Abstract
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.
Highlights
Protein tyrosine O-sulfation (PTS) plays a crucial role in extracellular biomolecular interactions that dictate various cellular processes
The genetic incorporation of sTyr into proteins in response to amber nonsense codon (TAG) was reported[27]. This method enables the synthesis of site- sulfated proteins in Escherichia coli (E. coli) using engineered Methanocaldococcus jannaschii tRNA (MjtRNATyr) and its cognate, sTyr-specific tyrosyl-tRNA synthetase pair
We report the synthesis of site- sulfated proteins in both live yeast and mammalian cells through the genetic incorporation of sTyr using an engineered tyrosyl-tRNA synthetase derived from E. coli (EcTyrRS)
Summary
Protein tyrosine O-sulfation (PTS) plays a crucial role in extracellular biomolecular interactions that dictate various cellular processes It involves in the development of many human diseases. 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. We report the synthesis of site- sulfated proteins in both live yeast and mammalian cells through the genetic incorporation of sTyr using an engineered tyrosyl-tRNA synthetase derived from E. coli (EcTyrRS). In addition to fundamental biochemical studies, this work opens a door to the production of sulfoproteins as therapeutic agents in eukaryotes
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