title: Preparation and Analysis of N-Terminal Chemokine Receptor Sulfopeptides Using Tyrosylprotein Sulfotransferase Enzymes.
abstract: In most chemokine receptors, one or multiple tyrosine residues have been identified within the receptor N-terminal domain that are, at least partially, modified by posttranslational tyrosine sulfation. For example, tyrosine sulfation has been demonstrated for Tyr-3, -10, -14, and -15 of CCR5, for Tyr-3, -14, and -15 of CCR8, and for Tyr-7, -12, and -21 of CXCR4. While there is evidence for several chemokine receptors that tyrosine sulfation is required for optimal interaction with the chemokine ligands, the precise role of tyrosine sulfation for chemokine receptor function remains unclear. Furthermore, the function of the chemokine receptor N-terminal domain in chemokine binding and receptor activation is also not well understood. Sulfotyrosine peptides corresponding to the chemokine receptor N-termini are valuable tools to address these important questions both in structural and functional studies. However, due to the lability of the sulfotyrosine modification, these peptides are difficult to obtain using standard peptide chemistry methods. In this chapter, we provide methods to prepare sulfotyrosine peptides by enzymatic in vitro sulfation of peptides using purified recombinant tyrosylprotein sulfotransferase (TPST) enzymes. In addition, we also discuss alternative approaches for the generation of sulfotyrosine peptides and methods for sulfopeptide analysis.
authors.affiliation: Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, USA.
authors.forename: Christoph
authors.initials: C
authors.lastname: Seibert
authors.affiliation: Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, USA.
authors.forename: Anthony
authors.initials: A
authors.lastname: Sanfiz
authors.affiliation: Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, USA.
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Sakmar
authors.affiliation: Department of Chemistry, University of Wisconsin-Whitewater, Whitewater, Wisconsin, USA. Electronic address: veldkamc@uww.edu.
authors.forename: Christopher T
authors.initials: CT
authors.lastname: Veldkamp
mesh_terms: D000818:Animals; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008322:Mammals; D013058:Mass Spectrometry / Q000379:methods; D015202:Protein Engineering / Q000379:methods*; D058849:Protein Refolding; D019707:Receptors, Chemokine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015238:Sulfotransferases / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D019707:Receptors, Chemokine; D011994:Recombinant Proteins; C005662:tyrosine O-sulfate; D014443:Tyrosine; D015238:Sulfotransferases; C039079:protein-tyrosine sulfotransferase
keywords: Biased agonism; Chemokine receptors; Chemokines; GPCR; Ligand bias; Peptide sulfation; Posttranslational modification; Sulfopeptide; Sulfotyrosine; Sulfotyrosine peptide; TPST; Tyrosine sulfation; Tyrosylprotein sulfotransferase