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CC chemokine receptor 5 (CCR5) is the receptor for several inflammatory chemokines and is a coreceptor for HIV-1. Posttranslational sulfation of tyrosines in the N-terminal regions of chemokine receptors has been shown to be important in the binding affinity for chemokine ligands. In addition, sulfation of CCR5 is crucial for mediating interactions with HIV-1 envelope protein gp120. The major sulfation pathway for peptides derived from the N-terminal domains of CCR5 and CCR8 and variations of the peptides were determined by in vitro enzymatic sulfation by tyrosylprotein sulfotranferase-2 (TPST-2), subsequent separation of products by RP-HPLC, and mass spectrometry analysis. It was found that the patterns of sulfation and the rates of sulfation for CCR5 and CCR8 depend on the number of amino acids N-terminal of Tyr-3. Results herein address previous seemingly contradictory studies and delineate the temporal sulfation of N-terminal chemokine receptor peptides.

Human tyrosylprotein sulfotransferases catalyze the transfer of a sulfuryl moiety from the universal sulfate donor PAPS to the hydroxyl substituent of tyrosine residues in proteins and peptides to yield tyrosine sulfated products and PAP. Tyrosine sulfation occurs in the trans-Golgi network, affecting an estimated 1% of the tyrosine residues in all secreted and membrane-bound proteins in higher order eukaryotes. In this study, an effective LC-MS-based TPST kinetics assay was developed and utilized to measure the kinetic properties of human TPST-2 and investigate its catalytic mechanism when G protein-coupled CC-chemokine receptor 8 (CCR8) peptides were used as acceptor substrates. Through initial rate kinetics, product inhibition studies, and radioactive-labeling experiments, our data strongly suggest a two-site ping-pong model for TPST-2 action. In this mechanistic model, the enzyme allows independent binding of substrates to two distinct sites, and involves the formation of a sulfated enzyme covalent intermediate. Some insights on the important amino acid residues at the catalytic site of TPST-2 and its covalent intermediate are also presented. To our knowledge, this is the first detailed study of the reaction kinetics and mechanism reported for human TPST-2 or any other Golgi-resident sulfotransferase. [ABSTRACT FROM AUTHOR]