A simple, sensitive in vitro assay for cytoplasmic deglycosylation by peptide: N-glycanase

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.     

Misfolding of glycoproteins is a prerequisite for peptide: N-glycanase mediated deglycosylation

Peptide:N-glycanase (PNGase) is a deglycosylating enzyme that catalyzes the hydrolysis of the beta-aspartylglycosylamine bond of aspargine-linked glycopeptides and glycoproteins. Earlier studies from our laboratory indicated that PNGase catalyzed de-N-glycosylation was limited to glycopeptide substrates, but recent reports have demonstrated that it also acts upon full-length misfolded glycoproteins. In this study, we utilized two glycoprotein substrates, yeast carboxypeptidase and chicken egg albumin (ovalbumin), to study the deglycosylation activity of yeast PNGase and its mutants. Our results provide further evidence that PNGase acts upon full-length glycoprotein substrates and clearly establish that PNGase acts only on misfolded or denatured glycoproteins.     

Fbs1 protects the malfolded glycoproteins from the attack of peptide:N-glycanase

Fbs1 is a cytosolic lectin putatively operating as a chaperone as well as a substrate-recognition subunit of the SCF(Fbs1) ubiquitin ligase complex. To provide structural and functional basis of preferential binding of Fbs1 to unfolded glycoproteins, we herein characterize the interaction of Fbs1 with a heptapeptide carrying Man3GlcNAc2 by nuclear magnetic resonance (NMR) spectroscopy and other biochemical methods. Inspection of the NMR data obtained by use of the isotopically labeled glycopeptide indicated that Fbs1 interacts with sugar-peptide junctions, which are shielded in native glycoprotein, in many cases, but become accessible to Fbs1 in unfolded glycoproteins. Furthermore, Fbs1 was shown to inhibit deglycosylation of denatured ribonuclease B by a cytosolic peptide:N-glycanase (PNGase). On the basis of these data, we suggest that Fbs1 captures malfolded glycoproteins, protecting them from the attack of PNGase, during the chaperoning or ubiquitinating operation in the cytosol.     

Cytoplasmic peptide:N-glycanase and catabolic pathway for free N-glycans in the cytosol

Peptide:N-glycanase (PNGase) releases N-glycans from glycoproteins/glycopeptides. Cytoplasmic PNGase is widely recognized as a component of machinery for ER-associated degradation (ERAD), i.e. proteasomal degradation of misfolded, newly synthesized (glyco)proteins that have been exported from the ER. The enzyme belongs to the “transglutaminase superfamily” that contains a putative catalytic triad of cysteine, histidine, and aspartic acid. The mammalian orthologues of PNGase contain the N-terminal PUB domain that serves as the protein–protein interaction domain. The C-terminus of PNGase was recently found to be a novel carbohydrate-binding domain. Taken together, these observations indicate that C-terminus of mammalian PNGase is important for recognition of the substrates while N-terminus of this enzyme is involved in assembly of a degradation complex.     

The N-terminus of yeast peptide: N-glycanase interacts with the DNA repair protein Rad23

Yeast peptide: N-glycanase (PNGase) is involved in the proteasomal degradation of misfolded glycoproteins where it interacts with the DNA repair protein Rad23 as first detected in a yeast two-hybrid assay and subsequently confirmed by biochemical in vivo analyses. Limited proteolysis of PNGase with trypsin led to the removal of both an N-terminal and a C-terminal stretch. Based on these truncations the N-terminal region of yeast PNGase was identified as being responsible for binding to Rad23. Secondary structure predictions of this region suggest that it is composed of a single, solvent-exposed alpha-helix. The interaction between PNGase and Rad23 was studied using surface plasmon resonance revealing an equilibrium binding constant of approximately 2.5 microM. The oligomeric nature of Rad23 was also investigated using sedimentation equilibrium analysis. Although Rad23 exists as a dimer in solution, the monomeric form of Rad23 associates with a PNGase monomer in a 1:1 stoichiometric ratio.     

A glycosylated type I membrane protein becomes cytosolic when peptide: N-glycanase is compromised

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.     

Systematic synthesis and inhibitory activity of haloacetamidyl oligosaccharide derivatives toward cytoplasmic peptide:<Emphasis Type="Italic">N</Emphasis>-glycanase

A series of glycosyl haloacetamides were synthesized as potential inhibitors of cytoplasmic peptide:N-glycanase (PNGase), an enzyme that removes N-glycans from misfolded glycoproteins. Chloro-, bromo-, and iodoacetamidyl chitobiose and chitotetraose derivatives exhibited a significant inhibitory activity. No inhibitory activity was observed with of fluoroacetamididyl derivatives. Moreover, N-acetylglucosamine derivatives, β-chloropropionamidyl chitobiose, and chloroacetamidyl cellooligosaccharide derivatives did not show any activity. These results underscore the importance of the N-acetyl groups of chitobiose for PNGase recognition. In addition, reactivity and position of the leaving group at the reducing end are also important factors.     

Dual enzymatic properties of the cytoplasmic peptide: N-glycanase in C. elegans

The endoplasmic reticulum-associated degradation (ERAD) of misfolded (glyco)proteins ensures that only functional, correctly folded proteins exit from the ER and that misfolded ones are degraded by the ubiquitin-proteasome system. During the degradation of misfolded glycoproteins, some of them are subjected to deglycosylation by the cytoplasmic peptide:N-glycanase (PNGase). The cytosolic PNGase is widely distributed throughout eukaryotes. Here we show that the nematode Caenorhabditis elegans PNG-1, the cytoplasmic PNGase orthologue in this organism, exhibits dual enzyme functions, not only as PNGase but also as an oxidoreductase (thioredoxin). Using an in vitro assay as well as an in vivo assay system in budding yeast, the N-terminal thioredoxin domain and the central transglutaminase domain were found to be essential for oxidoreductase activity and PNGase activity, respectively. Occurrence of a C. elegans mutation affecting a catalytic residue in the PNGase domain strongly suggests the functional importance of this protein in higher eukaryotes.     

PNG1, a yeast gene encoding a highly conserved peptide:N-glycanase

It has been proposed that cytoplasmic peptide:N-glycanase (PNGase) may be involved in the proteasome-dependent quality control machinery used to degrade newly synthesized glycoproteins that do not correctly fold in the ER. However, a lack of information about the structure of the enzyme has limited our ability to obtain insight into its precise biological function. A PNGase-defective mutant (png1-1) was identified by screening a collection of mutagenized strains for the absence of PNGase activity in cell extracts. The PNG1 gene was mapped to the left arm of chromosome XVI by genetic approaches and its open reading frame was identified. PNG1 encodes a soluble protein that, when expressed in Escherichia coli, exhibited PNGase activity. PNG1 may be required for efficient proteasome-mediated degradation of a misfolded glycoprotein. Subcellular localization studies indicate that Png1p is present in the nucleus as well as the cytosol. Sequencing of expressed sequence tag clones revealed that Png1p is highly conserved in a wide variety of eukaryotes including mammals, suggesting that the enzyme has an important function.     

Dual-gradient high-performance liquid chromatography for identification of cytosolic high-mannose-type free glycans

It has been shown that free oligosaccharides derived from N-linked glycans accumulate in the cytosol of animal cells. Most of the glycans have only a single GlcNAc at their reducing termini (Gn1 glycans), whereas the original N-glycans retain N,N′-diacetylchitobiose at their reducing termini (Gn2 glycans). Under the conditions of high-performance liquid chromatography (HPLC) mapping established for pyridylamine (PA)-labeled Gn2 N-glycans, Gn1 glycans are not well retained on reversed-phase HPLC, making simultaneous analysis of Gn1 and Gn2 glycans problematic. We introduced a dual gradient (i.e., pH and butanol gradient) for the separation of Gn1 and Gn2 glycans in a single reversed-phase HPLC. Determination of elution time for various standard Gn2 high-mannose-type glycans, as well as Gn1 glycans found in the cytosol of animal cells, showed that elution of Gn1 and Gn2 glycans could be separated. Sufficient separation for most of the structural isomers could be achieved for Gn1 and Gn2 glycans. This HPLC, therefore, is a powerful method for identification of the structures of PA-labeled glycans, especially Gn1-type glycans, isolated from the cytosol of animal cells.     

UBXD1 binds p97 through two independent binding sites

The chaperone-related p97 protein plays a central role in various cellular processes involving the ubiquitin-proteasome system. The diverse functions of p97 are controlled by a large number of cofactors that recruit specific substrates or influence their ubiquitylation state. Many cofactors bind through a UBX or PUB domain, two major p97 binding modules. However, the recently identified UBXD1 cofactor possesses both domains. To elucidate the molecular basis underlying the interaction between UBXD1 and p97, we analyzed the contribution of both domains to p97 binding biochemically and in living cells. The PUB domain mediated robust binding to the carboxy-terminus of p97, while the UBX domain did not contribute to p97 binding. Importantly, we identified an additional p97 binding site in UBXD1 that competed with the p47 cofactor for binding to the N domain of p97. This novel, bipartite binding mode suggests that UBXD1 could be an efficient regulator of p97 cofactor interactions.     

Non-lysosomal degradation pathway for N-linked glycans and dolichol-linked oligosaccharides

There is growing evidence that asparagine (N)-linked glycans play pivotal roles in protein folding and intra- or intercellular trafficking of N-glycosylated proteins. During the N-glycosylation of proteins, significant amounts of free oligosaccharides (fOSs) and phosphorylated oligosaccharides (POSs) are generated at the endoplasmic reticulum (ER) membrane by unclarified mechanisms. fOSs are also formed in the cytosol by the enzymatic deglycosylation of misfolded glycoproteins destined for proteasomal degradation. This article summarizes the current knowledge of the molecular and regulatory mechanisms underlying the formation of fOSs and POSs in mammalian cells and Saccharomyces cerevisiae.     

Molecular identification and characterization of peptide: N-glycanase from Schizosaccharomyces pombe

Peptide:N-glycanase (PNGase) is an enzyme responsible for deglycosylation of misfolded glycoproteins in so-called endoplasmic reticulum-associated degradation (ERAD) system. In this study, we reported the molecular identification and characterization of SpPNGase (Schizosaccharomyces pombe PNGase). Enzymatic analysis revealed that SpPNGase deglycosylated the misfolded glycoproteins and distinguished native and denatured high-mannose glycoproteins in vitro. The deglycosylation activity was lost with the addition of chelating agent EDTA and was not restored by re-addition of metal ions. By construction of deletion mutant, we confirmed that N-terminal α-helix of SpPNGase was responsible for the protein-protein interaction. Combining the results from ternary structure prediction and dendrogram analysis, we suggested that the N-terminal α-helices of PNGase are derived from evolutionary motif/peptide fusion.     

Kinetic comparison of peptide: N-glycosidases F and A reveals several differences in substrate specificity

The initial velocities of hydrolysis of nineteen glycopeptides by peptide: N-glycosidase F and A were determined. Substrates were prepared from bovine fetuin, hen ovalbumin, pineapple stem bromelain, bovine fibrin and taka-amylase. From these glycopeptides, several variants with regard to peptide and carbohydrate structure were prepared and derivatized with dabsyl chloride, dansyl chloride or activated resorufin. Tyrosine containing glycopeptides were also used without an additional chromophore. Enzymatic hydrolysis of glycopeptides was quantified by narrow bore, reversed phase HPLC with turnaround cycle times of down to 6 min, but usually 15 min.K _M values ranging from 30 to 64 µm and from 4 to 36 µm were found for N-glycosidase F and A, respectively. Relative velocities of hydrolysis of the different substrates by each enzyme varied considerably. Little, if any, similarity of the performance of N-glycosidase F and A with the different substrates was observed. The minimal carbohydrate structure released by peptide: N-glycosidase F was a di-N-acetylchitobiose. N-glycosidase A could release even a singleN-acetylglucosamine, albeit 3000 times slower than a di-N-acetylchitobiose or larger glycans. In general the structure of the intact glycan had little effect on activity, and with both enzymes the rate of hydrolysis appeared to be primarily governed by peptide structure and length. However, N-glycosidase F did not release glycans 1,3-fucosylated at the asparagine linkedN-acetylglucosamine irrespective of the presence of xylose in the substrate.Abbreviations CAMCys S-carboxamidomethyl cystein - CMCys S-carboxymethyl cystein - Fib, Fet, Ova, Taa and Brl glycopeptides derived from bovine fibrin, fetuin, ovalbumin, taka-amylase A, and bromelain, respectively - GlcNAc N-acetylglucosamine - PLA phospholipase A₂ - PNGase peptide N-glycosidase - RESOS N-(Resorufin-4-carbonyl)piperidine-4-carboxylic acidN-hydroxysuccinimide ester     

Characterization of a lympho-inhibitory peptide produced by Mycoplasma bovis

Mycoplasma bovis is able to inhibit the mitogen-induced proliferation of bovine lymphocytes. Herein is described the isolation of an immuno-inhibitory peptide from M. bovis. Using size exclusion chromatography, three lympho-suppressive fractions were isolated from M. bovis free supernatant. MALDI-TOF analysis revealed a common peak throughout the suppressive fractions. The purest of these fractions was subjected to N-terminal sequencing, revealing an 84% homologous match with the C-terminus of the M. bovis surface protein VspL (variable surface protein-L). A recombinant of the 26 amino acid peptide was also able to suppress Concanavalin A (ConA)-induced proliferation of bovine lymphocytes. This describes a unique immunosuppressive peptide produced by the bovine respiratory pathogen, M. bovis.     

Studies on a heterologous complex formed by human butyrylcholinesterase

An electrophoretic band with butyrylcholinesterase activity was detected in 71 CHE2 C5+ and 378 CHE2 C5– individuals and was named C_4/5 in view of its similar mobility to either C₄ or C₅, depending on the pH of the agar gel used. The present data suggest that C_4/5 is a heterologous complex of butyrylcholinesterase. Although the C_4/5 band may have the same mobility as C₅, depending on the conditions of electrophoresis, our hypothesis is that these two bands result from the association of BChE with different molecules.     

Features of a twin-arginine signal peptide required for recognition by a Tat proofreading chaperone

The twin-arginine translocation (Tat) system is a bacterial protein targeting pathway. Tat-targeted proteins display signal peptides containing a distinctive SRRxFLK ‘twin-arginine’ motif. The Escherichia coli trimethylamine N-oxide reductase (TorA) bears a bifunctional Tat signal peptide, which directs protein export and serves as a binding site for the TorD biosynthetic chaperone. Here, the physical interaction between TorD and the TorA signal peptide was investigated. A single substitution within the TorA signal peptide (L31Q) was sufficient to impair TorD binding. Screening of a random torD mutant library identified a variant TorD protein (Q7L) that displayed increased binding affinity for the TorA signal peptide.

Structured summary

MINT-6796225, MINT-6796279, MINT-6796298, MINT-6796315, MINT-6796332, MINT-6796350, MINT-6796371, MINT-6796391, MINT-6796410, MINT-6796429, MINT-6796446, MINT-6796460:

TorD (uniprotkb:P36662) physically interacts (MI:0218) with TorA (uniprotkb:P33225) by two-hybrid (MI:0018)

MINT-6796515, MINT-6796563, MINT-6796589, MINT-6796624, MINT-6796648, MINT-6796666, MINT-6796770, MINT-6796750:

TorA (uniprotkb:P33225) binds (MI:0407) to TorD (uniprotkb:P36662) by isothermal titration calorimetry (MI:0065)

     

Evidence for a synergistic salt-protein interaction -- complex patterns of activation vs. inhibition of nitrogenase by salt

The molybdenum nitrogenase enzyme system, comprised of the MoFe protein and the Fe protein, catalyzes the reduction of atmospheric N(2) to NH(3). Interactions between these two proteins and between Fe protein and nucleotides (MgADP and MgATP) are crucial to catalysis. It is well established that salts are inhibitors of nitrogenase catalysis that target these interactions. However, the implications of salt effects are often overlooked. We have reexamined salt effects in light of a comprehensive framework for nitrogenase interactions to offer an in-depth analysis of the sources of salt inhibition and underlying apparent cooperativity. More importantly, we have identified patterns of salt activation of nitrogenase that correspond to at least two mechanisms. One of these mechanisms is that charge screening of MoFe protein-Fe protein interactions in the nitrogenase complex accelerates the rate of nitrogenase complex dissociation, which is the rate-limiting step of catalysis. This kind of salt activation operates under conditions of high catalytic activity and low salt concentrations that may resemble those found in vivo. While simple kinetic arguments are strong evidence for this kind of salt activation, further confirmation was sought by demonstrating that tight complexes that have previously displayed little or no activity due to the inability of Fe protein to dissociate from the complex are activated by the presence of salt. This occurs for the combination Azotobacter vinelandii MoFe protein with: (a) the L127Delta Fe protein; and (b) Clostridium pasteurianum Fe protein. The curvature of activation vs. salt implies a synergistic salt-protein interaction.