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 post-translational 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 liability 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 from sulfopeptide analysis.

New tools for evaluating protein tyrosine sulfation: tyrosylprotein sulfotransferases (TPSTs) are novel targets for RAF protein kinase inhibitors

“…DNA encoding human TPST1 (residues Lys43–Leu360) and TPST2 (residues Gly43–Leu359) enzymes lacking the transmembrane domains was amplified by PCR and cloned into pOPINF (OPPF-UK) to produce recombinant protein containing an N-terminal 6xHis tag and a 3C protease cleavage site. Recombinant TPST1 and TPST2 proteins were expressed in BL21 (DE3) pLysS Escherichia coli (Novagen); expression was induced with 0.4 mM IPTG for 18 h at 37°C, and protein was isolated from inclusion bodies and refolded as described [ 44 ]. In brief, cells were resuspended in 3 ml of ice-cold lysis buffer [50 mM Tris–Cl (pH 8.0), 10 mM MgCl 2 , 1 mM DTT supplemented with cOmplete, EDTA-free, protease inhibitor cocktail tablets (Roche)] per gram of E. coli cell pellet and flash-frozen with liquid nitrogen.…”

Preparation of Tyrosylprotein Sulfotransferases for In Vitro One-Pot Enzymatic Synthesis of Sulfated Proteins/Peptides

“…Prokaryotic expressions of TPST reported previously resulted in an inactive form and refolding is needed. 35 , 36 TPSTs have been expressed in many eukaryotic systems such as HEK293-T, CHO, and SF9 insect cells and yeast. 14 , 15 , 20 , 37 The prokaryotic expression system generally provides high expression of the target protein; in addition, the plasmid can be easily constructed for the expression of heterologous enzymes.…”

New tools for evaluating protein tyrosine sulphation: Tyrosyl Protein Sulphotransferases (TPSTs) are novel targets for RAF protein kinase inhibitors

“…Human TPST1 (residues Lys43-Leu360) and TPST2 (residues Gly43-Leu359) enzymes lacking the transmembrane domains were amplified by PCR and cloned into pOPINF (OPPF-UK) to produce recombinant protein containing an N-terminal 6xHis tag and a 3C protease cleavage site. Recombinant TPST proteins were expressed in BL21 (DE3) pLysS E. coli (Novagen) with 0.4 mM IPTG for 18 h at 37°C and isolated from inclusion bodies and refolded as previously described [43]. In brief, cells were resuspended in 3 ml ice-cold lysis buffer (50 mM Tris-HCl, pH 8.0; 10 mM MgCl 2 ; and 1 mM DTT supplemented with cOmplete, EDTA-free protease inhibitor cocktail tablets (Roche) per gram of E. coli cell pellet, and flash frozen with liquid nitrogen.…”