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)

Atrial-natriuretic-peptide (ANP) receptor, previously identified as a 140 kDa protein with a disulphide-linked homodimeric structure, was purified from bovine lung by (NH4)2SO4 fractionation and affinity chromatography on ANP-Affi-Gel 10. The purified receptor had a binding capacity of 4.2 nmol of ANP/mg of protein and an affinity constant of 6.5 pM. The isoelectric point of the receptor was 5.8, consistent with the acidic nature of the protein (amino acid analysis revealed a predominance of glutamic acid and aspartic acid residues). Treatment with endoglycosidase H and glycopeptidase F revealed that the receptor has three complex types of oligosaccharide chains per 70 kDa subunit. Deglycosylation of the receptor did not affect its binding activity. Reduction with dithiothreitol and reoxidation by dialysis revealed a strong tendency of the receptor subunits to dimerize via disulphide cross-linking; however, carboxymethylation of the reduced receptor indicated that the intersubunit disulphide bond is not necessary for the ligand-binding activity.
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PMID:Purification and properties of active atrial-natriuretic-peptide receptor (type C) from bovine lung. 255 6

We investigated whether hepatitis C virus envelope glycoprotein E1 is transported from the endoplasmic reticulum (ER) to the cytoplasm of infected cells for class I MHC processing. Target cells expressing E1 were killed by CTL lines from a hepatitis C virus-infected chimpanzee, and synthetic peptides were used to define an epitope (amino acids 233-GNASRCWVA-241) presented by the Patr-B*1601 class I MHC molecule. An unusually high concentration (>100 nM) of this nonameric peptide was required for target cell lysis, but this could be reduced at least 1000-fold by replacing the asparagine at amino acid position 234 (Asn234) with aspartic acid (Asp), the anticipated anchor residue for NH2-terminal peptide binding to Patr-B*1601. Conspicuously, position 234 is part of an N-glycosylation motif (Asn-Xaa-Ser/Thr), suggesting that the Asn234 to Asp substitution might occur naturally within the cell due to deglycosylation/deamidation of this amino acid by the cytosolic enzyme peptide N-glycanase. In support of this model, we demonstrate that presentation of the epitope depended on 1) cotranslational synthesis of E1 in the ER, 2) glycosylation of the E1 molecule, and 3) a functional TAP transporter to shuttle peptide from the cytosolic to ER compartment. These results indicate for the first time that during infection of the host, viral envelope glycoproteins originating in the ER are processed in the cytoplasm for class I MHC presentation. That a posttranslational change in amino acid sequence from Asn to Asp alters the repertoire of peptides presented to CD8+ CTL has implications for the design of antiviral vaccines.
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PMID:Hepatitis C virus envelope glycoprotein E1 originates in the endoplasmic reticulum and requires cytoplasmic processing for presentation by class I MHC molecules. 991 84

This review covers the unique catalytic and molecular properties of three proteolytic enzymes and a glycosidase from Aspergillus. An aspartic proteinase from A. saitoi, aspergillopepsin I (EC 3.4.23.18), favors hydrophobic amino acids at P1 and P'1 like gastric pepsin. However, aspergillopepsin I accommodates a Lys residue at P1, which leads to activation of trypsinogens like duodenum enteropeptidase. Substitution of Asp76 to Ser or Thr and deletion of Ser78, corresponding to the mammalian aspartic proteinases, cathepsin D and pepsin, caused drastic decreases in the activities towards substrates containing a basic amino acid residue at 1. In addition, the double mutant T77D/G78(S)G79 of porcine pepsin was able to activate bovine trypsinogen to trypsin by the selective cleavage of the K6-I7 bond of trypsinogen. Deuterolysin (EC 3.4.24.39) from A. oryzae, which contains 1g atom of zinc/mol of enzyme, is a single chain of 177 amino acid residues, includes three disulfide bonds, and has a molecular mass of 19,018 Da. It was concluded that His128, His132, and Asp164 provide the Zn2+ ligands of the enzyme according to a 65Zn binding assay. Deuterolysin is a member of a family of metalloendopeptidases with a new zinc-binding motif, aspzincin, defined by the "HEXXH + D" motif and an aspartic acid as the third zinc ligand. Acid carboxypeptidase (EC 3.4.16.1) from A. saitoi is a glycoprotein that contains both N- and O-linked sugar chains. Site-directed mutagenesis of the cpdS, cDNA encoding A. saitoi carboxypeptidase, was cloned and expressed. A. saitoi carboxypeptidase indicated that Ser153, Asp357, and His436 residues were essential for the enzymic catalysis. The N-glycanase released high-mannose type oligosaccharides that were separated on HPLC. Two, which had unique structures of Man10 GlcNAc2 and Man11GlcNAc2, were characterized. An acidic 1,2-alpha-mannosidase (EC 3.2.1.113) was isolated from the culture of A. saitoi. A highly efficient overexpression system of 1,2-alpha-mannosidase fusion gene (f-msdS) in A. oryzae was made. A yeast mutant capable of producing Man5GlcNAc2 human-compatible sugar chains on glycoproteins was constructed. An expression vector for 1,2-alpha-mannosidase with the "HDEL" endoplasmic reticulum retention/retrieval tag was designed and expressed in Saccharomyces cerevisiae. The first report of production of human-compatible high mannose-type (Man5GlcNAc2) sugar chains in S. cerevisiae was described.
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PMID:Unique catalytic and molecular properties of hydrolases from Aspergillus used in Japanese bioindustries. 1083 Apr 77

A cytoplasmic peptide:N-glycanase has been implicated in the proteasomal degradation of newly synthesized misfolded glycoproteins exported from the endoplasmic reticulum. The gene encoding this enzyme (Png1p) has been identified in yeast. Based on sequence analysis, Png1p was classified as a member of the 'transglutaminase-like superfamily' that contains a putative catalytic triad of amino acids (cysteine, histidine, and aspartic acid). More recent studies in yeast indicate that Png1p can bind to the 26S proteasome through its interaction with the DNA repair protein Rad23p. A mouse homologue of Png1p (mPng1p) bound not only to the Rad23 protein, but also to various proteins related to ubiquitin and/or the proteasome through an extended amino-terminal domain. This NH2 terminus of mPng1p, which is not found in yeast, contains a PUB domain predicted to be involved in the ubiquitin-related pathway. This review will focus on the primary structure and potential functions of the cytoplasmic PNGases.
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PMID:Cytoplasmic peptide:N-glycanase (PNGase) in eukaryotic cells: occurrence, primary structure, and potential functions. 1197 27

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

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 the active sites of the proteases reside. The deglycosylation reaction by PNGase brings about two major changes on substrate proteins; one is a removal of N-glycan chains, and the other is the introduction of negative charge(s) into the core peptide by converting glycosylated asparagine residue(s) into aspartic acid residue(s). Therefore, PNGase action can be accurately monitored by detecting both changes using two different methods; that is, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for deglycosylation and isoelectric focusing for detection of introduction of negative charge(s) into core proteins. This chapter will describe the simple in vivo as well as in vitro assay method to detect PNGase activity.
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PMID:A cytoplasmic peptide: N-glycanase. 1711 67

Nephrin is a type-1 transmembrane glycoprotein and the first identified principal component of the glomerular filtration barrier. Ten potential asparagine (N)-linked glycosylation sites have been predicted within the ectodomain of nephrin. However, it is not known which of these potential sites are indeed glycosylated and what type of glycans are involved. In this work, we have identified the terminal sugar residues on the ectodomain of human nephrin and utilized a straightforward and reliable mass spectrometry-based approach to selectively identify which of the ten predicted sites are glycosylated. Purified recombinant nephrin was subjected to peptide-N-glycosidase F (PNGase F) to enzymatically remove all the N-linked glycans. Since PNGase F is an amidase, the asparagine residues from which the glycans have been removed are deaminated to aspartic acid residues, resulting in an increase in the peptide mass with 1 mass unit. Following trypsin digestion, deglycosylated tryptic peptides were selectively identified by MALDI-TOF MS and their sequence was confirmed by tandem TOF/TOF. The 1 Da increase in peptide mass for each asparagine-to-aspartic acid conversion, along with preferential cleavage of the amide bond carboxyl-terminal to aspartic acid residues in peptides where the charge is immobilized by an arginine residue, was used as a diagnostic signature to identify the glycosylated peptides. Thus, nine of ten potential glycosylation sites in nephrin were experimentally proven to be modified by N-linked glycosylation.
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PMID:Identification of N-linked glycosylation sites in human nephrin using mass spectrometry. 1721 72

We recently demonstrated that the mechanism of processing of an HLA-A*0201-restricted peptide epitope, Tyr(369)(D), derived from the membrane protein tyrosinase, involves retrotranslocation of glycosylated molecules from the endoplasmic reticulum to the cytosol, removal of an N-linked carbohydrate from Asn(371) by peptide N-glycanase, proteolysis by the proteasome and other proteases, and retransport of the resulting peptides into the endoplasmic reticulum for association with HLA-A*0201. Carbohydrate removal results in deamidation of Asn(371) to aspartic acid. The asparagine-containing homolog of this peptide, Tyr(369)(N), is not presented by tyrosinase-expressing cells, and this has been presumed to be due to quantitative glycosylation of Asn(371). Although examining cytosolic intermediates that accumulated in human melanoma cells treated with proteasome inhibitors, we were surprised to find both molecules that had been deglycosylated by peptide N-glycanase and a large number of molecules that had not been previously glycosylated. The failure of Tyr(369)(N) to be processed and presented from these latter molecules may be partially due to a process of deamidation independent of glycosylation. However, we also established that proteasomes degrade tyrosinase molecules that are still glycosylated, giving rise to a set of discrete intermediates that are not observed when unglycosylated molecules are degraded. We propose that Tyr(369)(N) fails to be presented because unglycosylated tyrosinase is degraded rapidly and relatively nonselectively. In contrast, glycosylation alters the selectivity of tyrosinase processing by the proteasome, enhancing the production or survival of Tyr(369)(D).
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PMID:N-glycosylation enhances presentation of a MHC class I-restricted epitope from tyrosinase. 1934 61

Mass spectrometry (MS) is used to quantify the relative distribution of glycans attached to particular protein glycosylation sites (micro-heterogeneity) and evaluate the molar site occupancy (macro-heterogeneity) in glycoproteomics. However, the accuracy of MS for such quantitative measurements remains to be clarified. As a key step towards this goal, a panel of related tryptic peptides with and without complex, biantennary, disialylated N-glycans was chemically synthesised by solid-phase peptide synthesis. Peptides mimicking those resulting from enzymatic deglycosylation using PNGase F/A and endo D/F/H were synthetically produced, carrying aspartic acid and N-acetylglucosamine-linked asparagine residues, respectively, at the glycosylation site. The MS ionisation/detection strengths of these pure, well-defined and quantified compounds were investigated using various MS ionisation techniques and mass analysers (ESI-IT, ESI-Q-TOF, MALDI-TOF, ESI/MALDI-FT-ICR-MS). Depending on the ion source/mass analyser, glycopeptides carrying complex-type N-glycans exhibited clearly lower signal strengths (10-50% of an unglycosylated peptide) when equimolar amounts were analysed. Less ionisation/detection bias was observed when the glycopeptides were analysed by nano-ESI and medium-pressure MALDI. The position of the glycosylation site within the tryptic peptides also influenced the signal response, in particular if detected as singly or doubly charged signals. This is the first study to systematically and quantitatively address and determine MS glycopeptide ionisation/detection strengths to evaluate glycoprotein micro-heterogeneity and macro-heterogeneity by label-free approaches. These data form a much needed knowledge base for accurate quantitative glycoproteomics.
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PMID:Quantitative mapping of glycoprotein micro-heterogeneity and macro-heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides. 2372 53

Recombinant protein expression using eukaryotic expression systems has certain advantages, such as addition of posttranslational modifications that help protein stability and activity. Asparagine-linked sugar attachment is one of the most common posttranslation modifications. However, sugar modification can impede the growth of high-quality protein crystals for structural studies using X-ray crystallography. To overcome this problem, consensus sites of N-linked attachments can be mutated into other similar residues, such as aspartic acid. Alternatively, enzymatic deglycosylation can be used to remove sugars. Peptide-N-Glycosidase F (PNGase F; EC 3.5.1.52) and Endoglycosidase H (Endo H; EC 3.2.1.96) are the most popular enzymes for this purpose.
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PMID:Enzymatic deglycosylation of glycoproteins. 2418 31


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