Mass spectrometry analysis of the oxidation states of the pro-oncogenic protein anterior gradient-2 reveals covalent dimerization via an intermolecular disulphide bond

David J Clarke, Euan Murray, Jakub Faktor, Aiman Mohtar, Borek Vojtesek, C Logan MacKay, Pat Langridge Smith, Ted R Hupp

Research output: Contribution to journalArticlepeer-review

Abstract

Anterior Gradient-2 (AGR2) is a component of a pro-oncogenic signalling pathway that can promote p53 inhibition, metastatic cell migration, limb regeneration, and cancer drug-resistance. AGR2 is in the protein-disulphide isomerase superfamily containing a single cysteine (Cys-81) that forms covalent adducts with its client proteins. We have found that mutation of Cysteine-81 attenuates its biochemical activity in its sequence-specific peptide docking function, reduces binding to Reptin, and reduces its stability in cells. As such, we evaluated how chemical oxidation of its cysteine affects its biochemical properties. Recombinant AGR2 spontaneously forms covalent dimers in the absence of reductant whilst DTT promotes dimer to monomer conversion. Mutation of Cysteine-81 to alanine prevents peroxide catalysed dimerization of AGR2 in vitro, suggesting a reactive cysteine is central to covalent dimer formation. Both biochemical assays and ESI mass spectrometry were used to demonstrate that low levels of a chemical oxidant promote an intermolecular disulphide bond through formation of a labile sulfenic acid intermediate. However, higher levels of oxidant promote sulfinic or sulfonic acid formation thus preventing covalent dimerization of AGR2. These data together identify the single cysteine of AGR2 as an oxidant responsive moiety that regulates its propensity for oxidation and its monomeric-dimeric state. This has implications for redox regulation of the pro-oncogenic functions of AGR2 protein in cancer cells.

Original languageEnglish
JournalBBA - Bioenergetics
Early online date10 Mar 2016
DOIs
Publication statusPublished - May 2016

Keywords

  • cancer
  • P53
  • Anterior Gradient-2
  • aptamers
  • therapeutics
  • protein mass spectrometry

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