created: 15th May 1996, last updated: 29th April 1998,© 1998 ABRF
The need to characterize sample-limited glycoprotein glycans continues to increase as more irrefutable examples of the essential role that covalent carbohydrates play in biological processes are identified (1). Specific carbohydrate structures are indispensable for the proper maturation, intracellular sorting, and function of many glycoproteins, and disease states arise in humans from a failure of glycoproteins to be properly processed, targeted and degraded. Thus, understanding the structure, enzymology and cell biology of protein glycosylation is requisite to pinpointing their roles in normal and pathological states. In addition to understanding the above natural phenomena, the ability to use recombinant DNA technology to produce úglyco-pharmaceuticalsú, such as hormones (TSH, FSH, HCG) and growth factors (EPO), has intensified the need to define more efficiently the contribution of heterologous expression systems in the biosynthesis of glycoprotein oligosaccharides. Although the peptide backbone of a protein is likely to be the same independent of the expression system, this is usually not true for glycosylation patterns, which can vary widely with cell type. Added to these considerations is the inherent complexity and heterogeneity displayed by covalently-linked carbohydrates. The three major classes of covalently-attached carbohydrate are either N-linked to asparagine residues in Asn-Xaa-Thr/Ser sequons, O-linked to a subset of Thr/Ser residues or linked to the C-terminus of some membrane proteins as glycosylphosphatidyl inositol or úGPIú anchors. There are chemical and enzymatic methods for determining which class or classes may be present on a given glycoprotein.
In recent years, the technology to analyze smaller amounts (ú 5 pmol) of glycoprotein oligosaccharides has advanced considerably through improvements in four areas----sample preparation, labeling, separations and mass spectrometry. Combining these newer methods with lectin and exoglycosidase analysis has produced a "critical mass" of techniques that are being used to unravel the glycobiology of proteins. This workshop presented an overview of the methods, which have been successfully applied to the analysis of sample-limited (< nmol) glycoproteins and which could be successfully imported into a core facility's repertoire of capabilities. The extant challenge of "glycotechnology" is to approach the low pmol level of sensitivity, routinely used for protein sequencing.
Detecting Glycoproteins in Gels and on Blots.
Determining whether a protein is in fact glycosylated is the initial step in glycoprotein glycan analysis. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) has become the method of choice as the final step prior to protein sequencing. Glycosylated proteins often migrate as diffuse bands by SDS-PAGE. A marked decrease in band width and change in migration position after treatment with peptide-N4-(N-acetyl-ú-D-glucosaminyl) asparagine amidase (PNGase F) is considered diagnostic of N-linked glycosylation. If the other types of glycosylation are predominant other approaches must be used (Diagram I). Lectin blotting methods provide an approach which is independent of the class of glycosylation (N versus O). Lectins, carbohydrate-binding proteins from various plant tissues, have both high affinity and narrow specificity for a wide range of defined sugar epitopes found on glycoprotein glycans (2). When conjugated with biotin or digoxigenin, they can be easily identified on membrane blots through a colorimetric reaction utilizing avidin or anti-digoxigenin antibodies conjugated with alkaline phosphatase (3), analogous to secondary antibody-alkaline phosphatase reactions employed in Western blotting. Screening with a panel of lectins with well-defined specificity can provide considerable information about a glycoprotein's carbohydrate complement. Importantly, the color development amplification is sufficiently high that 10-50 ng of a glycoprotein can easily be seen on a membrane blot of an SDS-PAGE. Although lectins exhibit very high affinity for their cognate ligands, some do reveal significant avidity for structurally-related epitopes. Thus, it is important to carefully note the possibility of cross-reactivity when choosing a panel of lectins, and apply those with the highest probability of individually distinguishing complex, hybrid and high mannose N-linked glycans from O-linked structures.
Monosaccharide analysis can also be used to determine whether a protein is glycosylated and as in the case of lectin analysis provides additional information on structural features.Quantitative monosaccharide composition analysis i) identifies glycosylated proteins, ii) gives the molar ratio of individual sugars to protein, iii) suggests, in some cases, the presence of oligosaccharide classes, iv) is the first step in designing a structural elucidation strategy, and v) provides a measure of production consistency for recombinant glycoprotein therapeutics. In recent years high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) has been extensively used to determine monosaccharide composition (reviewed in Ref. 4). More recently, fluorophore-based labeling methods have been introduced and many are available in kit form. A distinct advantage of fluorescent methods is an increase in sensitivity (ú 50-fold). One potential disadvantage is that different monosaccharides may demonstrate different selectivity for the fluorophore during the coupling reaction, either in the hydrolysate or in the external standard mixture. However, the increase in sensitivity and the ability to identify which monosaccharides are present from a small portion of the total amount of available glycoprotein, as well as the potential for greater sensitivity using laser induced fluorescence makes this approach attractive.
Monosaccharide composition analysis of small amounts of protein is best performed on PVDF (PSQ) membranes, after either electroblotting (5) or if smaller aliquots are to be analyzed on dot blots (Diagram I). PVDF is an ideal matrix for carbohydrate analysis since neither mono- or oligosaccharides bind to the membrane, once released by either acid or enzymatic hydrolysis (5).The usefulness of monosaccharide analysis in studying the glycobiology of proteins was demonstrated on the ú subunit of the gastric H,K ATPase (5,6). By monosaccharide analysis on PVDF membranes the ú subunit was found to be devoid of sialic acid (Neu5Ac) (5). These initial findings lead to further structural studies which identified unique oligosaccharide structures, all terminated in GalyGal, and has raised intriguing questions concerning their function (6).
Release of Oligosaccharides from PVDF Membranes
The next tier of analysis involves the release of oligosaccharides with either specific endoglycosidases (for reviews, see Refs. 7 and 8). Endoglycosidases are another set of tools that can provide information about glycoprotein glycans. Endoglycosidases can hydrolyse N-linked oligosaccharides from glycoproteins leaving one core N-acetylglucosamine attached to an asparagine on the peptide backbone and the other core N-acetylglucosamine as the reducing end of the released oligosaccharide. Several endoglycosidases isolated from Streptomyces plicatus and Flavobacterium meningosepticum are commercially available and have a substrate specificity allowing for the selective release of high-mannose, hybrid and various forms of multi-antennary complex oligosaccharides. A second family of glycohydrolases that remove intact N-linked oligosaccharides are peptide-N-glycosidases. These actually hydrolyze the amide nitrogen of asparagine yielding an aspartic acid residue on the protein and a 1-amino-N-acetylglucosamine on the released oligosaccharide's reducing-end. Under normal conditions, the amino group is hydrolyzed by water yielding an oligosaccharide with the usual N-acetylglucosamine reducing end and free ammonia.
Using changes in mobility by SDS-PAGE as a measure of the action of endoglycosidases can be misleading. For example, treatment of the ú subunit of the gastric H,K ATPase with endo-ú-N-acetylglucosaminidase (Endo H) had no apparent affect on the band morphology or migration position by SDS-PAGE. However, after analyzing the released oligosaccharides, oligomannosidic-type structures were found (6). Thus, to use endoglycosidases effectively in obtaining information on glycan structure, the oligosaccharides should be analyzed, either by HPLC or MS.
Release of oligosaccharides by PNGase F and endoglycosidases is protein dependent and may be glycosylation site specific. Although demonstrating complete release can be shown by SDS-PAGE (i.e. change in mobility to equal the Mr of the peptide), the optimized conditions may not be applicable to the blotted protein. Determining the release yield on pmol amounts of protein is now possible using the fluorophore-based monosaccharide methods. In one example shown at the workshop, the amount of oligosaccharides released from electroblotted fetuin with PNGase F was determined. Digests, with and without detergents and denaturants, were dried and after re-dissolving in DMSO were spotted onto carbohydrate adsorption disks (9). After washing with acetonitrile, the oligosaccharides were eluted with water and dried. The dried oligosaccharides were hydrolyzed (2N TFA, 100oC for 4h). The PNGase F-treated Coomassie-stained blot was similarly hydrolyzed after mincing. Dried hydrolyzates were then coupled to 2-aminoanthranilic acid (10). When the PNGase F reaction was carried out in phosphate buffer (50 mM, pH 8.0), ú 5% of the oligosaccharides were released. In the presence of 0.1% reduced Triton X100, 31% of the oligosaccharides were released, presumably the increase is due to the detergent preventing adsorption of the enzyme to the PVDF membrane. In the presence of ú-mercaptoethanol, SDS and NP40, 87% of the oligosaccharides were released. Thus, using fluorophore-based monosaccharide analysis, the optimal conditions for oligosaccharide release for different glycoproteins can be determined.
Characterization of Sample-limited Oligosaccharides
Judicious use of lectin blotting before and after endo-and peptide-N-glycosidase digestions can provide a wealth of information about a novel glycoprotein quickly and inexpensively with the equipment generally available in the modern protein or biochemistry core facility. Additional information can be generated by digesting glycoproteins with exoglycosidases, enzymes that remove specific terminal sugars from the non-reducing end of oligosaccharide chains. In addition, probing blots of glycoproteins with a panel of lectins before and after exoglycosidase digestion can reveal what sugar moieties have been uncovered, providing clues as to the underlying structures.
For further studies of the released oligosaccharides labeling with a fluorophore provides a sensitive, specific approach which is essential for most HPLC and CZE separations. Removing protein, salts, denaturants and detergents, as described above, is a necessary step prior to using coupling reactions. Examples of analyzing the "total pool" of released oligosaccharides (ú pmol), before and after exoglycosidase treatments, using matrix-assisted laser-desorption mass spectrometry were shown at the workshop (Diagram I, dotted line). Treatment of the 2-aminobenzamide-labeled oligosaccharides from the gastric H,K ATPase ú subunit with y-fucosidase resulted in a mass shift of all signals by m/z 146, strongly suggesting that all the oligosaccharides contained a Fuc residue linked to the chitobiose core. For exoglycosidase and MS analysis on specific structures, fractionation of the oligosaccharides, using mass-spectrometric compatible separations, (such as multi-mode hydrophilic liquid interaction HPLC (11) and reversed-phase HPLC (12) is required (Diagram I). The current separation methods usuallygive singular structures after two orthogonal chromatographies. These purified oligosaccharides can then be analyzed using MALDI/MS after exoglycosidase treatments and/or MALDI/MS with post source decay (13) analysis. These last analyses will provide molecular weights, sequence with residue identification (e.g. Gal Man etc.), some linkage information and anomericity.
In summary, the ABRF '96 workshop on carbohydrate analysis in gels and blots presented new general strategies for obtaining structural information starting with < 1 nmol of glycoprotein. Examples were given to show how structural information could be obtained at each level of analysis and used to address the biological significance of glycosylation. These methods should prove particularly useful in the analysis of membrane glycoproteins (e.g. receptors, cognate ligands of adhesion molecules, ion transporters and channels) which require solubilization with detergents and salts for purification.
References
1. Varki, A. (1993) Glycobiology, 3, 97-130.
2. Cummings, R. D. (1994) Methods in Enzymol. 230, 66-86.
3. Haselbeck, A. and Hîsel, W. (1993) Methods in Mol. Biol. 14, 161-173.
4. Townsend, R. R. (1995) in Carbohydrate Analysis: High-performance liquid chromatography and capillary electrophoresis (Z. El Rassi ed.). pp. 181-209.
5. Weitzhandler, M., Kadlecek, D., Avdalovic, N., Forte, J. G., Chow, D. and Townsend, R. R. (1993) J. Biol. Chem. 268, 5121-5130.
6. Tyagarajan, K., Townsend, R. R. and Forte, J. G. (1996) Biochemistry 35, 3238-3246
7. Tarentino, A. L. and Plummer Jr., T. H. (1994) Methods in Enzymol. 230, 44-57.
8. Tarentino, A. L., Trimble, R. B. and Plummer Jr., T. H. (1989) Methods in Cell Biol. 32, 111-139.
9. Hardy, M. R. "Glycan labeling with the fluorophores 2-aminobenzamide and anthranilic acid" in Techniques in Glycobiology. (R Townsend and A. Hotchkiss eds.) Marcel Dekker, New York, in press
10. Anumula, K. R. "Highly sensitive pre-column derivatization procedures for quantitative determination of monosaccharides, sialic acids and amino sugar alcohols of glycoproteins by reversed phase HPLC" in Techniques in Glycobiology. (R Townsend and A. Hotchkiss eds.) Marcel Dekker, New York, in press
11. Townsend, R. R., Lipniunas, P. H., Bigge, C., Ventom, A., and Parekh, R. "Multi-mode high-performance liquid chromatography of fluorescently- labeled oligosaccharides from glycoproteins." Anal. BIochem. in press
12. SchÅlter, M. "Aminobenzamide labeling of disialylated oligosaccharides--A sensitive method for monitoring lot-to-lot consistency of recombinant glycoproteins". in Techniques in Glycobiology. (R. Townsend and A. Hotchkiss eds.) Marcel Dekker, New York, in press
13. Rapp, U., Resemann, A., Mayer-Posner, F. J., SchÑfer, W., and Feichtinger, K. "The fragmentation behavior of glycopeptides using the PSD technique". in Techniques in Glycobiology. (R. Townsend and A. Hotchkiss eds.) Marcel Dekker, New York, in press
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