Visit our interactive pathways at /pathways User Protocol 362280 Rev. 23 February 2006 RFH Page 1 of 5 Glycoprotein Deglycosylation Kit Cat. No. 362280 Note that this user protocol is not lot-specific and is representative of the current specifications for this product. Please consult the vial label and the certificate of analysis for information on specific lots. Also note that shipping conditions may differ from storage conditions. Full details are available at. INTRODUCTION Asparagine-linked (N-linked) and serine/threonine-linked (O-linked) oligosaccharides are major structural components of many eukaryotic proteins. They are involved in protein sorting, immune recognition, receptor binding, inflammation, pathogenicity, and many other biological processes. The diversity of oligosaccharide structures often results in heterogeneity in the mass and net charge of glycoproteins. N-linked oligosaccharides may contribute 3.5 kda or more per structure to the mass of a glycoprotein. Structural variations and the degrees of saturation of available glycosylation sites contribute to mass heterogeneity of glycoproteins. The presence of N- acetylneuraminic acid (sialic acid) affects both the mass and the charge of a glycoprotein. Phosphorylation or sulfation of carbohydrates may also affect the charge. O-linked sugars, although usually smaller than N-linked structures, may be present in larger numbers and exhibit greater heterogeneity in structure. To study the structure and function of a glycoprotein, it is often desirable to remove all or a select class of oligosaccharides. This approach allows the assignment of specific biological functions to particular components of the glycoprotein. For example, the loss of ligand binding to a glycoprotein after removal of sialic acid may implicate this sugar in the binding process. Researchers may prefer to remove sugars from glycoproteins for several reasons, such as to: Enhance or reduce blood clearance rates of glycoprotein therapeutic agents Investigate ligand binding Investigate the role of carbohydrates in enzyme activity and solubility Perform quality control analysis of glycoprotein-based pharmaceuticals Remove carbohydrate epitopes from antigens Remove heterogeneity in glycoproteins for X-ray crystallographic analysis Simplify amino acid sequence determination of glycoproteins Study the peptide portion of the glycoprotein by SDS-PAGE ANALYTICAL STRATEGIES Deglycosylation Methods Both chemical and enzymatic methods are available for removing oligosaccharides from glycoproteins. Hydrazinolysis of glycoproteins, although capable of removing both N- and O-linked sugars, results in complete destruction of the protein component and is, therefore, not suitable if recovery of the protein is desired. Milder chemical methods such as trifluoromethanesulfonic acid (TFMS) treatment, even when optimized, result in incomplete sugar removal and partial protein destruction. Here the amino acid-linked sugar residue of both N- and O-linked oligosaccharides is retained. Only the enzymatic method provides complete sugar removal without any protein degradation.
User Protocol 362280 Rev. 23 February 2006 RFH Page 2 of 5 Enzymatic Removal of N-Linked Oligosaccharides Use of N-glycosidase F is the most effective method of removing virtually all N-linked oligosaccharides from glycoproteins. The oligosaccharide is left intact and therefore is suitable for further analysis (the asparagine residue from which the sugar was removed is deaminated to aspartic acid, the only modification to the protein). A tripeptide with the oligosaccharide-linked asparagine as the central residue is the minimal substrate for N- glycosidase F. However, oligosaccharides containing a fucose α1,3-linked to the asparagine-linked N- acetylglucosamine, commonly found in plant glycoproteins, are resistant to N-glycosidase F. N-Glycosidase A, isolated from almond meal, must be used in this situation. This enzyme, however, is ineffective when sialic acid is present on the N-linked oligosaccharide. Steric hindrance slows or inhibits the action of N-glycosidase F on certain residues of glycoproteins. Denaturation of the glycoprotein by heating with SDS and β-mercaptoethanol greatly increases the rate of deglycosylation. For some glyco-proteins, cleavage by N-glycosidase F does not occur unless the protein is denatured. For others, some or all of the oligosaccharides can be removed from the native protein after extensive incubations of three days or longer duration. N-Glycosidase F will remain active under reaction conditions for at least three days allowing extended incubations of native glycoproteins. In general, it appears that particular residues, due to their location in the native protein structure, are resistant to N-glycosidase F and cannot be removed unless the protein is denatured. NOTE: A non-ionic detergent such as TRITON X-100 must be added in excess to the SDS-denatured glycoprotein prior to the addition of N-glycosidase F to complex any free SDS. Reduction of the rate of the N-glycosidase F cleavage will result if this procedure is not followed. Other commonly used endoglycosidases, such as endoglycosidase H and the endoglyco-sidase F series, are less suitable for total deglycosylation of N-linked sugars because of their limited specificity and because they leave one N-acetylglucosamine residue attached to the asparagine. O-Linked Oligosaccharides There is no enzyme comparable to N-glycosidase F for removing intact O-linked sugars. Monosaccharides must be removed by a series of exoglycosidases until only the Galβ1,3GalNAc core remains attached to serine or threonine. Endo-α-N-acetylgalactosaminidase from Streptococcus pneumoniae can then remove the core structure intact without any modification of the serine or threonine residues. Denaturation of the glycoprotein does not appear to significantly enhance de-o-glycosylation. Any modification of the core structure will block the action of endo-α-n- acetylgalactosaminidase. By far the most common modification of the core Galβ1,3GalNAc is mono-, di-, or trisialylation. These residues are easily removed by a suitable neuraminidase. The trisialyl structure is best removed by the α2-3,6,8,9-neuraminidase from Arthrobacter ureafaciens, since this enzyme is efficient at cleaving the Neu5Acα2,8-Neu5Ac bond. A less common, but widely distributed O-linked hexasaccharide structure contains β1,4-linked galactose and β1,6- linked N-acetylglucosamine as well as sialic acid. Complete removal of this O-linked structure or its derivatives would require, in addition to neuraminidase, a β1,4-specific galactosidase and an N-acetylglucosaminidase. The galactosidase must be β1,4-specific since a non-specific galactosidase would remove the β1,3-galactose from the core Galβ1,3-GalNAc leaving O-linked GalNAc, which cannot be removed by endo-α-n-acetylgalactosaminidase. Calbiochem brand Deglycosylation Kit provides the appropriate additional enzymes for degrading these and any other O-linked structures containing β1,4-linked galactose and β-linked N-acetylglucosamine, such as polylactosamine. Other rare modifications that have been found on O-linked oligosaccharides include α-linked galactose and α- linked fucose. Directly O-linked N-acetylglucosamine (found on nuclear proteins) and α-linked N- acetylgalactosamine (found in mucins) have also been reported. Addition of the appropriate enzymes (not included in the kit) would be necessary for complete de-o-glycosylation, if these residues are present. Fucose and mannose, directly O-linked to proteins, cannot presently be removed enzymatically.
User Protocol 362280 Rev. 23 February 2006 RFH Page 3 of 5 Monitoring Deglycosylation The simplest method of assessing the extent of deglycosylation is by mobility shifts on SDS-polyacrylamide gels (SDS-PAGE). The amount of each enzyme added to a reaction in the kit is less than 200 ng. Bands corresponding to the enzymes in these gels should be barely visible compared to the glycoprotein. Sequential addition of each of the three enzymes N-glycosidase F, α2-3,6,8,9-neuraminidase, and endo-α-nacetyl-galactosaminidase results in a noticeable increase in mobility. The greatest shift occurs as a result of removal of N-linked sugars by N-glycosidase F. Removal of sialic acid from O-linked sugars by α2-3,6,8,9- neuraminidase results in an obvious shift. Finally, removal of the O-linked core Galβ1,3GalNAc by endo-α-nacetylgalactosaminidase is responsible for a small shift. The ability to detect obvious mobility shifts after removal of the disaccharide core structure will depend on the size of the protein and the relative mass contribution of the disaccharides removed. Thus, it may be difficult to establish the presence of O-linked sugars solely on the basis of mobility shifts following endo-α-n-acetylgalactosaminidase treatment of very large proteins with only a small numbers of O-linked sugars. Other methods may also be used in conjunction with SDS-PAGE gels to monitor deglycosylation. These methods, used in gel or blot formats, directly detect the carbohydrate portion of the glycoprotein. In each method, the carbohydrate is oxidized with periodate. The oxidized carbohydrate is then either directly stained (Alcian Blue or silver stain) or is reacted with biotin hydrazide that biotinylates the sugar. An enzyme-linked streptavidin conjugate and the appropriate indicator substrate are used to detect the carbohydrate in a blot. Completely deglycosylated protein produces no signal with these methods. Kit Components NOTE: The quantity of enzymes recommended in the protocols is sufficient to deglycosylate approximately 200 µg of an average glycoprotein in the given time. N-Glycosidase F cleavage is generally the rate-limiting reaction due to the slow removal of some sterically hindered N-linked residues, even when the glycoprotein is denatured. Since all enzymes retain activity under the recommended reaction condition for several days, a much larger quantity of glycoprotein may be deglycosylated if the incubation period is extended. Conversely, there is no need to use the recommended amounts of enzymes if less than 200 µg of glycoproteins are being cleaved. The enzymes can be diluted into 1X reaction buffer. They will remain stable in diluted form at 4 C. 1. N-Glycosidase F (5000 U/ml): 20 µl. This enzyme cleaves all asparagine-linked complex, hybrid, or high mannose oligosaccharides unless α1,3-core fucosylated. Asparagine must be peptide-bonded at both termini. M.W. 36,000. 2. Endo-α-N-acetylgalactosaminidase, Streptococcus pneumoniae, Recombinant, E. coli (1.25 U/ml): 20 µl. This enzyme cleaves serine-or threonine-linked unsubstituted Galβ1,3GalNAcα. M.W. approx. 180,000. 3. α2-3,6,8,9-neuraminidase, Arthrobacter ureafaciens, Recombinant, E. coli (5.0 U/ml): 20 µl. This enzyme cleaves all non-reducing terminal branched and unbranched sialic acids. M.W. approx. 60 kda and 69 kda. 4. β1,4-galactosidase, Streptococcus pneumoniae, Recombinant, E. coli (3.0 U/ml): 20 µl. This enzyme releases only β1,4-linked, non-reducing terminal galactose from complex carbohydrates and glycoproteins. M.W. approx. 350,000. 5. β-n-acetylglucosaminidase, Streptococcus pneumoniae, Recombinant, E. coli (45 U/ml): 20 µl. This enzyme cleaves all non-reducing terminal β-linked N-acetylglucosamine residues from complex carbohydrates and glycoproteins. M.W. approx. 140,000. 6. Bovine Fetuin, Lyophilized: 500 µg. This control contains sialylated N- and O-linked oligosaccharides. Reconstitute the 500 µg of fetuin with 50 µl deionized water to yield a 10 mg/ml solution. Commercial preparations of fetuin contain proteases that will eventually degrade the protein. The product provided has been treated at 90 C for 10 min to inactivate the proteases. Upon reconstitution the fetuin solution may be stored at 4 C. 7. 5X Reaction Buffer (250 mm sodium phosphate buffer, ph 7.0): 200 µl. 8. Denaturation Solution (2% SDS, 1 M β-mercaptoethanol): 100 µl. 9. TRITON X-100 Detergent (15% Solution): 100 µl.
User Protocol 362280 Rev. 23 February 2006 RFH Page 4 of 5 Protocols Denaturing Standard Protocol Note: Enzyme solutions are preservative-free and should be transferred with sterile pipet tips to avoid contamination. 1. Dissolve 200 µg or less of a glycoprotein in 30 µl of deionized water in a microcentrifuge tube. 2. Add 10 µl 5X Reaction Buffer and 2.5 µl Denaturation Solution. Mix gently. 3. Heat at 100 C for 5 min. NOTE: Some proteins may precipitate when heated with SDS. In such cases, omit the heat treatment and increase the incubation time to 24 h after adding enzymes. 4. Cool to room temperature. Add 2.5 µl TRITON X-100 detergent solution. Mix gently. NOTE: Failure to add TRITON X-100 will result in the reduction of activity of some enzymes. 5. Add 1 µl each of N-Glycosidase F, α2-3,6,8,9-neuraminidase, and Endo-α-N-acetylgalactosaminidase. If deglycosylating a glycoprotein with a complex Core 2 O-linked structure, add 1 µl of β1,4-galactosidase and 1 µl β-n-acetylglucosaminidase. 6. Incubate for 3 h at 37 C. 7. Analyze by method of choice. NOTE: Alternatively, the enzymes may be added individually or sequentially in order to determine what types of oligosaccharides are present on the glycoprotein. Non-Denaturing Standard Protocol Note: Enzyme solutions are preservative-free and should be transferred with sterile pipet tips to avoid contamination. 1. Dissolve 200 µg or less of a glycoprotein in 35 µl of deionized water in a microcentrifuge tube. 2. Add 10 µl 5X Reaction Buffer. 3. Add 1 µl each of N-Glycosidase F, α2-3,6,8,9-neuraminidase, and Endo-α-N-acetylgalactosaminidase. If deglycosylating a glycoprotein with a complex Core 2 O-linked structure, add 1 µl of β1,4-galactosidase and 1 µl β-n-acetylglucosaminidase. 4. Incubate for 1 to 5 days at 37 C. NOTE: An aliquot should be deglycosylated using a denaturing protocol to provide a gel standard for the fully deglycosylated protein. The position of the native protein can then be compared with this standard to judge the extent of deglycosylation. References Chiba, A., et al. 1997. J. Biol. Chem. 272, 2156. Jiang, M.S., and Hart, G.W. 1997. J. Biol. Chem. 272, 2421. Wilkins, P.P., et al. 1996. J. Biol. Chem. 271, 18732. Altmann, F., et al. 1995. Glycoconj. J. 12, 84. Szkudlinski, M.W., et al. 1995. Endocrinology 136, 3325. Pahlsson, P., et al. 1994. Glycoconj. J. 11, 43. Saito, S., et al. 1994. J. Biol. Chem. 269, 5644. Tarentino, A.L., and Plummer, T.H. 1994. Methods Enzymol. 230, 44. Iwase, H., Hotta, K. 1993. Methods. Mol. Biol. 14, 151. Stults, N.L., and Cummings, R.D. 1993. Glycobiology 3, 589. Hard, K., et al. 1992. Eur. J. Biochem. 205, 785. Kuraya, N., and Hase, S. 1992. J. Biochem. (Tokyo) 112, 122. Trimble, R.B., and Tarentino, A.L. 1991. J. Biol. Chem. 266, 1646. Fukuda, M., et al. 1987. J. Biol. Chem. 262, 11952. Sojar, H.T., and Bahl, O.P. 1987. Arch. Biochem. Biophys. 259, 52.
Taga, E.M., et al. 1984. Biochemistry 23, 815. Kobata, A. 1979. Anal. Biochem. 100, 1. Uchida, Y., et al. 1979. J. Biochem (Tokyo) 86, 1573. Glasgow, L.R., et al. 1977. J. Biol. Chem. 252, 8615. Spiro, R.G., and Bhoyroo, V.D. 1974. J. Biol. Chem. 249, 5704. Trademarks Calbiochem is a registered trademark of EMD Biosciences, Inc. Triton is a registered trademark of Dow Chemical Company User Protocol 362280 Rev. 23 February 2006 RFH Page 5 of 5 Prices and availability are subject to change. Copyright 2005 EMD Biosciences, Inc., an affiliate of Merck KGaA, Darmstadt,. All rights reserved. Each product is sold with a limited warranty which is provided with each purchase. Each product is intended to be used for research purposes only. It is not to be used for drug or diagnostic purposes nor is it intended for human use. EMD Biosciences products may not be resold, modified for resale, or used to manufacture commercial products without written approval of EMD Biosciences.