Evidence of post-translational modification of the tumor suppressor maspin under oxidative stress.
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 non-reducing/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.
Department of Obstetrics and Gynecology, Yamaguchi University School of Medicine, Ube, Japan. shugo@yamaguchi-u.ac.jp
Shugo
S
Nawata
Heidi Y
HY
Shi
Norihiro
N
Sugino
Ming
M
Zhang
D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects; D011485:Protein Binding; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D015843:Serpins / Q000378:metabolism*
D016877:Oxidants; C085344:SERPIN-B5; D015843:Serpins; D006861:Hydrogen Peroxide