, faces a harshly acidic environment, with some pepsin from neighboring chief cells, at its luminal surface. Its large catalytic alpha-subunit is mostly oriented cytoplasmically. The smaller beta-subunit (HKbeta), is mainly extracellular, with one transmembrane domain and a small cytoplasmic domain. Seven N-linked oligosaccharides in the extracellular domain of HKbeta are thought to contribute to protection of the H,K-ATPase, since previous work has shown that their complete removal, by peptide N-glycosidase F (PNGase F), greatly increased susceptibility of HKbeta to proteolysis. The possibility of graded protection by different numbers of oligosaccharides was investigated here with the use of mutant HKbeta cDNA, having various N-glycosylation sites mutated (Asn to Gln), transfected into HEK-293 cells. Membrane preparations, two days after transfection, were solubilized in 1% Triton X-100 and subjected to trypsinolysis (pH 8, 37 degrees C, trypsin:protein 1:10-1:25). Relative amounts of HKbeta remaining after 20 min trypsin were determined, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and probing of Western blots with an antibody to the HKbeta extracellular domain, by chemiluminescent development of blots and densitometry of resulting films. Maturely glycosylated HKbeta was made significantly more susceptible to trypsin than wild type when at least five oligosaccharides were deleted, while the high-mannose form (pre-beta), from the endoplasmic reticulum, became significantly more susceptible than wild-type pre-beta with removal of only two or more oligosaccharides. For each mutant, and wild type, pre-beta was consistently more susceptible than the mature form. While the number, and kind, of oligosaccharides seem to affect protection for HKbeta against trypsinolysis, other aspects of protein maturation, including proper folding of peptide domains and possible subtle alterations of conformation during Golgi processing, are also likely to contribute to this protection.
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PMID:Contribution of oligosaccharides to protection of the H,K-ATPase beta-subunit against trypsinolysis. 1530 Jul 79

A cytoplasmic peptide: N-glycanase (PNGase) has been implicated in the proteasomal degradation of aberrant glycoproteins synthesized in the endoplasmic reticulum. The reaction is believed to be important for subsequent proteolysis by the proteasome since bulky N-glycan chains on misfolded glycoproteins may impair their efficient entry into the interior of the cylinder-shaped 20S proteasome, where its active site resides. This cytoplasmic enzyme was first detected in 1993 by a simple, sensitive assay method using 14C-labeled glycopeptide as a substrate. The deglycosylation reaction by PNGase brings about two major changes on substrate the peptide; one is removal of the N-glycan chain and the other is the introduction of a negative charge into the core peptide by converting the glycosylated asparagine residue(s) into an aspartic acid residue(s). The assay method we developed monitors these major changes in the core peptide, and the respective changes were detected by distinct analytical methods: i.e., paper chromatography and paper electrophoresis. This chapter will describe the simple, sensitive in vitro assay method for PNGase.
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PMID:A simple, sensitive in vitro assay for cytoplasmic deglycosylation by peptide: N-glycanase. 1580 8

The deglycosylating enzyme, peptide:N-glycanase, acts on misfolded N-linked glycoproteins dislocated from the endoplasmic reticulum (ER) to the cytosol. Deglycosylation has been demonstrated to occur at the ER membrane and in the cytosol. However, the mechanism of PNGase association with the ER membrane was unclear, because PNGase lacked the necessary signal to facilitate its incorporation in the ER membrane, nor was it known to bind to an integral ER protein. Using HeLa cells, we have identified a membrane protein that associates with PNGase, thereby bringing it in close proximity to the ER and providing accessibility to dislocating glycoproteins. This protein, Derlin-1, has recently been shown to mediate retrotranslocation of misfolded glycoproteins. In this study we demonstrate that Derlin-1 interacts with the N-terminal domain of PNGase via its cytosolic C-terminus. Moreover, we find PNGase distributed in two populations; ER-associated and free in the cytosol, which suggests the deglycosylation process can proceed at either site depending on the glycoprotein substrate.
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PMID:The retrotranslocation protein Derlin-1 binds peptide:N-glycanase to the endoplasmic reticulum. 1605 2

Peptide N-glycanase (PNGase) is involved in the cleavage of oligosaccharide chains from misfolded glycoproteins that are destined for degradation by the proteasome. Earlier, a number of potential binding partners of mouse PNGase (mPNGase) were detected by using the yeast two-hybrid system. In the current study, an in vitro system was set up to investigate direct interactions between mPNGase and these candidate proteins. Although the yeast two-hybrid system suggested an interaction of six different proteins with mPNGase, only mHR23B and the proteasome subunit mS4 were found to interact with mPNGase. In fact, mS4 competes with mHR23B for binding to mPNGase. These results suggested two possible pathways for the interaction between mPNGase and the proteasome. In one pathway, mHR23B mediates the interaction between mPNGase and the proteasome. In an alternative pathway, mPNGase directly binds to the proteasome subunit, mS4. In either case, it is clear that PNGase is located in close proximity to the proteasome and is available for deglycosylation of glycoproteins destined for degradation. Surprisingly, mPNGase also was found to mediate binding of the cytoplasmic protein, p97, to the proteasome through the formation of a ternary complex made up of mHR23B, mPNGase, and p97. Because p97 is known to bind to the endoplasmic reticulum membrane protein AMFR (gp78), an E3 ligase, we propose a model in which p97, mPNGase, and mHR23B mediate interaction of the endoplasmic reticulum with the proteasome.
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PMID:Multiple modes of interaction of the deglycosylation enzyme, mouse peptide N-glycanase, with the proteasome. 1624 33

Peptide N-glycanase removes N-linked oligosaccharides from misfolded glycoproteins as part of the endoplasmic reticulum-associated degradation pathway. This process involves the formation of a tight complex of peptide N-glycanase with Rad23 in yeast and the orthologous HR23 proteins in mammals. In addition to its function in endoplasmic reticulum-associated degradation, HR23 is also involved in DNA repair, where it plays an important role in damage recognition in complex with the xeroderma pigmentosum group C protein. To characterize the dual role of HR23, we have determined the high resolution crystal structure of the mouse peptide N-glycanase catalytic core in complex with the xeroderma pigmentosum group C binding domain from HR23B. Peptide N-glycanase features a large cleft between its catalytic cysteine protease core and zinc binding domain. Opposite the zinc binding domain is the HR23B-interacting region, and surprisingly, the complex interface is fundamentally different from the orthologous yeast peptide N-glycanase-Rad23 complex. Different regions on both proteins are involved in complex formation, revealing an amazing degree of divergence in the interaction between two highly homologous proteins. Furthermore, the mouse peptide N-glycanase-HR23B complex mimics the interaction between xeroderma pigmentosum group C and HR23B, thereby providing a first structural model of how the two proteins interact within the nucleotide excision repair cascade in higher eukaryotes. The different interaction interfaces of the xeroderma pigmentosum group C binding domains in yeast and mammals suggest a co-evolution of the endoplasmic reticulum-associated degradation and DNA repair pathways.
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PMID:Structure of the mouse peptide N-glycanase-HR23 complex suggests co-evolution of the endoplasmic reticulum-associated degradation and DNA repair pathways. 1650 Sep 3

Endomembrane (endoplasmic reticulum, Golgi apparatus, plasma membrane) proteins of soybean (Glycine max) root cells are highly glycosylated. We investigated whether N-linked oligosaccharide moieties are essential for the correct intracellular transport of plant endomembrane glycoproteins. Excised roots were incubated with tunicamycin, to block cotranslational glycosylation of proteins, and dual labeled with [(3)H]glucosamine and [(35)S] (methionine, cysteine). In the presence of tunicamycin, the incorporation of glucosamine into membrane proteins was inhibited by 60 to 90% while amino acid incorporation was only slightly affected. Autoradiograms of two-dimensionally separated polypeptides from each endomembrane fraction revealed the presence of at least one new polypeptide in tunicamycin-treated tissue. The new polypeptide was of the same isoelectric point but lower molecular weight than a preexisting polypeptide. The new polypeptide was unreactive to concanavalin A, as opposed to the preexisting polypeptide, suggesting the absence of the glycan portion. Trifluoromethanesulfonic acid and N-glycanase were used to cleave the carbohydrate from the preexisting concanavalin A binding polypeptide. In each case a deglycosylated polypeptide of the same isoelectric point and molecular weight as the new polypeptide from tunicamycin-treated tissue resulted. Since the absence of carbohydrate from the new endomembrane polypeptide did not prevent its appearance on autoradiograms of Golgi and plasma membrane, intracellular transport and intercalation of newly synthesized glycoproteins into plant cell membranes may not require the presence of polysaccharide moieties.
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PMID:Effect of Inhibition of Glycosylation on the Appearance of a 60 kD Membrane Glycopolypeptide in Endomembrane Fractions of Soybean Root. 1666 30

Mouse peptide N-glycanase (mPNGase) cleaves the N-glycan chain from misfolded glycoproteins and glycopeptides. Previously, several proteins were found to directly interact with mPNGase; among them, both mHR23B and mS4 were found to link mPNGase to the proteasome. In this study, we found that the cytoplasmic protein mp97 participates in the formation of a ternary complex containing mouse autocrine motility factor receptor (mAMFR), mp97, and mPNGase. This assemblage recruits the cytosolic mPNGase close to the endoplasmic reticulum (ER) membrane, where the retrotranslocation of misfolded glycoproteins is thought to occur. In addition to the ER membrane-associated E3 ligase mAMFR, a cytosolic protein mY33K, containing both UBA and UBX domains, was found to also directly interact with mp97. Thus, a complex containing five proteins, mAMFR, mY33K, mp97, mPNGase, and mHR23B, is formed in close proximity to the ER membrane and serves to couple the activities of retrotranslocation, ubiquitination, and deglycosylation and, thereby, route misfolded glycoproteins to the proteasome.
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PMID:The AAA ATPase p97 links peptide N-glycanase to the endoplasmic reticulum-associated E3 ligase autocrine motility factor receptor. 1670 68

The endoplasmic-reticulum-associated degradation of misfolded (glyco)proteins ensures that only functional, correctly folded proteins exit from the endoplasmic reticulum and that misfolded ones are degraded by the ubiquitin-proteasome system. During the degradation of misfolded glycoproteins, they are deglycosylated by the PNGase (peptide:N-glycanase). The free oligosaccharides released by PNGase are known to be further catabolized by a cytosolic alpha-mannosidase, although the gene encoding this enzyme has not been identified unequivocally. The findings in the present study demonstrate that an alpha-mannosidase, Man2C1, is involved in the processing of free oligosaccharides that are formed in the cytosol. When the human Man2C1 orthologue was expressed in HEK-293 cells, most of the enzyme was localized in the cytosol. Its activity was enhanced by Co2+, typical of other known cytosolic alpha-mannosidases so far characterized from animal cells. The down-regulation of Man2C1 activity by a small interfering RNA drastically changed the amount and structure of oligosaccharides accumulating in the cytosol, demonstrating that Man2C1 indeed is involved in free oligosaccharide processing in the cytosol. The oligosaccharide processing in the cytosol by PNGase, endo-beta-N-acetylglucosaminidase and alpha-mannosidase may represent the common 'non-lysosomal' catabolic pathway for N-glycans in animal cells, although the molecular mechanism as well as the functional importance of such processes remains to be determined.
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PMID:Man2C1, an alpha-mannosidase, is involved in the trimming of free oligosaccharides in the cytosol. 1684 60


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