Evidence of post-translational modification of the tumor suppressor maspin under oxidative stress

Shugo Nawata*, Heidi Y. Shi, Norihiro Sugino, Ming Zhang

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

Maspin, identified as a 42 kDa unique tumor suppressive serine protease inhibitor (serpin), has multifaceted biological functions by interacting with various target molecules under physiological and pathological conditions including oxidative stress. However, the type of post-translational modification that confers the specific binding affinity of maspin to target molecules, such as glutathione S-transferase (GST), has not been determined. The aim of this study, therefore, is to analyze the molecular heterogeneity of maspin and to identify its modifications in the normal mammary epithelial cell line, MCF-10A, which is known to express the maspin protein abundantly, using electrophoretic analysis. Conventional SDS-PAGE analysis of MCF-10A cell extracts showed that endogenous maspin is composed of both an intact form observed as a 42 kDa band and a smaller form observed as a 36 kDa band. Interestingly, a brief exposure of MCF-10A cells to 10 mM hydrogen peroxide (H2O2) led to the appearance of a novel endogenous maspin form, as demonstrated by non-denaturing PAGE and non-reducing SDS-PAGE. Two-dimensional sequential nonreducing/reducing SDS-PAGE supported that this novel form was generated by intramolecular disulfide-bonded linkage under oxidative stress, and this oxidized maspin form also existed under physiological conditions. Furthermore, a glutathione (GSH) bead pull-down assay revealed that the intramolecular disulfide-bonded maspin lost its binding activity to endogenous GST, indicating that intramolecular disulfide-bonded maspin might have some distinct properties under oxidative stress, although the precise biological significance of this modification remains elusive. In conclusion, we have shown that maspin undertakes different modifications under oxidative stress.

Original languageEnglish (US)
Pages (from-to)249-254
Number of pages6
JournalInternational journal of molecular medicine
Volume27
Issue number2
DOIs
StatePublished - Feb 2011

Keywords

  • Maspin
  • Oxidative stress
  • Post-translational modification
  • Tumor suppressive serpin

ASJC Scopus subject areas

  • Genetics

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