High-Throughput Screening of Sulfated Proteins by Using a Genome-Wide Proteome Microarray and Protein Tyrosine Sulfation System.

Protein tyrosine sulfation (PTS) is a widespread posttranslational modification that induces intercellular and extracellular responses by regulating protein-protein interactions and enzymatic activity. Although PTS affects numerous physiological and pathological processes, only a small fraction of the total predicted sulfated proteins has been identified to date. Here, we localized the potential sulfation sites of Escherichia coli proteins on a proteome microarray by using a 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthase-coupled tyrosylprotein sulfotransferase (TPST) catalysis system that involves in situ PAPS generation and TPST catalysis. Among the 4256 E. coli K12 proteins, 875 sulfated proteins were identified using antisulfotyrosine primary and Cy3-labeled antimouse secondary antibodies. Our findings add considerably to the list of potential proteins subjected to tyrosine sulfation. Similar procedures can be applied to identify sulfated proteins in yeast and human proteome microarrays, and we expect such approaches to contribute substantially to the understanding of important human diseases.

Tyrosine sulfation as a protein post-translational modification.

Integration of inorganic sulfate into biological molecules plays an important role in biological systems and is directly involved in the instigation of diseases. Protein tyrosine sulfation (PTS) is a common post-translational modification that was first reported in the literature fifty years ago. However, the significance of PTS under physiological conditions and its link to diseases have just begun to be appreciated in recent years. PTS is catalyzed by tyrosylprotein sulfotransferase (TPST) through transfer of an activated sulfate from 3'-phosphoadenosine-5'-phosphosulfate to tyrosine in a variety of proteins and peptides. Currently, only a small fraction of sulfated proteins is known and the understanding of the biological sulfation mechanisms is still in progress. In this review, we give an introductory and selective brief review of PTS and then summarize the basic biochemical information including the activity and the preparation of TPST, methods for the determination of PTS, and kinetics and reaction mechanism of TPST. This information is fundamental for the further exploration of the function of PTS that induces protein-protein interactions and the subsequent biochemical and physiological reactions.

Fluorescence assay for protein post-translational tyrosine sulfation.

We developed a fluorescent assay to conveniently determine the kinetics of protein sulfation, which is essential for understanding interface between protein sulfation and protein-protein interactions. Tyrosylprotein sulfotransferase (TPST) catalyzes protein sulfation using 3'-phosphate 5'-phosphosulfate (PAPS) as sulfuryl group donor. In this report, PAPS was regenerated following sulfuryl group transfer between adenosine 3',5'-diphosphate and 4-methylumbelliferyl sulfate catalyzed by phenol sulfotransferase (PST). The TPST and PST coupled enzyme platform continuously generated fluorescent 4-methylumbelliferone (MU) that was used to real-time monitor protein sulfation. Using a recombinant N utilization substance protein A fused Drosophila melanogaster tyrosylprotein sulfotransferase, we demonstrated that the activity of TPST determined through MU fluorescence directly correlated with protein sulfation. Kinetic constants obtained with small P-selectin glycoprotein ligand-1 peptide (PSGL-1 peptide, MW 1541) or its large glutathione S-transferase fusion protein (GST-PSGL-1, MW 27833) exhibited significant variation. This assay can be further developed to a high-throughput method for the characterization of TPSTs and for the identification and screening of their protein substrates.

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

Protein tyrosine sulfation (PTS), catalyzed by membrane-anchored tyrosylprotein sulfotransferase (TPST), is one of the most common post-translational modifications of secretory and transmembrane proteins. PTS, a key modulator of extracellular protein-protein interactions, accounts for various important biological activities, namely, virus entry, inflammation, coagulation, and sterility. The preparation and characterization of TPST is fundamental for understanding the synthesis of tyrosine-sulfated proteins and for studying PTS in biology. A sulfated protein was prepared using a TPST-coupled protein sulfation system that involves the generation of the active sulfate 3'-phosphoadenosine-5'-phosphosulfate (PAPS) through either PAPS synthetase (PAPSS) or phenol sulfotransferase. The preparation of sulfated proteins was confirmed through radiometric or immunochemical assays. In this study, enzymatically active Drosophila melanogaster TPST (DmTPST) and human TPSTs (hTPST1 and hTPST2) were expressed in Escherichia coli BL21(DE3) host cells and purified to homogeneity in high yield. Our results revealed that recombinant DmTPST was particularly useful considering its catalytic efficiency and ease of preparation in large quantities. This study provides tools for high-efficiency, one-step synthesis of sulfated proteins and peptides that are useful for further deciphering the mechanisms, functions, and future applications of PTS.

Specific Unbinding Forces Between Mutated Human P-Selectin Glycoprotein Ligand-1 and Viral Protein-1 Measured Using Force Spectroscopy.

Protein tyrosine sulfation (PTS) is a key modulator of extracellular protein-protein interaction (PPI), which regulates principal biological processes. For example, the capsid protein VP1 of enterovirus 71 (EV71) specifically interacts with sulfated P-selectin glycoprotein ligand-1 (PSGL-1) to facilitate virus invasion. Currently available methods cannot be used to directly observe PTS-induced PPI. In this study, atomic force microscopy was used to measure the interaction between sulfated or mutated PSGL-1 and VP1. We found that the binding strength increased by 6.7-fold following PTS treatment on PSGL-1 with a specific antisulfotyrosine antibody. Similar results were obtained when the antisulfotyrosine antibody was replaced with the VP1 protein of EV71; however, the interaction forces of VP1 were only approximately one-third of those of the antisulfotyrosine antibody. We also found that PTS on the tyrosine-51 residue of glutathione S-transferases fusion-PSGL-1 was mainly responsible for the PTS-induced PPI. Our results contribute to the fundamental understanding of PPI regulated through PTS.

Mechanism of sulfotransferase pharmacogenetics in altered xenobiotic metabolism.

INTRODUCTION: Cytosolic sulfotransferases (SULTs), one of the vital enzymes of detoxication, catalyze the sulfation of native and exogenous hydrophobic molecules. Xenobiotic accumulation can induce a variety of diseases, including cancers. Sulfation facilitates the solubilization and removal of xenobiotics. However, sulfation may activate the pharmacological activities of xenobiotics. AREAS COVERED: The purpose of this review was to correlate the sequence, structure and function of SULTs. We focused on understanding the sulfation mechanisms of SULT through its sequence variation. We selectively reviewed SULT drug substrates, explained the enzyme-catalyzed sulfation reaction and its kinetic mechanisms, and the effect of amino acid sequence variation, such as single-nucleotide polymorphism, on the enzyme function. EXPERT OPINION: A wealth of information is available in the literature for understanding the detailed mechanisms underlying xenobiotic sulfation. We reviewed information regarding the sequence, structure and reaction mechanism of SULTs and explained how SULT activities altered. In addition to revealing the SULT kinetics, the mRNA expression of specific SULTs in tissues that revealed their distribution in tissues also affects overall SULT activities. Understanding of the structure-function relationship and the reaction mechanism of SULTs is valuable for understanding, preventing and treating diseases.