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.     

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.     

Phage-induced fucosidases hydrolysing the exopolysaccharide of Klebsiella aerogenes type 54[A3(S1)]

Several strains of bacteriophage have been isolated that induce the formation of a polysaccharide hydrolase after infection of Klebsiella aerogenes type 54 [A3(S1)]. The action of this enzyme on polysaccharide solutions was to decrease their viscosity and increase their reducing value. These effects were associated with the release of two oligosaccharides (O1 and O2) from the polysaccharide. These two substances are not identical with any of the four oligosaccharides isolated from autohydrolysates. The two enzymically isolated fractions have been tentatively identified as tetrasaccharides, and oligosaccharide O2 is probably an acetylated version of oligosaccharide O1. This latter oligosaccharide differs in some way, still unknown, from the tetrasaccharide cellobiosylglucuronosylfucose found in acid hydrolysates of the slime polysaccharide. The enzyme is limited in its activity to the polysaccharide excreted by the A3 strain of K. aerogenes type 54 or by similar strains. It is also active on the polysaccharides altered by acid or alkaline treatment. The enzyme has optimum activity at pH6.5. A study of the products released by enzyme action has shown it to be a fucosidase splitting the fucosylglucose linkages found in the intact polysaccharide.     

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.     

Rapid and sensitive screening of N-glycans as 9-fluorenylmethyl derivatives by high-performance liquid chromatography: a method which can recover free oligosaccharides after analysis

There are a large number of labeling methods for asparagine-type oligosaccharides with fluorogenic and chromophoric reagents. We have to choose the most appropriate labeling method based on the purposes such as mass spectrometry, high-performance liquid chromatography and capillary electrophoresis. Asparagine-type glycans are released from core proteins as N-glycosylamine at the initial step of the releasing reaction when glycoamidase F is employed as the enzyme. The N-glycosylamine-type oligosaccharides thus released by the enzyme are subjected to hydrolysis or mutarotation to form free-form oligosaccharides. In the detailed studies on the enzyme reaction, we found a condition in which the released N-glycosylamine-type oligosaccharides were exclusively present at least during the course of enzyme reaction, and developed a method for in situ derivatization of the glycosylamine-type oligosaccharides with 9-fluorenylmethyl chloroformate (Fmoc-Cl). The Fmoc labeled sialo- and asialo- (or high-mannose and hybrid) oligosaccharides were successfully analyzed on an amine-bonded polymer column and amide-silica column, respectively. The present method showed approximately 5 times higher sensitivities than that using 2-aminobenzoic acid (2-AA). The separation profile was similar to that observed using 2-AA method as examined by the analyses of carbohydrate chains derived from several glycoproteins including complex-type, high-mannose type and hybrid type of N-linked oligosaccharides. The labeled oligosaccharides were stable at least for several months when stored at -20 degrees C. Furthermore, it should be emphasized that the Fmoc-derivatized oligosaccharides could be easily recovered as free reducing oligosaccharides simply by incubation with morpholine in dimethylformamide solution. We obtained a pure triantennary oligosaccharide with 3 sialic acid residues as a free reducing form from fetuin in good yield after isolation of the corresponding Fmoc oligosaccharide followed by removing reaction of the Fmoc group. The proposed method will be useful for preparation of free oligosaccharides as standard samples at pmol-nmol scale from commercially available glycoproteins.     

Structural determination of the oligosaccharide side chains from a glycoprotein isolated from the mucus of the coral Acropora formosa

An extracellular mucous glycoprotein has been isolated from the hard coral Acropora formosa. The glycoprotein contains sulfated oligosaccharide side chains attached through O-glycosidic linkages to serine and threonine, the principal amino acids (77%) in the polypeptide. The oligosaccharide side chains consist of D-arabinose, D-mannose, and N-acetyl-D-glucosamine with smaller amounts of D-galactose, L-fucose, and N-acetyl-D-galactosamine, but no sialic or uronic acids. Alkaline borohydride reductive cleavage resulted in a mixture of oligosaccharide alditols. Six oligosaccharides were purified by high performance liquid chromatography. The structures of these oligosaccharides, which do not resemble those of any other glycoprotein so far examined, were determined by a combination of gas chromatography/mass spectrometry analysis of methylation products and NMR spectroscopy. All oligosaccharides contain a reducing terminal mannitol residue with N-acetylglucosamine linked to carbon 2, 4, or 6 of the mannitol. There is no evidence for linkage of N-acetylglucosamine to any other glycoses in the glycoprotein. Galactose was detected in two oligosaccharides linked to the 4-position of mannitol. Arabinose (Ara) was found in only one oligosaccharide. This was probably due to hydrolysis of the labile arabino-furanoside linkages. Evidence is presented which indicates the arabinose occurs primarily at the terminal position of oligosaccharide side chains. The structures of the oligosaccharides isolated from the glycoprotein were: (Formula: see text).     

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.     

Analyses of N-linked oligosaccharides using a two-dimensional mapping technique

We propose a two-dimensional sugar map method for the simple, reproducible, and sensitive analysis of the structures of N-linked oligosaccharides. The structure of an unknown oligosaccharide can be characterized from its position on the map. The data base for the sugar map is prepared by the use of 113 kinds of standard oligosaccharides, 58 of whose structures have been confirmed by 1H NMR spectroscopy. The present method involves six steps, (i) preparation of oligosaccharides from glycopeptides by N-oligosaccharide glycopeptidase (almond) digestion, (ii) derivatization of the reducing ends of oligosaccharides with a fluorescent reagent, 2-amino-pyridine, by using sodium cyanoborohydride, (iii) separation of oligosaccharide derivatives by high-performance liquid chromatography with an ODS-silica column, (iv) analysis of the size of each separated oligosaccharide on an amide-silica column, (v) plotting of the elution position of a sample on the two-dimensional sugar map obtained for the standard oligosaccharides, and (vi) structural analysis of the oligosaccharides by a combination of sequential exoglycosidase digestion and the steps (iii-v). The present method was applied to the identification of the structures of oligosaccharides in hen ovalbumin. It was found that two unusual oligosaccharides that have not yet been reported exist in ovalbumin.     

A high-performance liquid chromatography method for the analysis of picomole amounts of oligosaccharides

A method for the high-performance liquid chromatography separation of tritium-reduced, acetylated oligosaccharides is described. Their highly sensitive detection in column eluant is facilitated by the use of a flow radioactivity detector. The method differentiates some structural isomers and provides resolution of high-mannose oligosaccharides comparable or superior to that of other high-performance liquid chromatography methods. The detection limit is 0.3 pmol of oligosaccharide. For the detection of radioactive oligosaccharides this method is much less laborious than scintillation counting of collected peak fractions. Generation of a continuous chromatographic trace offers a particular advantage in the detection of partially resolved peaks and the visualization of peak shape. A study of some of the factors influencing acetylation and reduction has led to the development of a robust analytical method.     

Offline coupling of low-pressure anion-exchange chromatography with MALDI-MS to determine the elution order of human milk oligosaccharides

Pooled human milk oligosaccharides were separated into neutral and several acidic oligosaccharide fractions by preparative anion-exchange chromatography (AEC) using AG 1-X2. The oligosaccharides were eluted stepwise using deionized water and three different concentrations of ammonium acetate buffer, pH 6.8. The elution order of the compounds was determined directly by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of the AEC effluent without any cleanup or concentration steps. Up to a concentration of 500 mM ammonium acetate, the masses of acidic oligosaccharides could be detected by screening the fractions in an automated mode. The combination of the improved chromatographic procedure, the applied MALDI matrices, and operating parameters is suitable for the detection of neutral oligosaccharides as well as acidic oligosaccharides. The method provides high sensitivity and mass accuracy, including for the high-molecular-weight monosialylated oligosaccharides up to 2751.5 Da. The applied ionic strength of the anion-exchange eluents enables a rapid and an unambiguous composition assignment by MALDI-MS for neutral, monosialylated, and disialylated oligosaccharides from human milk. The acidic fractions have to be desalted by electrodialysis and were finally analyzed by HPAEC-PAD to get a high-resolution "fingerprint" of structures present in each fraction. From these analyses, it can be concluded that the isomeric variety of monosialylated oligosaccharides occurring in human milk is higher than estimated before.     

A note on the estimation of microbial glycosidase activities by dinitrosalicylic acid reagent

 In the estimation of glycosidase activity by dinitrosalicylic acid (DNS) reagent, the stoichiometry of DNS reduction was reported to increase proportionately with the increase in the number of glycosidic linkages present in oligosaccharides liberated by the enzyme. The relationship between increases in DNS reduction and increases in the number of glycosidic bonds was found to be represented by a part of a rectangular hyperbola. The increase was optimum with disaccharide and insignificant when the degree of polymerization (DP) was ≥10. The difference did not arise as a result of the DNSA discriminating between mono- and oligosaccharide oxidation. The relationship stemmed from the acidity of the hydroxyl group adjacent to the reducing group, which repressed DNS reduction. The acidity is likely to decrease with an increase in oligosaccharide chain length. It is suggested that DNS reduction is actually optimum and uniform for all oligosaccharides of DP ≥ 10 and that it is minimum for monosaccharide. Thus the introduction of rectification factors in the estimation of glycosidase activities by the DNS method appears to be justified. Received: 18 January 1999 / Received revision: 7 December 1999 / Accepted: 19 December 1999     

Structural characterization of N-linked oligosaccharides on monoclonal antibody cetuximab by the combination of orthogonal matrix-assisted laser desorption/ionization hybrid quadrupole-quadrupole time-of-flight tandem mass spectrometry and sequential enzymatic digestion

Cetuximab is a novel therapeutic monoclonal antibody with two N-glycosylation sites: a conserved site in the CH2 domain and a second site within the framework 3 of the variable portion of the heavy chain. The detailed structures of these oligosaccharides were successfully characterized using orthogonal matrix-assisted laser desorption/ionization hybrid quadrupole-quadrupole time-of-flight mass spectrometry (oMALDI Qq-TOF MS) and tandem mass spectrometry (MS/MS) in combination with exoglycosidase digestion. The N-linked oligosaccharides were released by treatment with N-glycanase F, reductively aminated with anthranilic acid, and fractionated by normal phase high-performance liquid chromatography (NP-HPLC). The fluorescent-labeled oligosaccharide pool and fractions were analyzed by oMALDI Qq-TOF MS and MS/MS in negative ion mode. Each fraction was further digested with an array of exoglycosidase mixtures, and subsequent MALDI TOF MS analysis of the resulting products yielded information about structural features of the oligosaccharide. The combined data revealed the presence of 21 distinct oligosaccharide structures in cetuximab. These oligosaccharides differ mainly in degree of sialylation with N-glycolyl neuraminic acid and extent of galactosylation (zero-, mono-, di-, and alpha(1-3)-galactosidase). The individual oligosaccharides were further assigned to the specific sites in the Fab and Fc regions of the antibody. This study represents a unique approach in that MS/MS data were used to identify and confirm the oligosaccharide structures of a protein.     

In vivo ligand specificity of E-selectin binding to multivalent sialyl Lewisx N-linked oligosaccharides.

The in vivo specificity for E-selectin binding to a panel of N-linked oligosaccharides containing a clustered array of one to four sialyl Lewisx (SLex; NeuAcalpha2-3Gal[Fucalpha1-3]beta1-4GlcNAc) determinants was studied in mice. Following intraperitoneal dosing with lipopolysaccharide, radioiodinated tyrosinamide N-linked oligosaccharides were dosed i.v. and analyzed for their pharmacokinetics and biodistribution. Specific targeting was determined from the degree of SLex oligosaccharide targeting relative to a sialyl oligosaccharide control. Oligosaccharides targeted the kidney with the greatest selectivity after a 4-h induction period following lipopolysaccharide dosing. Unique pharmacokinetic profiles were identified for SLex biantennary and triantennary oligosaccharides but not for monovalent and tetraantennary SLex oligosaccharides or sialyl oligosaccharide controls. Biodistribution studies established that both SLex biantennary and triantennary oligosaccharides distributed to the kidney with 2-3-fold selectivity over sialyl oligosaccharide controls, whereas monovalent and tetraantennary SLex oligosaccharides failed to mediate specific kidney targeting. Simultaneous dosing of SLex biantennary or triantennary oligosaccharide with a mouse anti-E-selectin monoclonal antibody blocked kidney targeting, whereas co-administration with anti-P-selectin monoclonal antibody did not significantly block kidney targeting. The results suggest that SLex biantennary and triantennary are N-linked oligosaccharide ligands for E-selectin and implicate E-selectin as a bivalent receptor in the murine kidney endothelium.     

Structural analysis of N-linked oligosaccharides by a combination of glycopeptidase, exoglycosidases, and high-performance liquid chromatography

A simple, sensitive, and rapid method for the analysis of structures of N-linked carbohydrates is reported. The method involves four steps: preparation of carbohydrate chains from glycopeptides by N-oligosaccharide glycopeptidase digestion; derivatization of the reducing ends of carbohydrate chains with a fluorescent reagent, 2-aminopyridine, by using sodium cyanoborohydride; separation of oligosaccharide derivatives by reverse-phase high-performance liquid chromatography; and structural analysis of oligosaccharides by sequential exoglycosidase digestion. The elution positions of 50 standard oligosaccharide derivatives were determined by HPLC. The structure of an unknown oligosaccharide can be characterized by comparison of its elution position with those of the standard compounds. The method was applied to elucidate the structures of oligosaccharides in the myeloma IgG protein, Yot.     

Profiling oligosaccharidurias by electrospray tandem mass spectrometry: quantifying reducing oligosaccharides

A method to semiquantify urinary oligosaccharides from patients suffering from oligosaccharidurias is presented. 1-Phenyl-3-methyl-5-pyrazolone has been used to derivatize urinary oligosaccharides prior to analysis by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Disease-specific oligosaccharides were identified for several oligosaccharidurias, including GM1 gangliosidosis, GM2 gangliosidosis, sialic acid storage disease, sialidase/neuraminidase deficiency, galactosialidosis, I-cell disease, fucosidosis, Pompe and Gaucher diseases, and alpha-mannosidosis. The oligosaccharides were referenced against the internal standard, methyl lactose, to produce ratios for comparison with control samples. Elevations in specific urinary oligosaccharides were indicative of lysosomal disease and the defective catabolic enzyme. This method has been adapted to enable assay of large sample numbers and could readily be extended to other oligosaccharidurias and to monitor oligosaccharide levels in patients receiving treatment. It also has immediate potential for incorporation into a newborn screening program.     

The initial stages of processing of protein-bound oligosaccharides in vitro.

Following the rapid enzymatic transfer of an oligosaccharide (GlcNAc2Man9Glc3) from a lipid carrier to endogenous protein acceptors in membrane preparations from NIL fibroblasts, the transferred oligosaccharide chain undergoes processing. Protein-bound oligosaccharides, released from the polypeptide backbone by treatment with endo-beta-N-acetylglucosaminidase H, were analyzed by gel filtration and by susceptibility to alpha-mannosidase digestion. The initial stages of this processing in vitro consist of sequential excision of 3 glucose residues prior to the removal of mannose residues. The array of oligosaccharides generated in vitro by membrane preparations from NIL cells appears to be identical with processed oligosaccharides derived in vivo in intact NIL cells.     

Deciphering the Glycan Preference of Bacterial Lectins by Glycan Array and Molecular Docking with Validation by Microcalorimetry and Crystallography

Recent advances in glycobiology revealed the essential role of lectins for deciphering the glycocode by specific recognition of carbohydrates. Integrated multiscale approaches are needed for characterizing lectin specificity: combining on one hand high-throughput analysis by glycan array experiments and systematic molecular docking of oligosaccharide libraries and on the other hand detailed analysis of the lectin/oligosaccharide interaction by x-ray crystallography, microcalorimetry and free energy calculations. The lectins LecB from Pseudomonas aeruginosa and BambL from Burkholderia ambifaria are part of the virulence factors used by the pathogenic bacteria to invade the targeted host. These two lectins are not related but both recognize fucosylated oligosaccharides such as the histo-blood group oligosaccharides of the ABH(O) and Lewis epitopes. The specificities were characterized using semi-quantitative data from glycan array and analyzed by molecular docking with the Glide software. Reliable prediction of protein/oligosaccharide structures could be obtained as validated by existing crystal structures of complexes. Additionally, the crystal structure of BambL/Lewis x was determined at 1.6 Å resolution, which confirms that Lewis x has to adopt a high-energy conformation so as to bind to this lectin. Free energies of binding were calculated using a procedure combining the Glide docking protocol followed by free energy rescoring with the Prime/Molecular Mechanics Generalized Born Surface Area (MM-GBSA) method. The calculated data were in reasonable agreement with experimental free energies of binding obtained by titration microcalorimetry. The established predictive protocol is proposed to rationalize large sets of data such as glycan arrays and to help in lead discovery projects based on such high throughput technology.     

The structure of carbohydrate chains of blood-group substance. Isolation and elucidation of the structure of higher oligosaccharides from blood-group substance H

Twenty individual higher reduced oligosaccharides, having from seven to eleven monosaccharide units, were isolated after sodium borohydride degradation of blood-group substance H from pig stomach linings. Anion-exchange high-pressure liquid chromatography appears to be a very convenient and effective method for this kind of higher oligosaccharide mixtures separation. The oligosaccharide structures were determined by means of periodate oxidation, methylation analysis, partial acid and enzymic hydrolysis. It has been found that all the oligosaccharides investigated can be divided into four series. The oligosaccharides belonging to each series have the common oligosaccharide fragment to which terminal L-fucose and/or N-acetyl-D-glucosamine residues are attached. Comparison of all the oligosaccharide structures, including tri, penta and hexasaccharides described earlier, shows that the lower oligosaccharides represent the structural element of the higher oligosaccharides.     

Structural characterization of oligosaccharides by high-performance liquid chromatography, fast-atom bombardment-mass spectrometry, and exoglycosidase digestion

A method for structural characterization of oligosaccharides after preparing uv-absorbing derivatives is described. The derivatives can be rapidly analyzed and purified by high-performance liquid chromatography, with separation of various structures determined primarily by size and sugar composition. Derivatization requires as little as 0.5-1.0 nmol of oligosaccharide, and detection of down to 50 pmol of oligosaccharide is possible by monitoring absorbance at 229 nm. In addition, the carbohydrate portion of the derivative was found to retain its sensitivity to exoglycosidases, allowing sequential enzymatic digestions for determination of sugar sequence and anomerity to be performed. The derivatives also possessed a site of potential positive charge, making them amenable to analysis by fast-atom bombardment-mass spectrometry. Permethylation of the derivatives permitted their separation by capillary gas chromatography, thus allowing investigation of their structures by gas chromatography-mass spectrometry. The combination of these techniques will allow almost the complete structure of small amounts of oligosaccharides to be determined.     

A GM2-specific beta-hexosaminidase from the roe of striped mullet (Mugil cephalus)

The roe of striped mullet (Mugil cephalus) was found to contain a beta-hexosaminidase different from the beta-hexosaminidases isolated from other sources. The enzyme from mullet roe is able to cleave GalNAc from GM2 without the assistance of either an activator protein or a detergent. It also cleaves the oligosaccharide derived from GM2 and other oligosaccharides containing the GM2 sequence GalNAc beta 4(NeuAc alpha 3)Gal-. However, it is not effective in hydrolyzing neutral glycosphingolipids containing terminal GalNAc or GlcNAc, such as GbOse4Cer, GgOse3Cer, or LcOse3Cer. These results indicate that mullet roe beta-hexosaminidase can specifically cleave GalNAc from the glycoconjugates containing the GM2 sequence. No beta-hexosaminidase with such specificity has been previously described. Thus, this unique enzyme should be very useful for the detection and analysis of glycoconjugates containing the oligosaccharide chains with GM2 sequence.