Protein tyrosine O-sulfation, a widespread post-translational modification, is mediated by two Golgi
enzymes, tyrosylprotein sulfotransferase-1 and -2. These
enzymes catalyze the transfer of
sulfate from the universal
sulfate donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to the
hydroxyl group of
tyrosine residues to form
tyrosine O-sulfate ester and PAP. More than 60
proteins have been identified to be
tyrosine sulfated including several
G protein-coupled receptors, such as
CC-chemokine receptor 8 (CCR8) that is implicated in allergic
inflammation,
asthma, and
atherogenesis. However, the kinetic properties of purified
tyrosylprotein sulfotransferase (TPST)-1 and -2 have not been previously reported. Moreover, currently there is no available quantitative
TPST assay that can directly monitor individual sulfation of a series of
tyrosine residues, which is present in most known substrates. We chose an MS-approach to address this limitation. In this study, a liquid chromatography electrospray ionisation mass spectrometry (LC/ESI-MS)-based
TPST assay was developed to determine the kinetic parameters of individual TPSTs and a mixture of both
isozymes using CCR8
peptides as substrates that have three
tyrosine residues in series. Our method can differentiate between mono- and disulfated products, and our results show that the K(m,app) for the monosulfated substrate was 5-fold less than the nonsulfated substrate. The development of this method is the initial step in the investigation of kinetic parameters of the sequential
tyrosine sulfation of
chemokine receptors by TPSTs and in determining its catalytic mechanism.