Use of Glycosidases to Study Protein Trafficking

Hudson H. Freeze1

1 The Burnham Institute, La Jolla, California
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 15.2
DOI:  10.1002/0471143030.cb1502s03
Online Posting Date:  May, 2001
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Abstract

As proteins transit through the cell secretory pathway, modification of their substituent sugar chains occurs in a stepwise fashion. In the course of this processing (maturation) of oligosaccharide chains, the chains acquire sensitivity or resistance to highly specific glycosidases. Thus it is possible to identify processing mileposts by analyzing the general structure of the carbohydrate chains. This unit describes reaction conditions for the family of glycosidases and analysis of the results of digestion reactions.

     
 
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Table of Contents

  • 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 Oligosaccharide Chains 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
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Endoglycosidase H Digestion

  Materials
  • Immunoprecipitated protein of interest (unit 7.2)
  • 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; Sigma, Glyko, or Boehringer Mannheim)
  • 10× SDS‐PAGE sample buffer ( appendix 2A)
  • Water baths, 30° to 37°C and 90°C
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1) and autoradiography (unit 6.3)

Basic Protocol 2: Endoglycosidase D Digestion

  Materials
  • Immunoprecipitated protein of interest (unit 7.2)
  • 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 ( appendix 7022) or Nonidet P‐40 (NP‐40)
  • 0.5 U/ml endoglycosidase D (endo D; Boehringer Mannheim)
  • 10× SDS‐PAGE sample buffer ( appendix 2A)
  • Water baths, 37° and 90°C
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1) and autoradiography (unit 6.3)

Basic Protocol 3: Endoglycosidase F2 Digestion

  Materials
  • Immunoprecipitated protein of interest (unit 7.2)
  • 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
  • 10% (w/v) Triton X‐100 ( appendix 2A) or Nonidet P‐40 (NP‐40)
  • 0.1 M 1,10‐phenanthroline in methanol
  • 200 mU/ml endoglycosidase F 2 (endo F 2; Glyko)
  • 4× SDS‐PAGE sample buffer ( appendix 2A)
  • Water baths, 30° to 37°C and 90°C
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1) and autoradiography (unit 6.3)

Basic Protocol 4: Endoglycosidase F3 Digestion

  Materials
  • Immunoprecipitated protein of interest (unit 7.2)
  • 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
  • 10% (w/v) Triton X‐100 ( appendix 2A) or NP‐40
  • 0.1 U/ml endoglycosidase F 3 (endo F 3; Glyko)
  • 10× SDS‐PAGE sample buffer ( appendix 2A)
  • Water baths, 37° and 90°C
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1) and autoradiography (unit 6.3)

Basic Protocol 5: Peptide: N‐Glycosidase F Digestion

  Materials
  • Immunoprecipitated protein of interest (unit 7.2)
  • 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 determined at 37°C ( appendix 2A)
  • 10% (w/v) Triton X‐100 ( appendix 2A) or Nonidet P‐40 (NP‐40)
  • 200 to 250 mU/ml peptide:N‐glycosidase F (PNGase F; Sigma or Glyko)
  • 10× SDS sample buffer ( appendix 2A)
  • Water baths, 30° to 37°C and 90°C
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1) and autoradiography (unit 6.3)

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

  Materials
  • Immunoprecipitated protein of interest (unit 7.2)
  • 0.1 M 2‐mercaptoethanol (2‐ME)/0.1% (w/v) SDS (ultrapure electrophoresis grade; prepare fresh)
  • 10% (w/v) Triton X‐100 ( appendix 2A) or Nonidet P‐40 (NP‐40)
  • 0.5 M sodium acetate, pH 5.0 ( appendix 2A)
  • 1 IU/ml neuraminidase from Arthrobacter ureafaciens (Sigma or Glyko)
  • 10× SDS sample buffer ( appendix 2A)
  • Water baths, 37° and 90°C
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1), IEF/SDS‐PAGE, or NEPHGE/SDS‐PAGE ( appendix 3A), and for autoradiography (unit 6.3)

Basic Protocol 6: Sialidase (Neuraminidase) Digestion

  Materials
  • Immunoprecipitated protein of interest (unit 7.2)
  • 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
  • 10% (w/v) Triton X‐100 ( appendix 2A) or Nonidet P‐40 (NP‐40)
  • 100 mU/ml endo‐β‐galactosidase (Boehringer Mannheim, Oxford GlycoSystems, Sigma)
  • 10× SDS‐PAGE sample buffer ( appendix 2A)
  • Water baths, 37° and 95°C
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1) and autoradiography (unit 6.3)

Basic Protocol 7: Endo‐β‐Galactosidase Digestion

  Materials
  • Immunoprecipitated protein of interest (unit 7.2)
  • 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 ( appendix 2A) or Nonidet P‐40 (NP‐40)
  • 300 mU/ml endo‐α‐N‐acetylgalactosaminidase (5× concentrate from Glyko; use according to directions)
  • 10× SDS‐PAGE sample buffer ( appendix 2A)
  • Water bath, 37° and 95°C
  • Additional reagents and equipment for SDS‐PAGE (unit 6.3) and autoradiography (unit 6.3)

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

  Materials
  • Immunoprecipitated protein of interest (unit 7.2)
  • 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 ( appendix 2A) or Nonidet P‐40 (NP‐40)
  • 2.4 mg/ml O‐sialoglycoprotease (O‐sialoglycoprotein endoglycoprotease; Accurate Chemical & Scientific; reconstituted according to directions)
  • 10× SDS‐PAGE sample buffer ( appendix 2A)
  • Water baths, 37° and 95°C
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1) and autoradiography (unit 6.3)
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Figures

Videos

Literature Cited

Literature Cited
   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 qoligosaccharides. 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 α 1→3 to the asparagine‐linked N‐acetylglucosamine residue. Eur.J. Biochem. 199:647‐652.
   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(11):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 III.
   Kornfeld and Kornfeld, 1985. See above.
  Landmark review of processing.
   Tarentino and Plummer, 1994. See above.
  Best and most recent review of the use of these enzymes.
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