Endo β-N-acetylglucosaminidase F cleavage specificity with peptide free oligosaccharides

Endo β-N-acetylglucosaminidase activities were determined based on conversion of oligosaccharides containing two N-acetylglucosamines to the oligosaccharides with a single N-acetylglucosamine at the reducing terminal and following their separation on a carbohydrate analyzer. The oligosaccharides eluted from the high performance anion exchange column in the order of fucosyl-N,N′ -diacetylchitobiose, N,N′ -diacetylchitobiose and N-acetylglucosamine containing reducing terminals. Using this assay, differences in cleavage specificity of the glycoproteins was determined. The commercial Endo F-peptide N-glycosidase/glycanyl amidase (PNGase)mixture readily leaved high mannose and complex oligosaccharides (neutral and sialyated) with common core α1–6 linked fucose found in porcine thyroglobulin including the trimannosyl-chitobiose core structure. However, the same Endo F mixture did not cleave the non-fucosylated complex oligosaccharides found in human transferrin and also the common core structure. Glycopeptide counterparts with and without fucose were good substrates for the endoglycosidases. These results show that the specificity of these enzymes is such that they can recognize the conformational differences between free oligosaccharides and glycopeptides with and without the common core α1–6 linked fucose. In contrast, highly purified Endo F cleaved only the high mannose type oligosaccharides and was unable to cleave ovalbumin hybrid type oligosaccharides. However, it was similar to Endo H when reduced ovalbumin oligosaccharides were used as substrates, consistent with the recently isolated Endo F subfraction F₁ being similar to Endo H [Trimble, R. B. and Tarentino, a. L. (1991). J. Biol. Chem. 266, 1646]. Results obtained in this study suggest that the complex oligosaccharides cleaving enzymes F₂ and F₃ show high specificity towards peptide free oligosaccharides with the core α1-6 linked fucose, unlike the glycopeptide substrates. Therefore PNGase free Endo F₁, F₂ and F₃ mixtures should be useful in the functional evaluation of the oligosaccharides in glycoproteins.     

2-Iminothiolane: a reagent for the introduction of sulphydryl groups into oligosaccharides derived from asparagine-linked glycans

A facile method for introducing reactive sulphydryl groups intooligosaccharides was developed. 1-Amino-oligo-saccharides generatedfrom asparagine-linked glycans by peptide-N⁴(N-acetyl-ß-D-glucosaminyl)asparagine amidase (PNGase F) digestion were monitored by high-performanceanion-exchange chromatography with pulsed amperometric detectionand derivatized under optimal conditions with 2-iminothiolane—HC1.The resulting mercapto-butyramido oligosaccharides, which wereobtained in high yield, were alkylated with a fluorescent reagentand used to selectively assay for endoglycosidases that hydrolysedi-N-acetyl-chitobiose linkages. 1-amino-oligosaccharides fluorescent oligosaccharides 2-iminothiolane mercapto-butyramido oligosaccharides     

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.     

Deglycosylation of asparagine-linked glycans by peptide:N-glycosidase F

Endo-beta-N-acetylglucosaminidase F (Endo F) and peptide:N-glycosidase F (PNGase F) were purified from cultures of Flavobacterium meningosepticum by ammonium sulfate precipitation followed by gel filtration on TSK HW-55(S). This system separated the two enzymes and provided PNGase F in a high state of purity, but the basis for the resolution appeared to be hydrophobic interaction and not molecular size. Studies using purified Endo F and PNGase F with defined glycopeptides demonstrated that Endo F was somewhat similar to Endo H in that it hydrolyzed many, but not all, high-mannose and hybrid oligosaccharides, as well as complex biantennary oligosaccharides. PNGase F, in contrast, hydrolyzed all classes of asparagine-linked glycans examined, provided both the alpha-amino and carboxyl groups of the asparagine residue were in peptide linkage. Deglycosylation studies with PNGase F revealed that many proteins in their native conformation were susceptible to this enzyme but that prior denaturation in sodium dodecyl sulfate greatly decreased the amount of enzyme required for complete carbohydrate removal.     

Use of resorufin-labelled N-glycopeptide in a high-performance liquid chromatography assay to monitor endoglycosidase activities during cultivation ofFlavobacterium meningosepticum

Peptide-N ⁴-(N-acetyl--glucosaminyl) asparagine amidase F (PNGase F) and endo--N-acetyl glucosaminidase F (Endo F) activities were monitored during cultivation ofFlavobacterium meningosepticum using a new fluorescence-HPLC procedure based on a commercially available substrate. The PNGase F activity reached a maximum level at the end of the log phase and remained constant during the stationary phase, while Endo F continuously increased until late stationary phase. PNGase F obtained at the end of the log phase was less contaminated by other proteins compared with late stationary phase.Abbreviations Con A concanavalin A - Endo F endo--N-acetyl glucosaminidase F (EC 3.2.1.96) - GlcNAc N-acetylglucosamine - PNGase F peptide-N ⁴-(N-acetyl--glucosaminyl) asparagine amidase F (EC 3.5.1.52).     

The mapping by high-pH anion-exchange chromatography with pulsed amperometric detection and capillary electrophoresis of the carbohydrate moieties of human plasma alpha 1-acid glycoprotein.

The reducing oligosaccharides released from alpha 1-acid glycoprotein (AGP) by conventional hydrazinolysis have been analyzed by two different mapping techniques, using high-pH anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) and capillary electrophoresis (CE) with uv detection at 190 nm. The CE measurements proved about 4000 times more sensitive than the measurements by HPAE-PAD. The N-glycan pool was fractionated by Mono Q anion-exchange chromatography, and individual fractions so obtained were desialylated using Vibrio cholerae neuraminidase. The resulting asialo-N-glycans were further analyzed by HPAE-PAD, revealing 2 major, 4 intermediate, and 4 small peaks and at least 3 spikes, which counted for at least 13 different asialo-N-glycans. The carbohydrate structures were tentatively assigned by comparison of the Mono Q-separated N-glycans with the known AGP carbohydrate structures and known structures contained in a mapping database that allows structural assignment of N-glycans by mere comparison of retention times. In addition to the hitherto known AGP carbohydrate structures, we have tentatively identified a number of sulfated N-glycans that are currently being analyzed in more detail. We have also compared the glycan pools recovered from AGP using hydrazinolysis and glycopeptidase F (PNGase F). Approximately 40 distinct peaks could be detected in the hydrazinolysis-derived N-glycan pool by either technique (HPAE-PAD and CE), while about 30 distinct peaks were detected in the N-glycan pool derived by PNGase F digestion of the tryptic AGP digest of the same batch of AGP. These differences were attributed to an increased desialylation (approximately 3 mol%) during hydrazinolysis, based on the detection by HPAE-PAD and CE of free sialic acid and monosialylated oligosaccharides in the glycan pool derived by conventional hydrazinolysis. The integrity of the N-glycans' chitobiose core was examined by 500-MHz 1H NMR spectoscopy. The hydrazinolysis procedure could be optimized such that the hydrazinolysis-derived N-glycan pool was chromatographically essentially identical to the PNGase F-derived N-glycan pool. Hydrazinolysis proved best, with practically no loss of N-acetlylneuraminic acid and the closest resemblance to the PNGase F-derived N-glycan pool, using an automated apparatus. Notably, it was recognized that, in our hands, PNGase F digestion in the presence of sodium dodecyl sulfate resulted in partial desialylation of the liberated N-glycans.     

Structural basis for the substrate specificity of endo-beta-N-acetylglucosaminidase F(3)

Endo-beta-N-acetylglucosaminidase F(3) cleaves the beta(1-4) link between the core GlcNAc's of asparagine-linked oligosaccharides, with specificity for biantennary and triantennary complex glycans. The crystal structures of Endo F(3) and the complex with its reaction product, the biantennary octasaccharide, Gal-beta(1-4)-GlcNAc-beta(1-2)-Man-alpha(1-3)[Gal-beta(1-4)-GlcNAc-be ta(1-2)-Man-alpha(1-6)]-Man-beta(1-4)-GlcNAc, have been determined to 1.8 and 2.1 A resolution, respectively. Comparison of the structure of Endo F(3) with that of Endo F(1), which is specific for high-mannose oligosaccharides, reveals highly distinct folds and amino acid compositions at the oligosaccharide recognition sites. Binding of the oligosaccharide to the protein does not affect the protein conformation. The conformation of the oligosaccharide is similar to that seen for other biantennary oligosaccharides, with the exception of two links: the Gal-beta(1-4)-GlcNAc link of the alpha(1-3) branch and the GlcNAc-beta(1-2)-Man link of the alpha(1-6) branch. Especially the latter link is highly distorted and energetically unfavorable. Only the reducing-end GlcNAc and two Man's of the trimannose core are in direct contact with the protein. This is in contrast with biochemical data for Endo F(1) that shows that activity depends on the presence and identity of sugar residues beyond the trimannose core. The substrate specificity of Endo F(3) is based on steric exclusion of incompatible oligosaccharides rather than on protein-carbohydrate interactions that are unique to complexes with biantennary or triantennary complex glycans.     

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.     

Molecular cloning and expression of endo-beta-N-acetylglucosaminidase D, which acts on the core structure of complex type asparagine-linked oligosaccharides

Endo-beta-N-acetylglucosaminidase D (Endo D) produced by Streptococcus pneumoniae cleaves the di-N-acetylchitobiose structure in asparagine-linked oligosaccharides. The enzyme generally acts on complex type oligosaccharides after removal of external sugars by neuraminidase, beta-galactosidase, and beta-N-acetylglucosaminidase. We cloned the gene encoding the enzyme and expressed it as a periplasm enzyme in Escherichia coli. The first 37 amino acids in the predicted sequence are removed in the mature enzyme, yielding a protein with a molecular mass of 178 kDa. The substrate specificity of the recombinant enzyme is indistinguishable from the enzyme produced by S. pneumoniae. Endo-beta-N-acetylglucosaminidase A (Endo A) from Arthrobacter protophormiae, the molecular mass of which is 72 kDa, had 32% sequence identity to Endo D, starting from the N-terminal sides of both enzymes, although Endo A hydrolyzes high-mannose-type oligosaccharides and does not hydrolyze complex type ones. Endo D is not related to endo-beta-N-acetylglucosaminidases H, F(1), F(2), or F(3), which share common structural motifs. Therefore, there are two distinct groups of endo-beta-N-acetylglucosaminidases acting on asparagine-linked oligosaccharides. The C-terminal region of Endo D shows homology to beta-galactosidase and beta-N-acetylglucosaminidase from S. pneumoniae and has an LPXTG motif typical of surface-associated proteins of Gram-positive bacteria. It is possible that Endo D is located on the surface of the bacterium and, together with other glycosidases, is involved in virulence.     

Transfer of glycerol by Endo-beta-N-acetylglucosaminidase F to oligosaccharides during chitobiose core cleavage

N-Linked oligosaccharides, when hydrolyzed by glycerol-containing preparations of endo-beta-N-acetylglucosaminidase (Endo) F from Flavobacterium meningosepticum were found to have glycerol attached to their reducing ends. The absence of a reducing end was confirmed by high-field 1H NMR spectroscopy, and the incorporated glycerol was verified through mass spectrometry and collisionally activated decomposition fast atom bombardment/mass spectrometry/mass spectrometry techniques. Periodate oxidation of [1(3)-14C]glycerol-labeled oligosaccharides indicated glycerol was glycosidically linked via its 1(3) carbon to the C1 of the reducing end N-acetylglucosamine. In a second, less favored reaction, the glycerol glycoside was hydrolyzed by Endo F using water as the terminal nucleophile, thus regenerating the N-acetylglucosamine reducing end. Glycerol could be removed from Endo F preparations without affecting enzyme stability, and chitobiosyl core hydrolysis in its absence provided intact oligosaccharides with normal N-acetylglucosamine reducing ends. The incorporation of labeled glycerol may provide a useful method for monitoring of Endo F release of oligosaccharides.     

Peptide-N 4-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity could explain the occurrence of extracellular xylomannosides in a plant cell suspension

We have previously isolated mannoside and xylomannoside oligosaccharides with one or two terminal reducingN-acetylglucosamine residues from the extracellular medium of white campion (Silene alba) suspension culture. We have now demonstrated the presence of peptide-N ⁴-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity in cell extracts as well in the culture medium that could explain the production of those compounds. An additional xylomannoside, (GlcNAc)Man₃(Xyl)GlcNAc(Fuc)GlcNAc, was characterized, and¹H- and¹³C-NMR assignments for the oligosaccharide Man₃(Xyl)GlcNAc(Fuc)GlcNAc were obtained using homonuclear and heteronuclear spectroscopy (COSY).Abbreviations Endo endo--N-acetylglucosaminidase - Fuc fucose - GlcNAc N-acetylglucosamine - Man mannose - NMR nuclear magnetic resonance - PNGase peptide-N ⁴-(N-acetylglucosaminyl)asparagine amidase - Xyl xylose     

Characterization of carbohydrates using highly fluorescent 2-aminobenzoic acid tag following gel electrophoresis of glycoproteins.

Application of the most sensitive fluorescent label 2-aminobenzoic acid (anthranilic acid, AA) for characterization of carbohydrates from the glycoproteins ( approximately 15 pmol) separated by polyacrylamide gel electrophoresis is described. AA label is used for the determination of both monosaccharide composition and oligosaccharide map. For the monosaccharide determination, bands containing the glycoprotein of interest are excised from the polyvinylidene fluoride (PVDF) membrane blots, hydrolyzed in 20% trifluoroacetic acid, derivatized, and analyzed by C-18 reversed-phase high-performance liquid chromatography. For the oligosaccharide mapping, bands were digested with peptide N-glycosidase F (PNGase F) in order to release the N-linked oligosaccharides, derivatized, and analyzed by normal-phase anion-exchange chromatography. For convenience, the PNGase F digestion was performed in 1:100 diluted ammonium hydroxide overnight. The oligosaccharide yield from ammonium hydroxide-PNGase F digestion was better or equal to all the other reported procedures, and the presumed "oligosaccharide-amine" product formed in the reaction mixture did not interfere with labeling of the oligosaccharides under the conditions used for derivatization. Sequencing of oligosaccharides can be performed using the same mapping method following treatment with an array of glycosidases. In addition, the mapping method is useful for determining the relative and simultaneous distribution of sialic acid and fucose.     

Echinococcus granulosus: antigen characterization by chemical treatment and enzymatic deglycosylation.

Parasite antigenic fractions obtained by biochemical purification of sheep hydatid fluid were subjected to enzymatic digestion. The relative mobilities of the 5 and B antigens, before and after treatment, were analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Antigenic fractions transferred to nitrocellulose were also treated with sodium metaperiodate and concanavalin A. The results indicate that antigen 5 contains a substantial amount of carbohydrates covalently linked to a polypeptide backbone, which strongly bind to concanavalin A and is removed by N-glycosidase F (PNGase F). Antigen 5 possesses complex N-linked oligosaccharides (PNGase F sensitive), without terminal N-acetyl-D-glucosamine residues (N-acetyl-D-glucosaminidase nonsensitive) and has no high-mannose oligosaccharides (endo-beta-N-acetylglucosaminidase H nonsensitive). In contrast, the antigen B of low molecular weight is not susceptible to either enzymatic digestions (PNGase F, Endo H, and N-acetyl-D-glucosaminidase) or sodium metaperiodate oxidation and it does not bind to concanavalin A. Polyclonal antibodies prepared against the two antigens reacted with the deglycosylated antigen 5 in Western blot. The dominant epitopes are, therefore, polypeptides, although the presence of carbohydrate epitopes in the native glycoproteins cannot be excluded.     

Single-step Purification by Lectin Affinity and Deglycosylation Analysis of Recombinant Human and Porcine Deoxyribonucleases I Expressed in COS-7 Cells

Human and porcine recombinant deoxyribonucleases I (DNases I) were expressed in COS-7 cells, and purified by a single-step procedure. Since affinities for concanavalin A (Con A) and wheatgerm agglutinin (WGA) were strong in these recombinant DNases I, purification using Con A–WGA mixture-agarose column was performed. By this method, the enzymes in culture medium could quickly be isolated to apparent homogeneity in approx. 10 min. From 1 ml of culture medium, about 20–30 μg of purified DNase I with a specific activity ranging from 22000 to 41000 units/mg were obtained. The purified DNases I were subjected to enzymatic deglycosylation by either peptide N-glycosidase F (PNGase F) or endoglycosidase H (Endo H). The recombinant enzyme was cleaved by PNGase F, but not by Endo H, indicating that the recombinant enzymes are modified by N-linked complex-type carbohydrate moieties. In the human recombinant DNase I, activity was decreased by PNGase F-treatment, while that of the porcine DNase I remained unaffected. The thermal stability of the human enzyme was extremely susceptible to heat following PNGase F-treatment, as was the porcine enzyme to a lesser extent. This study suggests that N-linked complex-type carbohydrate moieties may contribute to the enzymatic activity and/or thermal stability of recombinant DNases I.     

Enzymatic deglycosylation of thyroid-stimulating hormone with peptide N-glycosidase F and endo-beta-N-acetylglucosaminidase F

We investigated the ability of two enzymes, peptide N-glycosidase F (PNGase F) and endo-beta-N-acetylglucosaminidase F (Endo F), to deglycosylate microgram quantities of bovine TSH and its subunits under nondenaturing conditions. One oligosaccharide chain could be selectively removed from the alpha subunit by PNGase F, and all the oligosaccharide chains from both subunits could be removed by Endo F. These methods of enzymatic deglycosylation should permit study of the functional role of each N-linked carbohydrate chain of various glycoprotein hormones.     

Structural analysis of the glycoprotein allergen Art v II from the pollen of mugwort (Artemisia vulgaris L.).

The glycoprotein allergen Art v II, from the pollen of mugwort (Artemisia vulgaris L.) was treated with peptide:N-glycosidase F (PNGase F) to release asparagine-linked oligosaccharides. The oligosaccharides were isolated by gel permeation chromatography and their structures determined by 500-MHz 1H NMR spectroscopy, fast atom bombardment-mass spectrometry, and high-pH anion-exchange chromatography. The high-mannose oligosaccharides Man5GlcNAc2, Man6GlcNAc2, Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 were present in the ratios 2:49:19:24:6 and accounted for all the asparagine-linked oligosaccharides released from Art v II by PNGase F. The NH2-terminal amino acid sequences of Art v II and of four peptides generated by cyanogen bromide (CNBr) cleavage of deglycosylated Art v II were determined. The first 30 amino acid residues of Art v II did not contain any potential N-glycosylation sites. One potential N-glycosylation site was identified in one of the CNBr fragments. The native protein conformation was shown by enzyme-linked immunosorbent assay inhibition assays to be essential for the binding of rabbit IgG to Art v II and for the binding of human IgE to the major IgE-binding epitope(s) in this allergen. At least one minor IgE-binding epitope still bound IgE after denaturation of the allergen. Removal of the high-mannose chains from denatured Art v II had no significant effect on the binding of human IgE to the minor IgE-binding epitope(s).     

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.     

One-step purification of mammalian deoxyribonucleases I and differences among pancreas, parotid, and pancreas-parotid (mixed) types based on species- and organ-specific N-linked glycosylation

Mammalian deoxyribonucleases I (DNase I) are classified into three types, namely, pancreas, parotid, and pancreas-parotid (mixed), based on differences in their tissue concentrations. In this study, DNase I purification by concanavalin A-wheat germ agglutinin mixture-agarose column from rat (parotid type), rabbit (mixed type), and pig (pancreas type) is described. This method permits a relatively easy one-step purification of DNase I from rat and rabbit parotid glands, the rat submaxillary gland, and porcine pancreas. To elucidate differences among the three types, these DNases I were subjected to enzymatic deglycosylation either by peptide N-glycosidase F (PNGase F) or endoglycosidase H (Endo H). Following deglycosylation, digests were separated on DNA-casting polyacrylamide gel electrophoresis. PNGase F produced a single lower mobility product in all samples. Endo H produced a double band in rat and rabbit parotid glands and porcine pancreas, and a single band in the rabbit pancreas corresponding with the PNGase F product. DNase I activity of the porcine pancreas was completely extinguished by deglycosylation, while that of the parotid glands and rabbit pancreas was unaffected. Our results suggest that the distinct properties of DNase I exhibited by the three types may be attributed to differences in the extent of post-translational N-linked glycosylation of the enzyme.     

Carbohydrate analysis of glycoprotein hormones

Complete carbohydrate composition analysis of glycoprotein hormones, their subunits, and oligosaccharides isolated from individual glycosylation sites can be accomplished using high-pH anion-exchange chromatography combined with pulsed amperometric detection. Neutral and amino sugars are analyzed from the same hydrolyzate by isocratic chromatography on a Dionex CarboPAC PA1 column in 16 mM NaOH. Sialic acid is quantified following mild hydrolysis conditions on the same column in 150 mM sodium acetate in 150 mM NaOH. Ion chromatography on a Dionex AS4A column in 1.8 mM Na(2)CO(3)/1.7 mM NaHCO(3); postcolumn, in-line anion micromembrane suppression; and conductivity detection can be used to quantify sulfate, a common component of pituitary glycoprotein hormone oligosaccharides. Mass spectrometric analysis before and after elimination of oligosaccharides from a single glycosylation site can provide an estimate of the average oligosaccharide mass, which facilitates interpretation of oligosaccharide composition data. Following release by peptide N-glycanase (PNGase) digestion and purification by ultrafiltration, oligosaccharides can be characterized by a high-resolution oligosaccharide mapping technique using the same equipment employed for composition analysis. Oligosaccharide mapping can be applied to the entire hormone, individual subunits, or individual glycosylation sites by varying PNGase digestion conditions or substrates. Oligosaccharide release by PNGase is readily monitored by SDS-PAGE. Site-specific deglycosylation can be confirmed by amino acid sequence analysis. For routine isolation of oligosaccharides, addition of 2-aminobenzamide at the reducing terminus facilitates detection; however, the oligosaccharide retention times are altered. Composition analysis is also affected as the 2-aminobenzamide-modified GlcNAc peak overlaps the fucose peak.     

Differential release of high mannose structural isoforms by fungal and bacterial endo-β-N-acetylglucosaminidases

Endo-β-N-acetylglucosaminidases (ENGases) are widely used to remove N-linked oligosaccharides from glycoproteins for glycomic and proteomic studies and biopharmaceutical processes. Although several ENGases are widely available and their main oligosaccharide structural preferences are generally known (i.e. high mannose, hybrid or complex), the preferences of ENGases from different kingdoms for individual structural isoforms within the major classes of N-linked oligosaccharides have previously not been compared. In this work, a fungal ENGase (Endo Tv) was purified for the first time from a commercial Trichoderma viride chitinase mixture by sequential anion exchange and size exclusion chromatography, a commonly used strategy for purification of chitinases and endo enzymes. Oligosaccharides released from substrate glycoproteins by Endo Tv were identified and quantified by high pH anion exchange chromatography with pulsed amperometric detection and verified by mass spectrometric analysis. Unlike the widely-used bacterial ENGases, Endo H and Endo F1, Endo Tv released exclusively high mannose N-linked oligosaccharides from RNase B, ovalbumin, and yeast invertase. Endo Tv did not hydrolyze fucosylated, hybrid, complex type or bisecting N-acetylglucosamine-containing structures from bovine fetuin, ovalbumin and IgG. When compared to the bacterial ENGase, Endo H, the relative ratio of high-mannose oligosaccharide structural isoforms released from RNase B by Endo Tv was found to differ, with Endo Tv releasing more Man?GlcNAc and Man?GlcNAc isoform I and less Man(9)GlcNAc from RNase B. Based on these data, it is suggested that use of ENGases from multiple sources may serve to balance an introduced bias in quantitative analysis of released structural isoforms and may further prove valuable in biochemical structure-function studies.