Rat organic anion transporting protein 1A1 (Oatp1a1): purification and phosphopeptide assignment

Biochemistry
Yansen XiaoA W Wolkoff

Abstract

Rat organic anion transporting protein 1a1 (oatp1a1), a hepatocyte basolateral plasma membrane protein, mediates transport of various amphipathic compounds. Our previous studies indicated that serine phosphorylation of a single tryptic peptide inhibits its transport activity without changing its cell surface content. The site of phosphorylation is unknown and was the subject of the present study. Following immunoaffinity chromatographic purification from rat liver, oatp1a1 was subjected to trypsin digestion and MALDI-TOF. Except for predicted N-glycosylated peptides, 97% of oatp1a1 tryptic peptides were observed. A single tryptic phosphopeptide was found in the C-terminus (aa 626-647), existing in unphosphorylated or singly or doubly phosphorylated forms and sensitive to alkaline phosphatase treatment. The beta-elimination reaction resulted in a mass loss of 98 or 196 Da from this peptide, and subsequent Michael addition with cysteamine increased masses by the predicated 77 and 154 Da, indicating that oatp1a1 can be singly or doubly phosphorylated at serine or threonine residues in the C-terminal sequence SSATDHT (aa 634-640). Subsequent tandem MS/MS analysis revealed that phosphorylation at S634 accounted for all singly phosph...Continue Reading

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Citations

Jan 28, 2010·Pharmacological Reviews·Curtis D Klaassen, Lauren M Aleksunes
Dec 25, 2010·American Journal of Physiology. Gastrointestinal and Liver Physiology·Jo H ChoiAllan W Wolkoff
Dec 4, 2012·Biochimica Et Biophysica Acta·Kai-Wei HsuehChao-Hsiung Lin
Nov 16, 2010·Journal of Proteomics·María Jesús García-MurriaManuel M Sánchez del Pino
Apr 20, 2010·Protein Expression and Purification·German ReyesImogen R Coe
Apr 2, 2010·Journal of Clinical Neuroscience : Official Journal of the Neurosurgical Society of Australasia·Hao ZhangLu-Gang Yu
Apr 5, 2014·Hepatology : Official Journal of the American Association for the Study of Liver Diseases·M Sawkat Anwer
May 24, 2016·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Caixia YangGang Liu
Oct 12, 2013·Drug Metabolism and Disposition : the Biological Fate of Chemicals·Wen-Jun WangAllan W Wolkoff
Mar 1, 2008·American Journal of Physiology. Gastrointestinal and Liver Physiology·Pijun WangAllan W Wolkoff
Sep 4, 2018·Biological & Pharmaceutical Bulletin·Yoshinobu NakamuraIkumi Tamai

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