Functional genetic encoding of sulfotyrosine in mammalian cells

Protein tyrosine O-sulfation (PTS) plays a crucial role in extracellular biomolecular interactions that dictate various cellular processes. It also involves in the development of many human diseases. Regardless of recent progress, our current understanding of PTS is still in its infancy. To promote and facilitate relevant studies, a generally applicable method is needed to enable efficient expression of sulfoproteins with defined sulfation sites in live mammalian cells. Here we report the engineering, in vitro biochemical characterization, structural study, and in vivo functional verification of a tyrosyl-tRNA synthetase mutant for the genetic encoding of sulfotyrosine in mammalian cells. We further apply this chemical biology tool to cell-based studies on the role of a sulfation site in the activation of chemokine receptor CXCR4 by its ligand. Our work will not only facilitate cellular studies of PTS, but also paves the way for economical production of sulfated proteins as therapeutic agents in mammalian systems.

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Mycoplasma contamination
Reporter expression and enzyme assays were performed in triplicate. Sample size was determined on the basis of a large number of previously reported studies of similar proteins using similar methods.
No data were excluded from the analyses All experiments were reproduced three times. All attempts at replication were successful.
None of the experimental methods were required or appropriate for randomization.
Investigators were not blinded in this study because no clinical relevant experiments were performed. However, there is no bias for all the data collected in this study.
-For clone sulfo-1C-A2, this mouse anti-sulfotyrosine antibody is certified by MilliporeSigma for use in Immunoprecipitation, ELISA, and Western Blotting.