Abstract
Many indirect serum studies show changes in protein glycosylation in disease, but further progress will require direct investigation of oligosaccharide composition. Current methods of deglycosylation using PNGase-F often result in incomplete removal of oligosaccharides. This is an unsatisfactory situation because only small quantities of material are often available in clinical studies, glycosylation changes may occur in only a small proportion of the molecules and quantification of the released oligosaccharides may be unreliable. The ability of PNGase-F to deglycosylate haptoglobin (Hp) under different conditions has been investigated. Oligosaccharides were completely removed from 50μg of Hp by treatment for 24 h with PNGase-F in 50 mmol/l ammonium formate buffer, pH 8.6, in combination with sodium dodecyl sulphate, mercaptoethanol and Nonidet P40. This modified procedure was equally effective at removing oligosaccharides from other serum glycoproteins (α1 glycoprotein, α1, transferrin) and fetuin, and its efficiency was independent of the polymeric structure of the molecule or the amount of glycosylation. The method has the additional advantage of using 20% less enzyme than previous methods, which substantially reduces costs.
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References
Turner GA. N-glycosylation of serum proteins in disease and its investigation using lectins.Clin Chim Acta 1992:208: 149–71.
Gosselin S, Payie K, Viswantha T. Substrate specificity of peptide-N-glycosidase fromFlavobacterium meningosepticum.Glycobiology 1993:3, 419–21.
Nuck R, Zimmermann M, Sauvageot D, Josic D, Reutter W: Optimized deglycosylation of glycoproteins by peptide-N4-N-acetyl-β-glucosaminyl: — asparagine amidase fromflavobacterium meningosepticum.Glycoconjugate J 1990:7; 279–86.
Haselbeck A, Hosel W. Studies on the effects of the incubation conditions, various detergents and protein concentration on the enzymic activity of N-glycosidase F, glycopeptidase F and endoglycosidase F.Topics Biochem 1988:8; 1–4.
Tretter V, Altmann F, Marz L. Peptide-N4-N-acetyl-β-glucosaminyl: asparagine amidase-F cannot release glycans with fucose attached α[1–3] to the asparagine-linkedN-acetylglucosamine residue.Eur J Biochem 1991:199; 647–52.
Tarentino AL, Gomez CM, Plummer TH. Deglycosylation of asparagine-linked glycans by peptide-N-glycosidase-F.Biochemistry 1985:24; 4665–71.
Hirani S, Bernasconi RJ, Rasmussen JR. Use of N-glycanase to release asparagine-linked oligosaccharides for structural analysis.Anal Biochem 1987:162; 485–92.
Mann AC, Thompson S, Record CO, Self CH, Turner GA. Monosaccharide composition of haptoglobin purified from alcoholic cirrhotic and control sera determined by HPAE.Biochem Soc Trans 1993:21; 214S.
Mann AC, Record CO, Self CH, Turner GA, Monosaccharide composition of haptoglobin in liver diseases and alcohol abuse: large changes in glycosylation associated with alcoholic liver disease.Clin Chim Acta 1994:227; 69–78.
Thompson S. Simplified and reproducible silver staining of proteins in polyacrylamide gels.Med Sci Res 1987:15, 1253–1255.
Blake MS, Johnson KH, Russel-Jones GJ, Gotschlich EC. A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots.Anal Biochem 1984:136; 175.
Pharmacia AB.PhastSystem Separation Technique File No. 110. Uppsala, Sweden, Pharmacia, 1987.
Pharmacia AB.PhastGel Silver Kit Instruction Manual. Uppsala, Sweden: Pharmacia, 1990.
Barker WC, Putnam FW. Appendix: amino acid sequences of plasma proteins. In: Putnam FW, ed.The Plasma Proteins — Structure, Function and Genetic Control,Vol IV. 2nd edn. New York: Academic Press, 1984; 361–99.
Gosselin S, Martin BM, Murray GJ, Viswanatha T. Flavobacterium meningosepticum peptide-N-glycosidase — influence of ionic-strength on enzymic activity.J Biochem Biophys Methods 1992:24; 71–9.
Plummer TH, Elder JH, Alexander S, Phelan AW, Tarentino AL. Demonstration of peptide N glycosidase activity in endo-beta-acetylglcosaminidase F preparations.J Biol Chem 1984:259; 10700–4.
Steube K, Gross V, Hosel W, et al. Different susceptibilities of complex-mannose-type, hybridmannose-type and high-mannose-type alpha-1-proteinase inhibitor and alpha-1-acid glycoprotein to endo-β-N-acetylglucosaminidase-F and peptide-N-glycosidase-F.Glycoconjugate J 1986:3, 247–254.
Tarentino AL, Quinones G, Trumble A, et al. Molecular cloning and amino acid sequence of peptide N4-N-acetyl-β-D-glucosaminyl: asparagine amidase fromFlavobacterium meningosepticum.J Biol Chem 1990:265; 6961–6.
Barsomian, GD, Johnson TL, Borowski M, et al. Cloning and expression of peptide-N4-N-acetyl-β-D-glucosaminyl: asparagine amidase F inEscherichia coli.J Biol Chem 1990:265; 6967–6972.
Lemp D, Haselbeck A, Klebl F. Molecular cloning and heterologous expression of N-glycosidase-F fromFlavobacterium meningosepticum.J Biol Chem 1990:265; 15606–10.
Thompson S, Dargan E, Turner GA. Increased fucosylation and other carbohydrate changes in haptoglobin in ovarian cancer.Cancer Lett 1992:66; 43–8.
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Mann, A.C., Self, C.H. & Turner, G.A. A general method for the complete deglycosylation of a wide variety of serum glycoproteins using peptide-N-glycosidase-F. Glycosylation & Disease 1, 253–261 (1994). https://doi.org/10.1007/BF00919333
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DOI: https://doi.org/10.1007/BF00919333