Tyrosine O‐Sulfation

Denis Corbeil1, Christoph Thiele1, Wieland B. Huttner1

1 Max‐Planck‐Institute of Molecular Cell Biology and Genetics, Dresden, Germany
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 14.7
DOI:  10.1002/0471140864.ps1407s39
Online Posting Date:  March, 2005
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Abstract

The O-sulfation of tyrosine residues of plasma membrane and secretory proteins that transit through the secretory pathway of eukaryotic cells is a widespread post-translational modification. This enzymatic reaction is catalyzed by trans-Golgi-associated tyrosylprotein sulfotransferases, which recognize tyrosine residues located in a specific acidic amino acid sequence. Tyrosine sulfation promotes extracellular protein–protein interactions involved in diverse biological processes, ranging from the receptor binding of regulatory peptides to the interaction of viral envelope proteins with the cell surface. This unit outlines procedures to determine whether a protein of interest contains sulfated tyrosine residues, using methods based on labeling proteins with inorganic [35S]-sulfate, alkaline hydrolysis, and one-dimensional thin-layer electrophoresis.

Keywords: Sulfation; tyrosine residue; post-translational modification; sulfate labeling

     
 
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Table of Contents

  • Unit Introduction
  • Strategic Planning
  • Basic Protocol 1: Long-Term [35S]-Sulfate Labeling of Mammalian Cells in Culture and Immunoprecipitation
  • Support Protocol 1: Detection of [35S]-Sulfated Proteins
  • Basic Protocol 2: Tyrosine Sulfate Analysis—Alkaline Hydrolysis Method
  • Support Protocol 2: Thin-Layer Electrophoresis of Tyrosine [35S]-Sulfate
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Long-Term [35S]-Sulfate Labeling of Mammalian Cells in Culture and Immunoprecipitation

 Materials
  • Mammalian adherent cells expressing protein of interest and appropriate control cells lacking the protein of interest or expressing the protein of interest containing a point mutation in the tyrosine sulfation site
  • Complete tissue culture medium appropriate for cell line
  • Labeling medium with and without label (see recipe), prewarmed
  • PBS (appendix 2E), cold
  • Immunoprecipitation buffer (see recipe) with protease inhibitors, ice cold
  • RIPA lysis buffer (see recipe) with protease inhibitors, ice cold
  • Antibody
  • Protein A-agarose beads
  • Wash buffers A, B, and C (see recipes)
  • 2× SDS-PAGE sample buffer (see recipe)
  • Disposable pipets
  • Centricon concentrator (Amicon)
  • 2.0- and 1.5-ml microcentrifuge tubes
  • Cell scraper
  • 15-ml centrifuge tubes
  • End-over-end rotator
  • Additional reagents and equipment for immunoprecipitation (unit 13.2) and SDS-PAGE (unit 10.1)

NOTE: All reagents and equipment coming into contact with live cells must be sterile, and proper sterile technique should be used accordingly.

NOTE: All culture incubations are performed in a 37°C, 5% CO2 humidified incubator unless specified otherwise. Some media (e.g., DMEM) may require altered levels of CO2 to maintain the pH at 7.4.

NOTE: The size of the cultures to be labeled depends on the amount of the protein of interest present in the cells. Typically, 100- to 500-mm diameter culture dishes are required.

CAUTION: Radioactive materials require special handling. See appendix 2B concerning safe use of radioisotopes.

Support Protocol 1: Detection of [35S]-Sulfated Proteins

 Additional Materials (also see Basic Protocol 1)
  • Gel containing protein of interest (see Basic Protocol 1)
  • 1 M sodium salicylate, pH 5 to 7
  • 20% PPO (2,5-diphenyloxazole) in toluene (optional)
  • Whatman 3MM filter paper or nitrocellulose membrane
  • X-ray film (e.g., Kodak XAR-5 or equivalent) or phosphorimager
  • Additional reagents and equipment for staining and destaining gels (unit 10.5), fluorography (unit 10.2), and blotting (unit 10.7)

CAUTION: Solutions that come into contact with the gel may become radioactive. Gloves should be worn at all times. Sodium salicylate can cause allergic reactions and is readily absorbed through the skin.

Basic Protocol 2: Tyrosine Sulfate Analysis—Alkaline Hydrolysis Method

 Materials
  • Immunoprecipitate containing the protein of interest (see Basic Protocol 1)
  • Wash buffer D (see recipe)
  • Pronase solution: 100 µg/ml Pronase (Roche) in 5 mM NH4HCO3, pH 8
  • 0.2 and 0.02 M barium hydroxide (Ba(OH)2)
  • N2 source
  • 1 and 0.1 M sulfuric acid
  • Uncapped 3-ml glass centrifuge tube
  • 110°C oven

CAUTION: Barium hydroxide may be fatal if swallowed, harmful if inhaled, and causes irritation to skin, eyes, and respiratory tract. It affects muscles and the central nervous system. Use gloves when handling and perform this experiment in a chemical fume hood.

Support Protocol 2: Thin-Layer Electrophoresis of Tyrosine [35S]-Sulfate

 Materials
  • Lyophilized sample (see Basic Protocol 2)
  • Electrophoresis buffer, pH 3.5 (see recipe)
  • Acetone
  • Unlabeled l-tyrosine-O-sulfate (Bachem)
  • Phenol red
  • Unlabeled l-serine-O-sulfate (Bachem)
  • Unlabeled l-tyrosine (Bachem)
  • 0.2% (w/v) ninhydrin in acetone (as spray)
  • Cellulose-coated plastic-backed TL chromatography sheet (20 cm × 20 cm × 100 µm)
  • Whatman 3 MM filter paper
  • 100°C oven
  • Additional reagents and equipment for thin-layer electrophoresis (unit 13.3)
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Figures

  •  FigureFigure 14.7.1 The formation of a tyrosine-O4-sulfate residue is catalyzed by tyrosylprotein sulfotransferase (TPST) using 3¢-phosphoadenosine 5¢-phosphosulfate (PAPS) as co-substrate and sulfate donor. This enzymatic reaction occurs in the lumen of the trans-Golgi network (TGN).
    View Image
  •  FigureFigure 14.7.2 Thin-layer electrophoresis of tyrosine sulfate. Schematic diagram of a 1-D separation, showing the positions of sulfated and nonsulfated hydroxyamino acids. The sample (S) containing [35S]-labeled tyrosine sulfate, and standards (tyrosine, T; tyrosine sulfate, TS; serine sulfate, SS) are analyzed by ninhydrin staining (A) and autoradiography/fluorography (B). The position of phenol red is indicated.
    View Image

Videos

Literature Cited

Literature Cited
    Beisswanger, R., Corbeil, D., Vannier, C., Thiele, C., Dohrmann, U., Kellner, R., Ashman, K., Niehrs, C., and Huttner, W.B. 1998. Existence of distinct tyrosylprotein sulfotransferase genes: Molecular characterization of tyrosylprotein sulfotransferase-2. Proc. Natl. Acad. Sci. U.S.A. 95:11134-11139.
    Bettelheim, F.R. 1954. Tyrosine-O-sulfate in a peptide from fibrinogen. J. Am. Chem. Soc. 76:2838-2839.
    Bundgaard, J.R., Johnsen, A.H., and Rehfeld, J.F. 2002. Analysis of tyrosine-O-sulfation. Methods Mol Biol. 194:223-239.
    Corbeil, D. and Huttner, W.B. 2004. Tyrosine sulfation. Encyclopedia Biol. Chem. 4:294-297. (W. Lennarz and M.D. Lane, eds.) Elsevier, Oxford.
    Costagliola, S., Panneels, V., Bonomi, M., Koch, J., Many, M.C., Smits, G., and Vassart, G. 2002. Tyrosine sulfation is required for agonist recognition by glycoprotein hormone receptors. EMBO J. 21:504-513.
    Farzan, M., Mirzabekov, T., Kolchinsky, P., Wyatt, R., Cayabyab, M., Gerard, N.P., Gerard, C., Sodroski, J., and Choe, H. 1999. Tyrosine sulfation of the amino terminus of CCR5 facilitates HIV-1 entry. Cell 96:667-676.
    Friederich, E., Fritz, H.J., and Huttner, W.B. 1988. Inhibition of tyrosine sulfation in the trans-Golgi retards the transport of a constitutively secreted protein to the cell surface. J. Cell Biol. 107:1655-1667.
    Glombik, M.M., Krömer, A., Salm, T., Huttner, W.B., and Gerdes, H.H. 1999. The disulfide-bonded loop of chromogranin B mediates membrane binding and directs sorting from the trans-Golgi network to secretory granules. EMBO J. 18:1059-1070.
    Huttner, W.B. 1982. Sulphation of tyrosine residues—A widespread modification of proteins. Nature (London) 299:273-276.
    Huttner, W.B. 1984. Determination and occurrence of tyrosine O-sulfate in proteins. Methods Enzymol. 107:200-223.
    Huttner, W.B. and Baeuerle, P.A. 1988. Protein sulfation on tyrosine. Mod. Cell Biol. 6:97-140.
    Huttner, W.B., Niehrs, C., and Vannier, C. 1991. Bind or bleed. Curr. Biol. 1:309-310.
    Kehoe, J.W. and Bertozzi, C.R. 2000. Tyrosine sulfation: A modulator of extracellular protein-protein interactions. Chem. Biol. 7:R57-R61.
    Medzihradszky, K.F., Darula, Z., Perlson, E., Fainzilber, M., Chalkley, R.J., Ball, H., Greenbaum, D., Bogyo, M., Tyson, D.R., Bradshaw, R.A., and Burlingame, A.L. 2004. O-Sulfonation of serine and threonine: Mass spectrometric detection and characterization of a new posttranslational modification in diverse proteins throughout the eukaryotes. Mol Cell Proteomics. 3:429-443.
    Monigatti, F., Gasteiger, E., Bairoch, A., and Jung, E. 2002. The Sulfinator: Predicting tyrosine sulfation sites in protein sequences. Bioinformatics 18:769-770.
    Moore, K.L. 2003. The biology and enzymology of protein tyrosine O-sulfation. J. Biol. Chem. 278:24243-24246.
    Niehrs, C., Huttner, W.B., and Rüther, U. 1992. In vivo expression and stoichiometric sulfation of the artificial protein sulfophilin, a polymer of tyrosine sulfation sites. J. Biol. Chem. 267:15938-15942.
    Niehrs, C., Beiwanger, R., and Huttner, W.B. 1994. Protein tyrosine sulfation, 1993—An update. Chem.-Biol. Interact. 92:257-271.
    Önnerfjord, P., Heathfield, T.F., and Heinegard, D. 2004. Identification of tyrosine sulfation in extracellular leucine-rich repeat proteins using mass spectrometry. J. Biol. Chem. 279:26-33.
    Ouyang, Y-B., Lane, W.S., and Moore, K.L. 1998a. Tyrosylprotein sulfotransferase: Purification and molecular cloning of an enzyme that catalyzes tyrosine O-sulfation, a common posttranslational modification of eukaryotic proteins. Proc. Natl. Acad. Sci. U.S.A. 95:2896-2901.
    Ouyang, Y-B. and Moore, K.L. 1998b. Molecular cloning and expression of human and mouse tyrosylprotein sulfotransferase-2 and a tyrosylprotein sulfotransferase homologue in Caenorhabditis elegans. J. Biol. Chem. 277:24770-24774.
    Robbins, P. and Lippmann, F. 1956. Identification of enzymatically active sulfate as adenosine-3¢-phosphate-5¢-phospho-sulfate. J. Am. Chem. Soc. 78:2652-2653.
    Weigmann, A., Corbeil, D., Hellwig, A., and Huttner, W.B. 1997. Prominin, a novel microvilli-specific polytopic membrane protein of the apical surface of epithelial cells, is targeted to plasmalemmal protrusions of nonepithelial cells. Proc. Natl. Acad. Sci. U.S.A. 94:12425-12430.
    Wolfender, J.L., Chu, F., Ball, H., Wolfender, F., Fainzilber, M., Baldwin, M.A., and Burlingame, A.L. 1999. Identification of tyrosine sulfation in Conus pennaceus conotoxins alpha-PnIA and alpha-PnIB: further investigation of labile sulfo- and phosphopeptides by electrospray, matrix-assisted laser desorption/ionization (MALDI) and atmospheric pressure MALDI mass spectrometry. J. Mass Spectrom. 34:447-454.
 Key References
    Huttner, 1984. See above.

Describes methods for sulfate labeling and various procedures to detect tyrosine sulfate in proteins. The determination of the stoichiometry of tyrosine sulfation of proteins is also discussed.

    Bundgaard et al., 2002. See above.

Describes several analytical methods for tyrosine sulfate analysis.

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