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Endoglycosidase and Glycoamidase Release of N‐Linked Glycans

Hudson H. Freeze1,  Christian Kranz1

1Burnham Institute for Medical Research, La Jolla, California

Publication Name: 
Current Protocols in Molecular Biology
Unit Number: 
Unit 17.13A
DOI: 
10.1002/0471142727.mb1713as89
Online Posting Date: 
January, 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. Mol. Biol. 89:17.13A.1-17.13A.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: Estimating the Number of N-Linked Glycans on a Glycoprotein
  • Basic Protocol 6: Sialidase (Neuraminidase) Digestion
  • Basic Protocol 7: Endo--Galactosidase Digestion
  • Basic Protocol 8: Endo--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 10.16)
  • 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 10.2A)
  • Water baths, 30° to 37°C and 90°C
  • Additional reagents and equipment for SDS-PAGE (UNIT 10.2A) and autoradiography (APPENDIX 3A)

Basic Protocol 2: Endoglycosidase D Digestion

 Materials
  • Immunoprecipitated protein of interest (UNIT 10.16)
  • 0.1 M 2-mercaptoethanol (2-ME)/0.1% (w/v) SDS (ultrapure electrophoresis grade; prepare fresh)
  • 0.5 M NaH2PO4, 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 10.2A)
  • Water baths, 37° and 90°C
  • Additional reagents and equipment for SDS-PAGE (UNIT 10.2A) and autoradiography (APPENDIX 3A)

Basic Protocol 3: Endoglycosidase F2 Digestion

 Materials
  • Immunoprecipitated protein of interest (UNIT 10.16)
  • 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 2)
  • 10% (w/v) Triton X-100 or Nonidet P-40 (NP-40)
  • 0.1 M 1,10-phenanthroline in methanol
  • 200 mU/ml endoglycosidase F2 (endo F2)
  • 4 × SDS sample buffer (UNIT 10.2A)
  • Water baths, 30° to 37°C and 90°C
  • Additional reagents and equipment for SDS-PAGE (UNIT 10.2A) and autoradiography (APPENDIX 3A)

Basic Protocol 4: Endoglycosidase F3 Digestion

 Materials
  • Immunoprecipitated protein of interest (UNIT 10.16)
  • 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 2)
  • 10% (w/v) Triton X-100 or NP-40
  • 0.1 U/ml endoglycosidase F3 (endo F3)
  • 10× SDS sample buffer (UNIT 10.2A)
  • Water baths, 37° and 90°C
  • Additional reagents and equipment for SDS-PAGE (UNIT 10.2A) and autoradiography (APPENDIX 3A)

Basic Protocol 5: Peptide: N-Glycosidase F Digestion

 Materials
  • Immunoprecipitated protein of interest (UNIT 10.16)
  • 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 2)
  • 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 10.2A)
  • Water baths, 30° to 37°C and 90°C
  • Additional reagents and equipment for SDS-PAGE (UNIT 10.2A) and autoradiography (APPENDIX 3A)

Basic Protocol 6: Sialidase (Neuraminidase) Digestion

 Materials
  • Immunoprecipitated protein of interest (UNIT 10.16)
  • 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 2)
  • 1 IU/ml neuraminidase from Arthrobacter ureafaciens
  • 10× SDS sample buffer (UNIT 10.2A)
  • Water baths, 37° and 90°C
  • Additional reagents and equipment for SDS-PAGE (UNIT 10.2A), for IEF/SDS-PAGE or NEPHGE/SDS-PAGE (UNIT 10.3), and for autoradiography (APPENDIX 3A)

Basic Protocol 7: Endo--Galactosidase Digestion

 Materials
  • Immunoprecipitated protein of interest (UNIT 10.16)
  • 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 2)
  • 10% (w/v) Triton X-100 or Nonidet P-40 (NP-40)
  • 100 mU/ml endo--galactosidase
  • 10× SDS sample buffer (UNIT 10.2A)
  • Water baths, 37° and 95°C
  • Additional reagents and equipment for SDS-PAGE (UNIT 10.2A) and autoradiography (APPENDIX 3A)

Basic Protocol 8: Endo--N-Acetylgalactosaminidase Digestion

 Materials
  • Immunoprecipitated protein of interest (UNIT 10.16)
  • 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--N-acetylgalactosaminidase (use according to manufacturer's directions)
  • 10× SDS sample buffer (UNIT 10.2A)
  • Water bath, 37° and 95°C
  • Additional reagents and equipment for SDS-PAGE (UNIT 10.2A) and autoradiography (APPENDIX 3A)

Basic Protocol 9: O-Sialoglycoprotease Digestion

 Materials
  • Immunoprecipitated protein of interest (UNIT 10.16)
  • 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 10.2A)
  • Water baths, 37° and 95°C
  • Additional reagents and equipment for SDS-PAGE (UNIT 10.2A) and autoradiography (APPENDIX 3A)
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Figures

  • Figure 17.13A.1
    Symbol structures for the core region and precursor of N-linked sugar chains. Each sugar is given a symbol and abbreviation at the bottom of the figure. Each one except sialic acid uses its anomeric carbon (C-1) for linking to other sugars. Sialic acid uses C-2 for glycosidic linkage to other sugars. Glycosidases and glycosyltransferases are anomeric specific and distinguish or configurations of each sugar. The core structure (A) is common to all N-linked chains and is composed of three Man and two GlcNAc residues. The or configuration of each sugar is indicated, and the OH group to which that sugar is linked is shown on the bar linking the two symbols. Thus, GlcNAc1-4GlcNAc is represented by two filled squares with and 4 between them. When a structure is first presented, it will have full display such as that on the left side; if it is repeated, only the symbols will be used, as shown immediately to the right. The precursor glycan (B) for all N-linked chains is synthesized in the ER and transferred cotranslationally to the peptide containing an available Asn-X-Thr/Ser sequon.

    View Image
  • Figure 17.13A.2
    N-linked glycan maturation pathway for high-mannose and hybrid types, and sensitivities to various enzymes. Brackets (top) show the structures designated as high-mannose and hybrid chains. The boxes indicate ER or Golgi localization. The pathway begins with the precursor glycan (see Fig. 17.13A.1). Each successive numbered step in circles represents a glycosidase or glycosyl transferase that generates a new sugar chain with different sensitivities to the various endoglycosidases or PNGase F. (1) precursor glycan is trimmed by -glucosidases I and II, removing three Glc. (2) ER mannosidase removes one Man. (3) -Mannosidase I in Golgi complex removes two Man to make Man6GlcNAc2, with a single remaining 1-2Man. (4) The final 1-2Man is removed by a Golgi complex -mannosidase I. (5) GlcNAc transferase I adds GlcNAc to Man5GlcNAc2. (6) -Mannosidase II or -mannosidase IIx (MX) removes the 1-3 and 1-6Man units to make GlcNAc1Man3GlcNAc2. Sensitivity to various enzymes (bottom) changes when moving from left to right, but remains the same within vertical columns. NOTE: This continued maturation to form complex chains is shown in Figure 17.13A.3. Additionally, these figures are not comprehensive; many glycosylation steps have not been included, but they do not affect the sensitivities to the enzymes listed.

    View Image
  • Figure 17.13A.3
    N-linked glycan maturation pathway for complex types, and sensitivities to various enzymes (see Fig. 17.13A.2 for additional details). (8) GlcNAc transferase II adds a second GlcNAc to initiate a biantennary chain. (9) GlcNAc transferase IV adds a third GlcNAc to initiate a triantennary chain. (10) GlcNAc transferase V adds a fourth GlcNAc to initiate a tetraantennary chain. (11) Fucosyltransferase adds 1-6Fuc to the core region of complex chains. (12) 1-4Gal is added to available GlcNAc residues of hybrid and complex chains. (13) 2-3 or 2-6Sia is added to Gal residues of hybrid and complex chains.

    View Image
  • Figure 17.13A.4
    A small portion of the O-GalNAc pathway. The first step of the O-linked pathway occurs in the early Golgi complex with the addition of -GalNAc. There are at least six other sugars that can be added at this point in this complex pathway. Often 1-3Gal is added (1), quickly followed by 2-3Sia (2). The presence of these structures can be detected with a combination of O-glycosidase and sialidase. Additional sugars can be added as shown. 2-6Sia (3) or 1-6GlcNAc (4) followed by 1-4Gal (5) and 2-3Sia (6) on Gal. Each of these sugars must be removed before O-glycosidase can cleave the disaccharide.

    View Image
  • Figure 17.13A.5
    PNGase F and endoglycosidase-sensitive bonds in the core of N-linked glycans. PNGase F is a glycoamidase that severs the bond between GlcNAc and Asn, liberating the entire sugar chain and converting Asn into Asp. The endoglycosidases (H, D, and Fs) cleave the bond between the two GlcNAc residues in the core region, leaving one GlcNAc still bound to the protein. The differential specificity of the endoglyosidases is based on the structure of the sugar chain in a fully denatured protein. Incomplete denaturation may not expose all sensitive linkages. X and Y are unspecified sugar residues.

    View Image
  • Figure 17.13A.6
    Data from the estimation of the number of glycosylation sites on lysosome-associated membrane protein 1 (LAMP-1; Viitala et al., 1988). LAMP-1 contains eighteen potential N-linked sites. Graded digestion with increasing amounts of PNGase F was used to generate this ladder of glycoforms. Each band contains at least one less N-linked chain than the band above it. An average N-linked carbohydrate chain has an apparent mass of ~1.5 to 3 kDa. Lysosomal membrane glycoprotein was immunoprecipitated from [35S]Met-labeled cells and the sample was digested with PNGase F for 0 min (lane 1), 5 min (lane 2), 20 min (lane 3), 45 min (lane 4), and 24 hr (lane 5). Figure courtesy of Dr. Minoru Fukuda.

    View Image
  • Figure 17.13A.7
    Endo--galactosidase-sensitive linkages in poly-N-acetyllactosamines. Linear, unsubstituted poly-N-acetyllactosamine units (GlcNAc1-3Gal1-4) are sensitive to digestion with endo--galactosidase, while substitutions—such as sulfate esters (S) or branches starting with GlcNAc (not shown)—completely block digestion. Substitution of neighboring GlcNAc with Fuc or sulfate esters slows the rate, but does not block cleavage. Sensitive sites are shown with bold arrows, slowly hydrolyzed sites with a lighter arrow, and resistant bonds are struck out. Various substitutions are possible, leading to broad bands on gels. This will create variable sensitivities, but even partial sensitivity should give a sharper, more defined band.

    View Image
  • Figure 17.13A.8
    Schematic diagram showing results that could be obtained for a hypothetical protein with two N-linked glycosylation sites as it moves through the Golgi complex. Assume that the protein has been biosynthetically labeled with an amino acid precursor (such as [35S]Met) for 10 min and chased in the absence of label for 45 min and 120 min. The protein is then precipitated with a specific antibody. At each time point, equal amounts of the sample are analyzed by fluorography after one-dimensional SDS-PAGE, either without any digestion (control; C) or following digestion with endo H (H), endo D (D), endo F2 (F2), PNGase F (PNG), or sialidase (Sia). Glycan structures consistent with the banding patterns are shown below the schematic gel pattern.

    View Image

Literature Cited

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 -mannosidase II and -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--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--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--glucosaminyl) asparagine amidase F cannot release glycans with fucose attached 13 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 -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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