Bovine Pituitary Membrane Glycoproteins Contain β-N-Acetylgalactosaminylated N-Linked Sugar Chains

Abstract: Bovine pituitary glycoprotein hormones contain unique N-linked sugar chains with GalNAcβ1 → 4GlcNAc structure in their outer chain moieties. In the present study, whether bovine pituitary membrane glycoproteins contain the sugar chains with the disaccharide structure was investigated. Western blot analysis of the membrane glycoproteins using Wistaria floribunda agglutinin (WFA), which binds oligosaccharides terminating with β- N -acetylgalactosamine residue(s), showed that most protein bands detected with Coomassie Brilliant Blue staining bind to WFA. However, no WFA binding was observed for the bands after treatment of the blotted filter with jack bean β- N -acetylhexosaminidase or N -Glycanase. The WFA-positive bands were also detected in membrane glycoprotein samples from bovine cerebrum, cerebellum, and medulla oblongata, although their expression levels were low. Structural analysis of the oligosaccharides released by hydrazinolysis from the pituitary membrane glycoproteins by serial lectin column chromatography and sequential exoglycosidase digestion revealed that the major oligosaccharides, which bound to a WFA-agarose column, are of biantennary complex type with one and two GalNAcβ1 → 4GlcNAc groups in their outer chain moieties. These results indicate that the β- N -acetylgalactosaminylation is not unique to the glycoprotein hormones but occurs to most bovine pituitary glycoproteins.     

Direct carbohydrate analysis of glycoproteins electroblotted onto polyvinylidene difluoride membrane from sodium dodecyl sulfate-polyacrylamide gel.

A procedure for the carbohydrate analysis of glycoproteins electrotransferred to a polyvinylidene difluoride membrane is described. The glycoproteins (plant lectins, transferrin, and vitronectin) were first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotted onto a membrane. Each of the glycoprotein bands visualized by staining with Coomassie brilliant blue R-250 was excised from the membrane and subjected to direct hydrolysis either in 2.5 M trifluoroacetic acid at 100 degrees C for 6 h for neutral sugars and hexosamines, or in 0.05 M H2SO4 at 80 degrees C for 1 h for sialic acids. The hydrolysate obtained was analyzed for neutral sugars, hexosamines, and sialic acids independently by three different systems of high-performance liquid chromatography. The analytical values were reproducible with reasonable accuracy and agreed with those expected with recoveries of 57-66%. The method was successfully applied to a mannose-specific lectin of Sophora japonica bark, which is composed of four different subunits that aggregate sugar specifically. Because the four subunits could be separated by SDS-PAGE alone, the method proved useful for determining their carbohydrate compositions. Three of them were shown to contain carbohydrates typical of N-linked oligosaccharides of plant origin, which agreed well with the results of the binding assay carried out on a membrane using various horseradish peroxidase-labeled lectins.     

Analysis of serine/threonine-linked oligosaccharides derived by alkaline-borohydride treatment of mucin glycoproteins electroblotted onto membranes: comparison of the saccharide profiles of the 390 kDa and 350 kDa forms of epitectin

Alkaline borohydride treatment is widely used for the release of carbohydrate moieties from O-glycosylated glycoproteins and mucins. We have adapted this procedure to micro quantities of glycoproteins blotted on membranes. After electrophoresis and transfer to nitrocellulose, nylon or polyvinylidene difluoride membrane, alkaline borohydride treatment was done directly on glycoprotein containing areas of membrane which were cut out with the aid of guide strips stained with Coomassie Blue or lectin-digoxigenin. In combination with standard saccharide fractionation techniques, this procedure can be used to characterize the oligosaccharides of mucins or mucin-type glycoproteins that are separated by gel electrophoresis from crude sources. Using this approach we have characterized the saccharides derived from the two species of epitectin, a malignancy-associated mucin type glycoprotein, isolated from metabolically labelled H.Ep2 cells.     

Amino acid analysis on polyvinylidene difluoride membranes

A procedure for the amino acid analysis of proteins electrotransferred to polyvinylidene difluoride (PVDF) membranes is described. The proteins are first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotted onto a PVDF membrane. After staining with Coomassie brilliant blue, the visualized protein bands are excised from the membrane. Each band is placed in a vial and subjected to gas-phase hydrolysis in 6 N HCl in a vacuum desiccator at 110 degrees C. The amino acids are extracted from the membrane into 0.1 N HCl/30% CH3OH and analyzed by reverse-phase HPLC using postcolumn o-phthalaldehyde-derivatizing reagent. The method was shown to give reproducible and reasonably accurate compositions for several proteins, as well as to provide an estimate of protein content. As little as 10 pmol of a 67-kDa protein can be determined.     

Identification and characterization of glycoproteins after extraction of bovine chromaffin-granule membranes with lithium di-iodosalicylate. Purification of glycoprotein II from the soluble fraction.

Chromaffin-granule membranes were separated into insoluble and soluble fractions after extraction with lithium di-iodosalicylate (LDIS). These fractions were characterized by one- and two-dimensional gel electrophoresis, and glycoproteins were detected after electroblotting with peroxidase-labelled concanavalin A and wheat-germ agglutinin (WGA). The LDIS-insoluble fraction contained components identified as glycoproteins III, H, J and K (carboxypeptidase H). Microsequence analysis indicated that component J is an N-terminally extended form of glycoprotein K. A major glycoprotein, GpII (Mr 80,000-100,000), present in the LDIS-soluble fraction was purified by affinity chromatography on WGA-Sepharose. This was characterized by one- and two-dimensional gel electrophoresis with Coomassie Blue staining, by amino acid analysis and automated N-terminal sequence analysis. Extraction of chromaffin-granule membranes with LDIS is a simple and rapid procedure that facilitates studies concerned with the structure and function of membrane glycoproteins from these and other secretory granules.     

Quantitative analysis of oligosaccharide structure of glycoproteins

A sensitive and quantitative method for the structural analysis of oligosaccharide was established for the glycoform analysis of glycoproteins. In this study,N-linked oligosaccharides of human IgG and bovine transferrin were analyzed for the evaluation of the method. Carbohydrate moiety of glycoprotein was released by hydrazinolysis and purified by paper chromatography. The oligosaccharides were labeled with a fluorescent dye, 2-aminobenzamide, for the enhancement of detection sensitivity. Sialylated (acidic) oligosaccharides were separated from neutral oligosaccharide by employing a strong anion-exchange column (MonoO) followed by the treatment with sialidase. Enzymatically desialyated fractions and neutral fractions of oligosaccharides were applied to normal-phase HPLC to resolve the peaks according to glucose unit (GU). The structure of separated molecules was further determined by sequential digestion with exoglycosidases. As a result, disialylated biantennary complextype oligo saccharide was found to be a major sugar chain in bovine transferrin (63%). In human IgG, core fucosylated asialobiantennary complex oligosaccharides were dominant. These results coincided well with reported results.     

Poly(N-Acetyllactosaminyl) Oligosaccharides of Chromaffin Granule Membrane Glycoproteins

Poly(N-acetyllactosaminyl) oligosaccharides have been identified, on the basis of their susceptibility to endo-beta-galactosidase, in a large-molecular-size glycopeptide fraction derived from chromaffin granule membrane glycoproteins. The glycoproteins containing poly(N-acetyl-lactosaminyl) oligosaccharides were selectively labeled by treatment of chromaffin granule membranes with endo-beta-galactosidase to expose N-acetylglucosamine residues, followed by incubation with galactosyltransferase and UDP-[14C]galactose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography demonstrated specific labeling in the 41-47 kilodalton (kD) region and in a distinct band at 90 kDa. Two-dimensional SDS-PAGE revealed that the poly(N-acetyllactosaminyl) oligosaccharides are predominantly present in glycoprotein IV, together with lesser labeling of glycoproteins II and III, whereas they are absent from dopamine beta-hydroxylase and carboxypeptidase H, which are the major glycoproteins of chromaffin granule membranes.     

Microassay analyses of protein glycosylation

To accurately characterize the carbohydrate moieties of oligosaccharide chains in glycosylated proteins, it is necessary to distinguish exactly which types of oligosaccharides are present at which site. We describe lectin overlay assays, which take advantage of the ability of lectins to distinguish between different types of glycoproteins via recognition of terminal sugars, thus allowing the chain type and peripheral antigenic components to be determined. Three microassays involving lectins are reported in this paper: non-proteasetreated intact glycoproteins; glycopeptides released by prior digestion of the glycoprotein and then separated by HPLC; and release of sugars from glycoproteins by hydrazinolysis and then coupling them to a multivalent support.     

Behaviour of dehydroalanine derivatives under hydrazinolysis conditions. Possible relevance to glycoprotein hydrazinolysis

Reactions that are relevant to the cleavage, by hydrazinolysis, of O‐linked oligosaccharides from glycoproteins were studied using dehydroalanine derivatives as models of the intermediates formed from O‐glycosylated serine residues. Conjugate addition of hydrazine followed by cyclisation to form pyrazolidinones, if occurring during glycoprotein hydrazinolysis, could reduce the yield of released oligosaccharide. However, N‐acetyldehydroalanine amide derivatives, which modelled the dehydroalanine derivatives believed to be intermediates in the hydrazinolysis of glycoproteins containing O‐linked oligosaccharides, underwent conjugate addition but no cyclisation. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

A high-throughput microscale method to release N-linked oligosaccharides from glycoproteins for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis

This report describes a convenient method for the rapid and efficient release of N-linked oligosaccharides from low microgram amounts of glycoproteins. A 96-well MultiScreen assay system containing a polyvinylidene difluoride (PVDF) membrane is employed to immobilize glycoproteins for subsequent enzymatic deglycosylation. Recombinant tissue-type plasminogen activator (rt-PA) is used to demonstrate the deglycosylation of 0.1-50 micrograms of a glycoprotein. This method enabled the recovery of a sufficient amount of N-linked oligosaccharides released enzymatically with peptide N-glycosidase F (PNGaseF) from as little as 0.5 microgram rt-PA for subsequent analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI- TOF) mass spectrometry. The immobilization of rt-PA to the PVDF membrane did not sterically inhibit the PNGaseF-mediated release of oligosaccharides from rt-PA as determined by tryptic mapping experiments. Comparison of the oligosaccharides released from 50 micrograms of rt-PA by either the 96-well plate method or by a standard solution digestion procedure showed no significant differences in the profiles obtained by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Both neutral and sialylated oligosaccharide standards spiked into wells were recovered equally as determined by HPAEC-PAD. One advantage of this approach is that reduction and alkylation can be performed on submicrogram amounts of glycoproteins with easy removal of reagents prior to PNGaseF digestion. In addition, this method allows 60 glycoprotein samples to be deglycosylated in 1 day with MALDI-TOF or HPAEC-PAD analysis being performed on the following day.      

Structures of the carbohydrate chains of membrane glycoproteins IIb and IIIa of human platelets

Human platelet membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa), which have been proposed to be subunits of a receptor for fibrinogen, were purified from Triton X-100-solubilized platelet membranes by affinity chromatography on a concanavalin A (Con A)-Sepharose column followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Compositional analyses of the purified glycoproteins showed that GPIIb and GPIIIa contain 15% and 18% carbohydrate by weight, respectively, which consists of galactose, mannose, glucosamine, fucose, and sialic acid. This suggested that these glycoproteins contained N-linked carbohydrate chains. The carbohydrate chains were released from each glycoprotein by hydrazinolysis and then fractionated by ion-exchange chromatography on a Mono Q column. From each glycoprotein, mono-, di-, and trisialylated and neutral oligosaccharide fractions were obtained. The structures of these oligosaccharides were investigated by means of compositional and methylation analyses and digestion by exoglycosidase, and their reactivities to immobilized lectins were also examined. The neutral oligosaccharides, which comprised about 14% of the total oligosaccharides released from GPIIb and about 52% of that from GPIIIa, were found to be of the high mannose-type, in that they contained 5 or 6 mannose residues. On the other hand, a major part of the acidic oligosaccharides was found to consist of typical bi- and triantennary complex-type sugar chains, and much smaller amounts of tetraantennary complex-type sugar chains, and complex-type sugar chains with a fucosyl residue at a N-acetylglucosamine residue in the peripheral portion or a bisecting N-acetylglucosamine at a beta-mannosyl residue in the core portion were also detected. In conclusion, we found that GPIIb contained mainly complex-type sugar chains, whereas high mannose-type sugar chains were the predominant carbohydrate units in GPIIIa, and that the detected differences in the carbohydrate moieties of GPIIb and GPIIIa were quantitative but not qualitative.     

The carbohydrate moiety of band-3 glycoprotein of human erythrocyte membranes.

Band-3 glycoprotein was purified from human blood-group-A erythrocyte membranes by selective solubilization and gel chromatography on Sepharose 6B in the presence of sodium dodecyl sulphate. The purified glycoprotein was subjected to hydrazinolysis in order to release the carbohydrate moiety. The released oligosaccharides were N-acetylated and applied to a column of DEAE-cellulose. Most of the band-3 oligosaccharides obtained were found to be free of sialic acids. When this neutral fraction was subjected to gel chromatography on a column of Sephadex G-50, two broad peaks were observed indicating that the band-3 glycoprotein was heterogeneous in the size of the oligosaccharide moieties. All fractions from gel chromatography were found to contain galactose, mannose, N-acetylglucosamine and fucose. The higher-molecular-weight (mol.wt. 3000-8000) peak consisted of fucose, mannose, galactose, N-acetylglucosamine and N-acetylgalactosamine in a molar proportion of 1.6:3.0:8.4:10.5:0.2. Most of these oligosaccharides were digested with a mixture of beta-galactosidase and beta-N-acetylhexosaminidase after alpha-L-fucosidase treatment to give a small oligosaccharide with the structure alpha Man2-beta Man-beta GlcNAc-GlcNAc. Methylation studies and limited degradation by nitrous acid deamination showed that the oligosaccharides contained the repeating disaccharide Gal beta 1----4GlcNAc beta 1----3, with branching points at C-6 of some of the galactose residues. These results indicate that a major portion of the band-3 oligosaccharide has a common core structure, with heterogeneity in the numbers of the repeating disaccharides, and contains fucose residues both in the peripheral portion and in the core portion. Haemagglutination tests were also carried out to determine the blood-group specificities of the glycoprotein and the results demonstrated the presence of both blood-group-H and I antigenic activities.     

水稻可溶性糖蛋白的纯化与鉴定研究

从水稻鲜叶中提取总蛋白,对总蛋白中的蛋白质含量进行了测定;通过硫酸铵沉淀将总蛋白提取液进行分级,从而达到了总蛋白细分和放量的目的。四级份的分级蛋白分别通过ConA-Sepharose 4B 亲和层析进行糖蛋白纯化,按吸收峰收集的各级糖蛋白混合物进行冷冻干燥,得到干粉;结合PAS法染色和考马斯亮蓝R-250染色对四级份的糖蛋白样品鉴定,在其中3个级份中均检测出糖蛋白;由于感度的差异,按考马斯亮蓝R-250染色可检测出近30种糖蛋白(包括部分糖肽),按PAS法染色可检测到7种糖蛋白;对3种含量较高的糖蛋白进行了胶上纯化,3种糖蛋白的PAS法染色均证实了3种样品为单一的糖蛋白或者糖肽,分别命名为RG1、RG2和RG3。     

Application of high-performance capillary electrophoresis to analysis of carbohydrates, especially those in glycoproteins

Methods for the component monosaccharide analysis and oligosaccharide mapping for glycoprotein research, based on HPCE of reductively pyridylaminated (PA) derivatives, are described. the component monosaccharides released from glycoproteins by acid hydrolysis are converted to PA derivatives and analyzed by HPCE as borate complexes. They can be quantified in the picomole range (introduced amount) with high reproducibility. The oligosaccharides released by hydrazinolysis are similarly converted to PA derivatives. Two-dimensional mapping of the relative mobilities of these derivatives, obtained in an acidic phosphate buffer and an alkaline borate buffer, ensures reliable identification of the oligosaccharides.     

Further characterization of HeLa S3 plasma membrane ghosts

A plasma membrane fraction of HeLa S3 cells, consisting of ghosts, is characterized more fully. A simple procedure is described which permits light and electron microscope study of the plasma membrane fraction through the entire depth of the final product pellet and through large areas parallel to the surface. Contamination by nuclei is 0.14%, too little for DNA detection by the diphenylamine reaction. Contamination by rough endoplasmic reticulum and ribosomes is small, a single ghost containing about 3% of the RNA in a single cell. Mitochondria were not encountered. Electron microscopy also shows (a) small vesicles associated with the outer surface of the ghosts, and (b) a filamentous web at the inner face of the ghost membrane. Sodium dodecyl sulfate (SDS)-polyacrylamide gel analysis shows that of the many Coomassie Blue-stained bands two were prominent. One, 43,000 daltons, co-migrated with purified rabbit muscle actin and constituted about 7.5% of the plasma membrane protein. The other major band, 34,000 daltons, was concentrated in the plasma membrane fraction. Two major glycoproteins detected by autoradiography of [14C]fucose-labeled glycoproteins on the gels, had apparent molecular weights of 35,000 daltons and 32,000 daltons. These major bands did not stain with Coomassie Blue. There were many other minor glycoprotein bands in the 200,000- to 80,000-dalton range. Ouabain-sensitive, Na+, K+-adenosine triphosphatase (ATPase) activity of the ghost fraction is purified 9.1 (+/- 2.2) times over the homogenate; recover of the activity is 12.0 (+/- 3.8%) of the homogenate. Enrichment and recovery of fucosylglycoprotein parallel those for ouabain-sensitive Na+, K+-ATPase activity. Fucosyl glycoprotein is recovered more than the enzyme activity in a smooth membrane vesicle fraction probably containing the bulk of plasma membrane not recovered as ghosts.     

Use of recombinant endomannosidase for evaluation of the processing of N-linked oligosaccharides of glycoproteins and their oligosaccharide-lipid precursors

Although glucose residues in a triglucosyl sequence are essential for the N-glycosylation of proteins and in their monoglucosyl form have been implicated in lectin-like interactions with chaperones, their removal is required for the formation of mature carbohydrate units and represents the initial steps in the glycoprotein processing sequence. In order to provide a probe for the glucosylation state of newly synthesized glycoproteins obtained from normal or altered cells, we have evaluated the usefulness of recombinant endo-alpha-mannosidase employing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to monitor the change in molecular mass brought about by the release of glucosylated mannose (Glc(1-3)Man). With this approach the presence of two triglucosylated-N-linked oligosaccharides in vesicular stomatis virus (VSV) G protein formed by castanospermine-treated CHO cells or the glucosidase I deficient Lec23 mutant could be clearly demonstrated and an even more pronounced change in migration was observed upon endomannosidase treatment of their more heavily N-glycosylated lysosomal membrane glycoproteins. Furthermore, the G protein of the temperature sensitive VSV ts045 mutant was found to be sensitive to endomannosidase, resulting in a change in electrophoretic mobility consistent with the presence of mono-glucosylated-N-linked oligosaccharides. The finding that endomannosidase also acts effectively on oligosaccharide lipids, as assessed by SDS-PAGE or thin layer chromatography, indicated that it would be a valuable tool in assessing the glucosylation state of these biosynthetic intermediates in normal cells as well as in mutants or altered metabolic states, even if the polymannose portion is truncated. Endomannosidase can also be used to determine the glucosylation state of the polymannose oligosaccharides released during glycoprotein quality control and when used together with endo-beta-N- acetylglucosaminidase H can distinguish between those terminating in a single N-acetylglucosamine or in a di-N-acetylchitobiose sequence.     

Structural study of N-linked sugar chains of sheep erythrocyte membrane glycoproteins

Our previous study showed that non-reducing terminal galactose residues of N-linked sugar chains present in sheep erythrocyte membrane glycoproteins are important for rosette formation with T lymphoblastic cells [Ogasawara et al. (1995) Immunol Lett 48: 35–38]. As a first step to elucidate the significant structures of sugar chains involved in rosette formation, we analysed N-linked sugar chains released from the membrane glycoproteins by hydrazinolysis. The oligosaccharides were labeled with NaB3H4 and fractionated using columns of Aleuria aurantia lectin-Sepharose, MonoQ and Bio-Gel P-4. Structural analyses of oligosaccharides by sequential exoglycosidase digestion in combination with methylation analysis revealed that the membrane glycoproteins contain bi- (19%), tri- (33%), and tetraantennary (44%) complex-type oligosaccharides and that the oligosaccharides having exposed galactose residues amount to 40% of the total.     

N-Glycosylation of membrane glycoproteins in retinol-deficient rat liver

The effect of vitamin A deficiency onN-linked oligosaccharides of membrane glycoproteins was studied in rat liver in order to evaluate the suggested role of retinol in proteinN-glycosylation. First, oligosaccharides of newly synthesized glycoproteins from rough endoplasmic reticulum of vitamin A deficient liver were compared with that of pair-fed controls. Oligosaccharides were metabolically labelled withd-[2-³H]mannose, released from the glycoproteins with endoglycosidase H, purified by reversed phase HPLC and ion exchange chromatography, and were reduced with sodium borohydride. HPLC fractionation of the oligosaccharide alditols showed that the glycoproteins carried mainly four oligosaccharide species, Glc₁Man₉GlcNAc₂, Man₉GlcNAc₂, Man₈GlcNAc₂ and Man₇GlcNAc₂, in identical relative amounts in the vitamin A deficient and the control tissue. In particular, no increase in the proportion of short chain oligosaccharides was noted in vitamin A deficient liver. Second, the number ofN-linked oligosaccharides was estimated in dipeptidylpeptidase IV (DPP IV), a major glycoprotein constituent of the hepatic plasma membrane, comparing the newly synthesized glycoprotein from rough endoplasmic reticulum and the mature form of DPP IV from the plasma membrane. No evidence was obtained that retinol deficiency caused incomplete glycosylation of this membrane glycoprotein. From these data, the suggested role of retinol as a cofactor involved in the synthesis ofN-linked oligosaccharides of glycoproteins must be questioned.Abbreviations DolP Dolichyl phosphate - DolPP dolichyl pyrophosphoryl - RetPMan retinyl phosphate mannose - DPP IV dipeptidyl peptidase IV (EC 3.4.14.5) - endo H endo--N-acetylglucosaminidase H (EC 3.2.1.96) - endo F endo--N-acetylglucosaminidase F (EC 3.2.1.96) - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Involvement of Lipid-linked Oligosaccharides in Synthesis of Storage Glycoproteins in Soybean Seeds

Membrane preparations from developing soybean (var. Prize) cotyledon tissue, at the time of synthesis of storage glycoproteins, catalyze the sequential assembly of lipid-linked oligosaccharides from uridine-5'-diphospho-N-acetyl-d-[6-(3)H] glucosamine and guanosine-5'diphospho-d-[U-(14)C]mannose. The maximum size of lipid-linked oligosaccharide that accumulates contains the equivalent of 10 saccharide units on the basis of Bio-Gel P-2 gel filtration studies. These lipid-linked oligosaccharides show similar characteristics to polyisoprenyl diphosphate derivatives on diethylaminoethyl-cellulose chromatography and are potential intermediates in glycoprotein biosynthesis in this tissue. These glycolipids do not appear to turn over in pulse-chase experiments and no completed storage glycoproteins were detected among the products of these incubations.Tissue slices from cotyledons at the same stage of development synthesize lipid-linked oligosaccharides from [(3)H]mannose and [(3)H]glucosamine with sizes equivalent to 1, 7, 10, and approximately 15 saccharide units. In pulse-chase experiments, the lipid-linked saccharides with the equivalent of 1 and 10 units rapidly turnover, whereas those with 7 and 15 units do not. Examination of the higher oligosaccharide peaks (10 and 15) by Bio-Gel P-4 gel filtration shows them to comprise 2 distinct subsets of oligosaccharides containing different proportions of glucosamine and mannose units. Tissue slices synthesize products which resemble the completed 7S storage glycoproteins as judged by similarity of molecular weight and precipitation with specific antisera. Analysis of the oligosaccharides obtained by hydrazinolysis of glycoproteins shows the presence of a similar size "high-mannose" type N-linked oligosaccharides as in other glycoproteins from animal and plant cells.     

Chromatographic separation of asparagine-linked oligosaccharides labeled with an ultravioletabsorbing compound,p-aminobenzoic acid ethyl ester

We have expanded on the suitability ofp-aminobenzoic acid ethyl ester as an ultraviolet-absorbing reagent [Wanget al., (1984) Anal Biochem 141:366–81] for the analysis of asparagine-linked oligosaccharides derived from glycoproteins. The oligosaccharides released from glycoproteins by hydrazinolysis/N-reacetylation were derivatized withp-aminobenzoic acid ethyl ester and the derivatives were purified and separated into neutral and acidic oligosaccharides on a PRE-SEP C₁₈ cartridge. The acidic oligosaccharides could be further separated into a few species by high-voltage paper electrophoresis. p-Aminobenzoic acid ethyl ester derivatives of neutral oligosaccharides were analyzed by gel permeation chromatography on Bio-Gel P-4 and HPLC on a silica-based amide column. The elution profile and the proportion of the oligosaccharides were in agreement with literature values. The overall yield of oligosaccharides from glycoproteins was approximately 70%. Fifty pmol of oligosaccharide were detectable on Bio-Gel P-4 and 4–5 pmol on HPLC.Abbreviations HPLC high performance liquid chromatography - NABEE p-aminobenzoic acid ethyl ester - FAB-MS fast-atom bombardment mass spectrometry - (GlcNAc)₂, (GlcNAc)₃, (GlcNAc)₄, (GlcNAc)₅ and (GlcNAc)₆ chito-oligosaccharides containing 2,3,4,5 and 6 residues ofN-acetylglucosamine