Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F cannot release glycans with fucose attached alpha 1----3 to the asparagine-linked N-acetylglucosamine residue

The ability of peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F (PNGase F) from Flavobacterium meningosepticum and PNGase A from sweet almonds to deglycosylate N-glycopeptides and N-glycoproteins from plants was compared. Bromelain glycopeptide and horseradish peroxidase-C glycoprotein, which contain xylose linked beta 1----2 to beta-mannose and fucose linked alpha 1----3 to the innermost N-acetylglucosamine, were used as substrates. In contrast to PNGase A, the enzyme from F. meningosepticum did not act upon these substrates even at concentrations 100-fold higher than required for complete deglycosylation of commonly used standard substrates. After removal of alpha 1----3-linked fucose from the plant glycopeptide and glycoprotein by mild acid hydrolysis, they were readily degraded by PNGase F at moderate enzyme concentrations. Hence we conclude that alpha 1----3 fucosylation of the inner N-acetylglucosamine impedes the enzymatic action of PNGase F. Knowledge of this limitation of the deglycosylation potential of PNGase F may turn it from a pitfall into a useful experimental tool.     

α1–6(α1–3)-Difucosylation of the asparagine-boundN-acetylglucosamine in honeybee venom phospholipase A2

Chymotryptic glycopeptides were prepared from a honeybee (Apis mellifica) venom phospholipase A2 (E.C. 3.1.1.4) fraction, with high affinity towards lentil (Lens culinaris) lectin. Treatment of the glycopeptide mixture with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase A, followed by HPLC fractionation, yielded two oligosaccharides, which were analysed by 500 MHz 1H-NMR spectroscopy to give the following structures [formula: see text] This is the first report on a naturally occurring glycoprotein N-glycan with two fucose residues linked to the asparagine-bound N-acetylglucosamine.     

Use of N-glycanase to release asparagine-linked oligosaccharides for structural analysis

An enzymatic procedure for releasing asparagine-linked oligosaccharides from glycoproteins by treatment with N-glycanase (peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase) has been investigated. Ribonuclease B, transferrin, fetuin, and alpha 1-acid glycoprotein were treated with N-glycanase and the released oligosaccharides were radiolabeled with NaB3H4. Lectin staining of the N-glycanase-treated proteins indicated that the deglycosylation reactions had proceeded to completion. The labeled carbohydrate chains were analyzed by HPLC on Micro-Pak AX-5 and AX-10 columns. The proportion of high-mannose and bi-, tri-, and tetraantennary complex chains obtained from each glycoprotein was in agreement with literature values. These results demonstrate that N-glycanase provides a simple method to release all common classes of asparagine-linked oligosaccharides from a glycoprotein in a form that can be radiolabeled directly for structural analysis.     

Characterization of cellular oligosaccharides from normal and cystic fibrotic fibroblasts using sequential endoglycosidase digestions

A method was developed for obtaining detailed oligosaccharide profiles from [2-3H]mannose- or [6-3H]fucose-labeled cellular glycoproteins. The oligosaccharides were segregated first according to class, using endo-beta-N-acetylglucosaminidase H (Endo H) to release the high mannose species, and then with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase (PNGase F), which provided a complete array of complex oligosaccharide chains. The high mannose and complex oligosaccharides were fractionated subsequently according to net negative charge on QAE-Sephadex. High resolution gel filtration on TSK HW-40(S) resolved the neutral high mannose population into species of the type Man9-5 N-acetylglucosamine. Desialylation of the complex chains with neuraminidase allowed resolution of these oligosaccharides into their corresponding asialo bi-, tri-, and tetraantennary species. Fibroblasts from normal and cystic fibrosis cells were analyzed for differences in their glycosylation patterns using these techniques. Over 95% of the [2-3H]mannose-labeled glycoproteins were susceptible to the combined glycosidase digestions, but no difference in either the high mannose or complex oligosaccharides were observed. Nonetheless, the methodology developed in this study provides an important new approach for investigating oligosaccharides of different cell types and variants of the same type. Metabolic changes induced in cellular glycoproteins, as illustrated by use of the processing inhibitor swainsonine, demonstrated the versatility of this procedure for investigating questions relating to glycoprotein structure and enzyme specificity. Thus, by employing a variation of this method, it was possible to confirm the location of fucose in the core of PNGase F-released hybrid oligosaccharides by the subsequent release with Endo H of the disaccharide, fucosyl-N-acetylglucosamine.     

Selective organic precipitation/extraction of released N-glycans following large-scale enzymatic deglycosylation of glycoproteins

A major difficulty with isolating enzymatically or chemically released oligosaccharides from large-scale glycoprotein deglycosylation reactions is the time-consuming chromatography, desalting, and concentration steps required to prepare a glycan fraction of manageable proportions. To overcome these time and preparative chromatography equipment requirements, we have developed a rapid organic solvent precipitation/extraction procedure that allows sequential isolation of endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96)-released high-mannose and hybrid, peptide-N(4)-(N-acetyl-beta-glucosaminyl) Asn amidase (EC 3.5.1. 52)-released complex, and beta-eliminated O-linked glycans without the need for intermediate chromatography, desalting, or concentration steps. The method involves precipitation of protein and released glycans at -20 degrees C in 80% acetone and extraction of the glycans from the pellet with 60% aqueous methanol after each deglycosylation step. Three pools of essentially salt- and detergent-free oligosaccharides (high-mannose/hybrid, complex, and O-linked) can be isolated in a high yield in 4 days with this protocol, which has been extensively tested using bovine RNase B, human bile salt-stimulated lipase expressed in Pichia pastoris, hen ovalbumin, bovine fetuin, bovine thyroglobulin, and several invertase preparations from wild-type and mutant yeast strains.     

Distinct N-glycan fucosylation potentials of three lepidopteran cell lines.

The fucosyltransferase activities of three insect cell lines, MB-0503 (from Mamestra brassicae), BM-N (from Bombyx mori) and Sf-9 (from Spodoptera frugiperda), were investigated and compared with that of honeybee venom glands. Cell extracts and venom gland extracts were incubated with GDP-[14C]fucose and glycopeptides isolated from human IgG and from bovine fibrin. The labeled oligosaccharide products were released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase A, fluorescence marked with 2-aminopyridine and analyzed both by reversed-phase and size-fractionation HPLC. They were identified by their elution positions before and after exoglycosidase treatment in comparison with standard oligosaccharides. These experiments revealed distinct fucosylation potentials in the three cell lines tested. While MB-0503 cells, like honeybee venom glands, are able to transfer fucose into alpha 1-3 and alpha 1-6 linkage to the innermost N-acetylglucosamine, only alpha 1-6-fucosyl linkages were detected with BM-N and Sf-9 cells.     

A New Peptide-N 4-(acetyl-β-glucosaminyl)asparagine Amidase from Soybean (Glycine max) Seeds: Purification and Substrate Specificity

We report here the isolation and characterization of a peptide-N ⁴-(acetyl-β-glucosaminyl) asparagine amidase (peptide: N-glycanase) from soybean (Glycine max) seeds. The enzyme was purified to homogeneity with 6.5% yield from defatted soybean meal extract by ion-exchange chromatography, gel filtration, hydroxyapatite chromatography, and hydrophobic chromatography. The purified enzyme, designated PNGase-GM, had the apparent molecular mass of 93 kDa by SDS-PAGE and 90 kDa by gel filtration, indicating this PNGase is a monomeric protein. The enzyme showed maximal activity at pH 4.5-5.0. PNGase-GM was capable of hydrolyzing the β-aspartylglycosylamine linkage (GlcNAcβ1→Asn) of various glycopeptide substrates bearing high-mannose type, hybrid type, and xylose/fucose-containing plant complex type N-glycan units, while this amidase was far less active on the glycopeptides bearing sialylated animal complex-type glycans.     

Analysis of N-acetyl-4-O-acetylneuraminic-acid-containing N-linked carbohydrate chains released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. Application to the structure determination of the carbohydrate chains of equine fibrinogen

The carbohydrate chains of equine fibrinogen were enzymatically released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F. The oligosaccharides obtained were fractionated by a combination of FPLC and HPLC and analyzed by 500-MHz 1H-NMR spectroscopy. Four monosialo and four disialo diantennary N-acetyllactosamine type of carbohydrate chains occur: (formula; see text)     

Carbohydrate structure of Marburg virus glycoprotein.

Marburg virus was propagated in E6 cells, a cloned cell line of Vero cells, in the presence of [6-3H]glucosamine. Radiolabelled viral glycoprotein was digested with trypsin, and oligosaccharides were liberated by sequential treatment with endo-beta-N-acetylglucosaminidase H, peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F and O-glycosidase, by beta-elimination, and by alkaline hydrolysis. After fractionation by HPLC and gel filtration, glycans were characterized chromatographically, by digestion with exoglycosidases and, in part, by methylation analysis and liquid secondary ion mass spectrometry. The oligosaccharide structures thus established include oligomannosidic and hybrid-type N-glycans, as well as neutral fucosylated bi-, tri- and tetraantennary species, most of which carry an additional bisecting N-acetylglucosamine. In addition, high amounts of neutral mucin-type O-glycans with type-1 and type-2 core structures were detected. None of the glycans present in this viral glycoprotein carried sialic acid residues.     

Occurrence of alpha 1-2-fucosylation in membrane glycoproteins of Morris hepatoma 7777 but not in liver. Aberrant type of fucosylation in a malignant tissue.

A comparative study was undertaken to characterize the linkages of L-fucose in N-glycans of plasma membrane glycoproteins from Morris hepatoma 7777, host liver and kidney cortex, as well as from rat serum. After in-vivo radiolabelling of rats with L-[6-3H]fucose, the asparagine-linked carbohydrate chains were released from delipidated plasma membrane glycoproteins, as well as from serum glycoproteins, by enzymic digestion with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase from Flavobacterium meningosepticum. They were then converted to their corresponding oligosaccharide alditols by reduction with sodium borohydride. Two specific alpha-L-fucosidases from almond emulsin and from Aspergillus niger, combined with affinity HPLC on immobilized Aleuria aurantia lectin were used to study the linkage of L-fucose in the oligosaccharide chains. Fucose alpha 1-2 linked to galactose, was present only in the plasma membrane of hepatoma 7777 (18% of total L-[3H]fucose in N-glycans), but was not expressed in host liver, kidney cortex and serum. None of the investigated sources contained an appreciable amount of fucose alpha 1-3/4 linked to N-acetyl-D-glucosamine. All the radioactively labelled oligosaccharides from host liver, kidney cortex and serum, but only 82% of these oligosaccharides from hepatoma, contained alpha-fucosyl residues linked at the C6 position of the proximal N-acetyl-D-glucosamine.     

Guinea-pig kidney beta-N-acetylgalactosaminyltransferase towards Tamm-Horsfall glycoprotein. Requirement of sialic acid in the acceptor for transferase activity.

A beta-N-acetylgalactosaminyltransferase that preferentially transferred N-acetylgalactosamine to Sd(a-) Tamm-Horsfall glycoprotein was found in guinea-pig kidney microsomal preparations. This enzyme was kidney-specific and was able to transfer the sugar to other glycoproteins, such as fetuin and alpha 1-acidic glycoprotein. The presence of sialic acid in the acceptors was essential for the transferase activity when either glycoproteins or their Pronase glycopeptides were used as acceptors. Two glycopeptides (Tamm-Horsfall glycopeptides I and II) with a different carbohydrate composition were separated by DEAE-Sephacel chromatography from Pronase-digested Tamm-Horsfall glycoprotein. The amount of N-acetylgalactosamine transferred to glycopeptides by the enzyme correlated with their degree of sialylation. Enzymic digestion of N-[14C]acetylgalactosamine-labelled Tamm-Horsfall glycopeptide II showed that the transferred sugar was susceptible to beta-N-hexosaminidase. The amount of sugar cleaved by beta-hexosaminidase was strongly increased when the labelled Tamm-Horsfall glycopeptide II was pretreated with mild acid hydrolysis, a procedure that removed the sialic acid residues. Alkaline borohydride treatment of the labelled Tamm-Horsfall glycopeptide II did not release radioactivity, thus indicating that enzymic glycosylation took place at the N-asparagine-linked oligosaccharide units of Tamm-Horsfall glycoprotein.     

Molecular cloning and heterologous expression of N-glycosidase F from Flavobacterium meningosepticum

N-Glycosidase F (peptide-N4-(N-acetyl-beta-glycosaminyl)asparagine amidase; EC 3.5.1.52) catalyzes the cleavage of N-glycosidically linked carbohydrate chains between N-acetylglucosamine and asparagine. The structural gene was isolated by screening a Flavobacterium meningosepticum genomic DNA library in lambda gt10 with oligonucleotides, deduced from partial amino acid sequences of the protein. A clone with an open reading frame of 1062 bases was obtained. The amino acid sequence reveals a 42-residue-long leader peptide, which shows similarities to the endoglycosidase H-leader with respect to the cleavage site of the signal peptide, but is distinct from the ones known from other Gram-positive or -negative bacteria. The molecular weight of the native protein, derived from the DNA sequence, is in agreement with the molecular weight of the purified protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (35,000). Escherichia coli, transformed with a plasmid containing this DNA sequence, expresses N-glycosidase F activity. The enzyme with its natural Flavobacterium promoter and leader peptide is not secreted in E. coli but seems to be associated with cell membranes.     

Chemo-enzymatic synthesis of a bioactive peptide containing a glutamine-linked oligosaccharide and its characterization

A bioactive peptide containing a glutamine-linked oligosaccharide was chemo-enzymatically synthesized by use of the solid-phase method of peptide synthesis and the transglycosylation activity of endo-β-N-acetylglucosaminidase. Substance P, a neuropeptide, is an undecapeptide containing two l-glutamine residues. A substance P derivative with an N-acetyl-d-glucosamine residue attached to the fifth or sixth l-glutamine residue from the N-terminal region was chemically synthesized. A sialo complex-type oligosaccharide derived from a glycopeptide of hen egg yolk was added to the N-acetyl-d-glucosamine moiety of the substance P derivative using the transglycosylation activity of endo-β-N-acetylglucosaminidase from Mucor hiemalis, and a substance P derivative with a sialo complex-type oligosaccharide attached to the l-glutamine residue was synthesized. This glycosylated substance P was biologically active, although the activity was rather low, and stable against peptidase digestion. The oligosaccharide moiety attached to the l-glutamine residue of the peptide was not liberated by peptide-N⁴-(N-acetyl-β-d-glucosaminyl) asparagine amidase F.     

Purification of the oligosaccharide-cleaving enzymes of Flavobacterium meningosepticum.

Four oligosaccharide chain-cleaving enzymes, including two new endoglycosidases distinct from endo-beta-acetylglucosaminidase (Endo) F1, have been identified and purified to homogeneity from cultural filtrates of Flavobacterium meningosepticum. FPLC-directed hydrophobic-interaction chromatography in conjunction with high-resolution ion-exchange chromatography provided a more simple, rapid method for the isolation of endoglycosidase F1, F2 and F3, and the amidase, peptide-N4-N-acetyl-beta-D-glucosaminyl)-asparagine amidase (PNGase F), in greater than 50% yield. The specificity of PNGase F and Endo F1 are well established. Endo F2 and Endo F3 represent new distinct endoglycosidases that prefer complex as compared to high-mannose asparagine-linked glycans. Endo F2 cleaved biantennary oligosaccharides, whereas Endo F3 cleaved both bi- and triantennary oligosaccharides.     

Release of O-linked sugar chains from glycoproteins with anhydrous hydrazine and pyridylamination of the sugar chains with improved reaction conditions.

A method for preparation of pyridylamino (PA-) derivatives of O-linked sugar chains from glycoproteins was developed. A glycopeptide containing O-linked Gal beta 1-3GalNAc was prepared from fetuin and treated with anhydrous hydrazine followed by N-acetylation of free amino groups. Sugar chains released were pyridylaminated with improved reaction conditions and excess reagents were removed by gel filtration. Gal beta 1-3GalNAc-PA obtained together with PA-Gal as a by-product was quantified by HPLC. Conditions for the hydrazine treatment were investigated and the treatment at 40 degrees C for 350 h gave the best results for releasing O-linked sugar chains. The total yield of Gal beta 1-3GalNAc-PA from the glycopeptide was 53% under the established conditions and that of PA-Gal was 18%. The present method was applied to a glycoprotein, and the expected PA-O-linked sugar chains were obtained. Under these conditions, N-linked sugar chains were also released.     

Rapid and sensitive analyses of glycoprotein-derived oligosaccharides by liquid chromatography and laser-induced fluorometric detection capillary electrophoresis

Asparagine-type oligosaccharides are released from core proteins as N-glycosylamines in the initial step of the action of the peptide N(4)-(N-acetyl-β-D-glucosaminyl)asparagine amidase F (PNGase F). The released N-glycosylamine-type oligosaccharides (which are exclusively present at least during the course of the enzyme reaction) could therefore be derivatized with amine-labeling reagents. Here we report a method using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) as a labeling reagent for glycosylamine-type oligosaccharides. We applied the method for the sensitive analysis of some oligosaccharide mixtures derived from well-characterized glycoproteins including human transferrin, α(1)-acid glycoprotein, bovine fetuin, and ribonuclease B. NBD-labeled oligosaccharides were successfully separated on an amide-bonded column or a diol-silica column. This labeling method included the release of oligosaccharides from glycoproteins and derivatization of oligosaccharides in a one-pot reaction and was completed within 3h. The method showed approximately fivefold higher sensitivity than that involving labeling with ethyl p-aminobenzoate (ABEE) in HPLC using fluorometric detection and a one order of magnitude higher response in ESI-LC/MS. We also applied this method for the sensitive analysis of glycoprotein-derived oligosaccharides by capillary electrophoresis with laser-induced fluorometric detection (LIF-CE). The limit of detection in HPLC and LIF-CE were 100fmol and 4fmol, respectively.     

Characterization of the carboxypeptidase N secreted by Hep G2 cells

Human hepatoma (Hep G2) cells secrete nanogram quantities of carboxypeptidase enzymes which are capable of hydrolyzing COOH-terminal lysine and arginine residues. A carboxypeptidase with a neutral pH optimum (greater than pH 7.0) was partially purified from the conditioned medium and compared with pure plasma carboxypeptidase N. The two enzymes behaved in a similar manner on gel filtration (apparent Mr = 280,000), DE52 ion exchange chromatography, and concanavalin A-affinity chromatography and were indistinguishable enzymatically and immunologically. Immunoblots of the Hep G2 and plasma carboxypeptidase N before and following deglycosylation with peptide-N4-[N-acetyl-beta-glucosaminyl]asparagine amidase F revealed a similar, if not identical, multimeric structure. A second carboxypeptidase with a lower molecular weight and a pH optimum of 5.0 was also detected in the Hep G2 medium.     

N-linked oligosaccharides are responsible for rat striatal dopamine D2 receptor heterogeneity

The glycoprotein nature of the binding subunit of the dopamine D2 receptor in rat striatum has been examined by photoaffinity labeling receptor preparations with N-(p-azido-m-[125I]iodophenethyl)spiperone followed by treatment of crude membrane receptor or receptor fractions isolated from sodium dodecyl sulfate (SDS) polyacrylamide gels with endo- and exoglycosidases. The major photoaffinity labeled protein migrates as a heterogeneous species on 10% SDS polyacrylamide gels and ranges from 130,000 to 75,000 relative molecular mass (Mr). This heterogeneity can be explained by glycosylation of the receptor by complex-type N-linked oligosaccharides. Three fractions of labeled receptor were isolated from SDS polyacrylamide gels over a range of 130,000 to 75,000 Mr; after digestion with peptide-N4-[N-acetyl-beta-glucosaminyl] asparagine amidase, all fractions yielded a single peptide approximately 40,000 Mr. Treatment of photoaffinity labeled membranes with alpha-mannosidase was without effect. The dopamine D2 receptor appears to contain substantial amounts of sialic acid as treatment of photoaffinity labeled membranes with neuraminidase increased the receptor mobility on SDS polyacrylamide gels to a species of 50,000-54,000 Mr. Treatment of the receptor with neuraminidase followed by endo-alpha-N-acetylgalactosaminidase did not change the electrophoretic migration pattern from that seen after neuraminidase treatment alone, suggesting that the binding peptide contains no serine- or threonine-linked oligosaccharides. A smaller binding peptide of approximately 31,000 Mr is also apparent in crude photoaffinity labeled membranes. This material also contains N-linked oligosaccharide. Complete removal of N-linked oligosaccharide from the dopamine D2 receptor did not change the rank order potency of agonist and antagonist compounds to compete for [3H]spiperone binding to crude membrane fractions. The dopamine D2 receptor represents a highly glycosylated neural receptor.     

Glycoprotein enzymes secreted by Aspergillus niger: purification and properties of alpha-glaactosidase.

An alpha-galactosidase (alpha-D-galactoside galactohydrolase [EC 3.2.1.22]) was purified to homogeneity from the culture filtrate of Aspergillus niger. The enzyme had an apparent molecular weight of 45,000 and was a glycoprotein. Radioactive enzyme was prepared by growing cells in [14C]fructose and this enzyme was used to prepare 14C-labeled glycopeptides. The glycopeptides emerged from Sephadex G-50 between stachyose and the glycopeptide from ovalbumin. Based on calibration of the column with various-sized dextran oligosaccharides, the glycopeptides appeared to have a molecular weight of 1,200 to 1,400. Analysis of the glycopeptide(s) indicated that it contained mannose and N-acetylglucosamine (GlcNAc) in an approximate ratio of 3 or 4 to 1. Assuming that there are two GlcNAc residues in the oligosaccharide and based on the molecular weight of the glycopeptide, the oligosaccharide probably contains eight to nine sugar residues. Alks probably attached to the protein by a GlcNAc leads to asparagine linkage. The purified alpha-galactosidase was most active on raffinose (Km = 5 x 10--4 M, Vmax = 3 mumol/min per mg of protein), but also showed good activity on p-nitrophenyl-alpha-D-galactoside ans somewhat less activity on stachyose and melibitol. The enzyme also hydrolyzed guar flour and locust bean gum, but did not attack the p-nitrophenyl glycosides of beta-galactose, alpha- or beta-glucose, or alpha- or beta-mannose.     

Demonstration of a new amidase acting on glycopeptides

An enzyme preparation from almond emulsin cleaved the peptide-carbohydrate linkage of stem bromelain glycopeptide, Asn-Asn (oligosaccharide)-Glu-Ser-Ser. The resulting products were determined to be an intact peptide, Asn-$\text{Asp}$-Glu-Ser-Ser, and an intact oligosaccharide unit with two moles of N-acetylglucosamine. So far as tested the enzyme hydrolyzed glycopeptides with 3 to 10 amino acids, while both asparagine-oligosaccharide from ovalbumin and Asn-GlcNAc were not. Thus, the enzyme is a new amidase capable of hydrolyzing aspartylglycosylamine linkage in glycopeptides with multiple amino acid residues.