Endoglycosidase and Glycoamidase Release of N‐Linked Glycans

Hudson H. Freeze1, Christian Kranz1

1 Sanford‐Burnham Medical Research Institute, La Jolla, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 8.15
DOI:  10.1002/0471142735.im0815s89
Online Posting Date:  April, 2010
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Abstract

Nearly all proteins entering the lumen of the endoplasmic reticulum (ER) become glycosylated en route to a cellular organelle, the plasma membrane, or the extracellular space. Many glycans can be attached to proteins, but the most common are the N‐linked glycans (oligosaccharides). These chains are added very soon after a protein enters the ER, but they undergo extensive remodeling (processing), especially in the Golgi. Processing changes the sensitivity of the N‐glycan to enzymes that cleave entire sugar chains or individual monosaccharides, which also changes the migration of the protein on SDS gels. These changes can be used to indicate when a protein has passed a particular subcellular location. This unit details some of the methods used to track a protein as it trafficks from the ER to the Golgi toward its final location. Curr. Protoc. Immunol. 89:8.15.1‐8.15.25. © 2010 by John Wiley & Sons, Inc.

Keywords: ER/Golgi; oligosaccharide; glycan; N‐glycosylation; glycosidase; intracellular trafficking

     
 
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Table of Contents

  • Introduction
  • The N‐Linked Pathway
  • The O‐Linked Pathway
  • Basic Protocol 1: Endoglycosidase H Digestion
  • Basic Protocol 2: Endoglycosidase D Digestion
  • Basic Protocol 3: Endoglycosidase F2 Digestion
  • Basic Protocol 4: Endoglycosidase F3 Digestion
  • Basic Protocol 5: Peptide: N‐Glycosidase F Digestion
  • Support Protocol 1: Estimating the Number of N‐Linked Glycans on a Glycoprotein
  • Basic Protocol 6: Sialidase (Neuraminidase) Digestion
  • Basic Protocol 7: Endo‐β‐Galactosidase Digestion
  • Basic Protocol 8: Endo‐α‐N‐Acetylgalactosaminidase Digestion
  • Basic Protocol 9: O‐Sialoglycoprotease Digestion
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Endoglycosidase H Digestion

  Materials
  • Immunoprecipitated protein of interest (unit 8.3)
  • 0.1 M 2‐mercaptoethanol (2‐ME)/0.1% (w/v) SDS (ultrapure electrophoresis grade; prepare fresh)
  • 0.5 M sodium citrate, pH 5.5
  • 1% (w/v) phenylmethylsulfonyl fluoride (PMSF) in isopropanol
  • 0.5 U/ml endoglycosidase H (endo H; natural or recombinant)
  • 10× SDS sample buffer (unit 8.4)
  • Water baths, 30° to 37°C and 90°C
  • Additional reagents and equipment for SDS‐PAGE (unit 8.4) and autoradiography ( appendix 3J)

Basic Protocol 2: Endoglycosidase D Digestion

  Materials
  • Immunoprecipitated protein of interest (unit 8.3)
  • 0.1 M 2‐mercaptoethanol (2‐ME)/0.1% (w/v) SDS (ultrapure electrophoresis grade; prepare fresh)
  • 0.5 M NaH 2PO 4, pH 6.5
  • 10% (w/v) Triton X‐100 or Nonidet P‐40 (NP‐40)
  • 0.5 U/ml endoglycosidase D (endo D)
  • 10× SDS sample buffer (unit 8.4)
  • Water baths, 37° and 90°C
  • Additional reagents and equipment for SDS‐PAGE (unit 8.4) and autoradiography ( appendix 3J)

Basic Protocol 3: Endoglycosidase F2 Digestion

  Materials
  • Immunoprecipitated protein of interest (unit 8.3)
  • 0.1 M 2‐mercaptoethanol (2‐ME)/0.1% (w/v) SDS (ultrapure electrophoresis grade; prepare fresh)
  • 0.5 M sodium acetate, pH 4.5 ( appendix 2A)
  • 10% (w/v) Triton X‐100 or Nonidet P‐40 (NP‐40)
  • 0.1 M 1,10‐phenanthroline in methanol
  • 200 mU/ml endoglycosidase F 2 (endo F 2)
  • 4 × SDS sample buffer (unit 8.4)
  • Water baths, 30° to 37°C and 90°C
  • Additional reagents and equipment for SDS‐PAGE (unit 8.4) and autoradiography ( appendix 3J)

Basic Protocol 4: Endoglycosidase F3 Digestion

  Materials
  • Immunoprecipitated protein of interest (unit 8.3)
  • 0.1 M 2‐mercaptoethanol (2‐ME)/0.1% (w/v) SDS (ultrapure electrophoresis grade; prepare fresh)
  • 0.5 M sodium acetate, pH 4.5 ( appendix 2A)
  • 10% (w/v) Triton X‐100 or NP‐40
  • 0.1 U/ml endoglycosidase F 3 (endo F 3)
  • 10× SDS sample buffer (unit 8.4)
  • Water baths, 37° and 90°C
  • Additional reagents and equipment for SDS‐PAGE (unit 8.4) and autoradiography ( appendix 3J)

Basic Protocol 5: Peptide: N‐Glycosidase F Digestion

  Materials
  • Immunoprecipitated protein of interest (unit 8.3)
  • 0.1 M 2‐mercaptoethanol (2‐ME)/0.1% (w/v) SDS (ultrapure electrophoresis grade; prepare fresh)
  • 0.5 M Tris⋅Cl, pH 8.6 as determined at 37°C ( appendix 2A)
  • 10% (w/v) Triton X‐100 or Nonidet P‐40 (NP‐40)
  • 200 to 250 mU/ml peptide:N‐glycosidase F (PNGase F)
  • 10× SDS sample buffer (unit 8.4)
  • Water baths, 30° to 37°C and 90°C
  • Additional reagents and equipment for SDS‐PAGE (unit 8.4) and autoradiography ( appendix 3J)

Support Protocol 1: Estimating the Number of N‐Linked Glycans on a Glycoprotein

  Materials
  • Immunoprecipitated protein of interest (unit 8.3)
  • 0.1 M 2‐mercaptoethanol (2‐ME)/0.1% (w/v) SDS (ultrapure electrophoresis grade; prepare fresh)
  • 10% (w/v) Triton X‐100 or Nonidet P‐40 (NP‐40)
  • 0.5 M sodium acetate, pH 5.0 ( appendix 2A)
  • 1 IU/ml neuraminidase from Arthrobacter ureafaciens
  • 10× SDS sample buffer (unit 8.4)
  • Water baths, 37° and 90°C
  • Additional reagents and equipment for SDS‐PAGE (unit 8.4), for IEF/SDS‐PAGE or NEPHGE/SDS‐PAGE (unit 8.5), and for autoradiography ( appendix 3J)

Basic Protocol 6: Sialidase (Neuraminidase) Digestion

  Materials
  • Immunoprecipitated protein of interest (unit 8.3)
  • 0.1 M 2‐mercaptoethanol (2‐ME)/0.1% (w/v) SDS (ultrapure electrophoresis grade; prepare fresh)
  • 0.5 M sodium acetate buffer, pH 5.8 ( appendix 2A)
  • 10% (w/v) Triton X‐100 or Nonidet P‐40 (NP‐40)
  • 100 mU/ml endo‐β‐galactosidase
  • 10× SDS sample buffer (unit 8.4)
  • Water baths, 37° and 95°C
  • Additional reagents and equipment for SDS‐PAGE (unit 8.4) and autoradiography ( appendix 3J)

Basic Protocol 7: Endo‐β‐Galactosidase Digestion

  Materials
  • Immunoprecipitated protein of interest (unit 8.3)
  • 0.1 M 2‐mercaptoethanol (2‐ME)/0.1% (w/v) SDS (ultrapure electrophoresis grade; prepare fresh)
  • 0.5 M sodium citrate phosphate buffer, pH 6.0, containing 500 µg/ml BSA (complete buffer supplied with enzyme)
  • 10% (w/v) Triton X‐100 or Nonidet P‐40 (NP‐40)
  • 300 mU/ml endo‐α‐N‐acetylgalactosaminidase (use according to manufacturer's directions)
  • 10× SDS sample buffer (unit 8.4)
  • Water bath, 37° and 95°C
  • Additional reagents and equipment for SDS‐PAGE (unit 8.4) and autoradiography ( appendix 3J)

Basic Protocol 8: Endo‐α‐N‐Acetylgalactosaminidase Digestion

  Materials
  • Immunoprecipitated protein of interest (unit 8.3)
  • 0.1 M 2‐mercaptoethanol (2‐ME)/0.1% (w/v) SDS (ultrapure electrophoresis grade; prepare fresh)
  • 0.4 M HEPES buffer, pH 7.4
  • 10% (w/v) Triton X‐100 or Nonidet P‐40 (NP‐40)
  • 2.4 mg/ml O‐sialoglycoprotease (O‐sialoglycoprotein endoglycoprotease; reconstituted according to manufacturer's directions)
  • 10× SDS sample buffer (unit 8.4)
  • Water baths, 37° and 95°C
  • Additional reagents and equipment for SDS‐PAGE (unit 8.4) and autoradiography ( appendix 3J)
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Figures

Videos

Literature Cited

   Akama, T.O., Nakagawa, H., Wong, N.K., Sutton‐Smith, M., Dell, A., Morris, H.R., Nakayama, J., Nishimura, S., Pai, A., Moremen, K.W., Marth, J.D., and Fukuda, M.N. 2006. Essential and mutually compensatory roles of α‐mannosidase II and α‐mannosidase IIx in N‐glycan processing in vivo in mice. Proc. Natl. Acad. Sci. U.S.A. 13;103:8983‐8988.
   Alexander, S. and Elder, J.H. 1989. Endoglycosidases from Flavobacterium meningosepticum: Application to biological problems. Methods Enzymol. 179:505‐518.
   Beckers, C.J.M., Keller, D.S., and Balch, W.E. 1987. Semi‐intact cells permeable to macromolecules: Use in reconstitution of protein transport from the endoplasmic reticulum to the Golgi complex. Cell 50:523‐534.
   Chui, D., Oh‐Eda, M., Liao, Y.‐F., Panneerselvam, K., Lal, A., Marek, K.W., Freeze, H.H., Moremen, K.W., Fukuda, M.N., and Marth, J.D. 1997. Alpha‐mannosidase‐II deficiency results in dyserythropoiesis and unveils an alternate pathway in oligosaccharide biosynthesis. Cell 90:157‐167.
   Davidson, H.W. and Balch, W.E. 1993. Differential inhibition of multiple vesicular transport steps between the endoplasmic reticulum and trans Golgi network. J. Biol. Chem. 268:4216‐4226.
   Elder, J.H. and Alexander, S. 1982. Endo‐β‐N‐acetylglucosaminidase F: Endoglycosidase from Flavobacterium meningosepticum that cleaves both high mannose and complex glycoproteins. Proc. Natl. Acad. Sci. U.S.A. 79:4540‐4544.
   Kornfeld, R. and Kornfeld, S. 1985. Assembly of asparagine‐linked oligosaccharides. Annu. Rev. Biochem. 54:631‐664.
   Mellors, A. and Lo, R.Y. 1995. O‐Sialoglycoprotease from Pasteurella haemolytica. Methods Enzymol. 248:728‐740.
   Norgard, K.E., Moore, K.L., Diaz, S., Stults, N.L., Ushiyama, S., McEver, R.P., Cummings, R.D., and Varki, A. 1993. Characterization of a specific ligand for P‐selectin on myeloid cells. A minor glycoprotein with sialylated O‐linked oligosaccharides. J. Biol. Chem. 268:12764‐12774.
   Plummer, T.H. Jr. and Tarentino, A.L. 1991. Purification of the oligosaccharide‐cleaving enzymes of Flavobacterium meningosepticum. Glycobiology 1:257‐263.
   Plummer, T.H. Jr., Elder, J.H., Alexander, S., Phelan, A.W., and Tarentino, A.L. 1984. Demonstration of peptide: N‐glycosidase F activity in endo‐β‐N‐acetylglucosaminidase F preparations. J. Biol. Chem. 259:10700‐10704.
   Tarentino, A.L. and Plummer, T.H. Jr. 1994. Enzymatic deglycosylation of asparagine‐linked glycans: Purification, properties, and specificity of oligosaccharide‐cleaving enzymes from Flavobacterium meningosepticum. Methods Enzymol. 230:44‐57.
   Tarentino, A.L., Trimble, R.B., and Plummer, T.H. Jr. 1989. Enzymatic approaches for studying the structure, synthesis, and processing of glycoproteins. Methods Cell Biol. 32:111‐139.
   Tretter, V., Altmann, F., and März, L. 1991. Peptide‐N4‐(N‐acetyl‐β‐glucosaminyl) asparagine amidase F cannot release glycans with fucose attached α1→3 to the asparagine‐linked N‐acetylglucosamine residue. Eur. J. Biochem. 199:647‐652.
   Varki, A., Cummings, R.D., Esko, J.D., Freeze, H.H., Stanley, P., Bertozzi, C.R., Hart, G.W., and Etzler, M.E. 2008. Essentials of Glycobiology. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=glyco2).
   Viitala, J., Carlsson, S.R., Siebert, P.D., and Fukuda, M. 1988. Molecular cloning of cDNAs encoding lamp A, a human lysosomal membrane glycoprotein with apparent Mr approximately equal to 120,000. Proc. Natl. Acad. Sci. U.S.A. 85:3743‐3747.
Key References
   Beckers et al., 1987. See above.
  Describes the use of Lec 1 CHO cells and endo D to study processing.
   Chui et al., 1997. See above.
  Demonstrates the importance of α‐mannosidase IIx.
   Kornfeld and Kornfeld, 1985. See above.
  Landmark review of processing.
   Tarentino and Plummer, 1994. See above.
  Best review of the use of these enzymes.
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