Mass spectrometric kinetic analysis of human tyrosylprotein sulfotransferase-1 and -2.
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.
Danan
Lieza M
LM
Department of Chemistry, University of California, Davis, California 95616, USA.
Yu
Zhihao
Z
Hoffhines
Adam J
AJ
Moore
Kevin L
KL
Leary
Julie A
JA
D002384:Catalysis; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010724:Phosphoadenosine Phosphosulfate / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D015238:Sulfotransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D008565:Membrane Proteins; D011994:Recombinant Proteins; D010724:Phosphoadenosine Phosphosulfate; D015238:Sulfotransferases; C114740:TPST2 protein, human; C039079:protein-tyrosine sulfotransferase