Mass spectrometric kinetic analysis of human tyrosylprotein sulfotransferase-1 and -2

Abstract

Protein tyrosine O-sulfation, a widespread post-translational modification, is mediated by two Golgi enzymes, tyrosylprotein sulfotransferase-1 and Ϫ2. 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 Ϫ2 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. (J

New Targets and Inhibitors of Mycobacterial Sulfur Metabolism

“…Non-radioactive assays using spectrophotometry and mass spectrometry have also been reported [166–168]. Recently, Prather et al developed a universal phosphatase-coupled sulfotransferase assay.…”

Incorporating support vector machine for identifying protein tyrosine sulfation sites

“…12 In contrast, Danan et al demonstrated that TPST2 is more catalytically efficient than TPST1 in sulfating CCR8 substrates. 15 The two tyrosylprotein sulfotransferases, TPST-1 and TPST-2, have overlapping but not identical substrate specificities in vitro and in vivo. 2,12,15 However, the relative abundances and distribution of the two isoenzymes have not yet been examined because of lack of suitable analytical reagents and tools.…”

“…15 The two tyrosylprotein sulfotransferases, TPST-1 and TPST-2, have overlapping but not identical substrate specificities in vitro and in vivo. 2,12,15 However, the relative abundances and distribution of the two isoenzymes have not yet been examined because of lack of suitable analytical reagents and tools. 10,12 Tyrosine-sulfated protein is important to cellular control, but no clear-cut acceptor sequence of TPSTs, which can be used to predict sulfotyrosine sites.…”

Simultaneous Identification of Tyrosine Phosphorylation and Sulfation Sites Utilizing Tyrosine-Specific Bromination

“…The Br signature in monobrominated peptide should be most evident for peptides up to around 1500 Da. For peptides over 2500 Da, the doublet shape of mono-brominated peptide completely disappears as the second isotopic peak originating from 13 C becomes stronger than the monoisotopic peak. Di-Br signature has a similar limitation.…”

“…First, tyrosine phosphorylation regulates protein's activity, localization, and interaction with other proteins in the cytosol [1][2][3]. Second, tyrosine sulfation modulates protein-protein interactions in the extracellular region [4][5][6][7][8][9], which is mediated by two tyrosyl protein sulfotransferases (TPST1 and TPST2) in the trans-Golgi network [10][11][12][13][14][15], resulting in many secretory proteins and Golgi membrane-anchored proteins being sulfated on tyrosine residues [16]. Tyrosine Oglycosylation has been recently investigated in the bacterial proteins [17,18].…”