The Endoplasmic Reticulum Chaperone Calnexin Is a NADPH Oxidase NOX4 Interacting Protein

Kim-Kristin Prior,Ilka Wittig,Matthias S Leisegang,Jody Groenendyk,Norbert Weissmann,Marek Michalak,Pidder Jansen-Dürr,Ajay M Shah,Ralf P Brandes

doi:10.1074/jbc.m115.710772

Abstract

Within the family of NADPH oxidases, NOX4 is unique as it is predominantly localized in the endoplasmic reticulum, has constitutive activity, and generates hydrogen peroxide (H2O2). We hypothesize that these features are consequences of a so far unidentified NOX4-interacting protein. Two-dimensional blue native (BN) electrophorese combined with SDS-PAGE yielded NOX4 to reside in macromolecular complexes. Interacting proteins were screened by quantitative SILAC (stable isotope labeling of amino acids in cell culture) co-immunoprecipitation (Co-IP) in HEK293 cells stably overexpressing NOX4. By this technique, several interacting proteins were identified with calnexin showing the most robust interaction. Calnexin also resided in NOX4-containing complexes as demonstrated by complexome profiling from BN-PAGE. The calnexin NOX4 interaction could be confirmed by reverse Co-IP and proximity ligation assay, whereas NOX1, NOX2, or NOX5 did not interact with calnexin. Calnexin deficiency as studied in mouse embryonic fibroblasts from calnexin−/− mice or in response to calnexin shRNA reduced cellular NOX4 protein expression and reactive oxygen species formation. Our results suggest that endogenous NOX4 forms macromolecular complexes with calnexin, which are needed for the proper maturation, processing, and function of NOX4 in the endoplasmic reticulum.

Highlights

From the ‡Institut für Kardiovaskuläre Physiologie, Goethe-Universität, Frankfurt am Main, 60590 Germany, the ¶Functional Proteomics, SFB 815 Core Unit, Goethe-Universität, 60590 Frankfurt am Main, Germany, the ʈCluster of Excellence “Macromolecular Complexes,” Goethe-Universität, 60590 Frankfurt am Main, Germany, the **Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada, the ‡‡Excellence Cluster Cardio-Pulmonary System, Justus-LiebigUniversity Member of the German Center for Lung Research (DZL), 60590 Giessen, Germany, the §§Institute for Biomedical Ageing Research and Center for Molecular Biosciences Innsbruck (CMBI), Universität Innsbruck, 6020 Insbruk, Austria, the ¶¶King’s College London British Heart Foundation Centre, Cardiovascular Division, London WC2R 2LS, United Kingdom, and the §German Center for Cardiovascular Research (DZHK), Partner site RheinMain, 60590 Frankfurt am Main, Germany
Our results suggest that endogenous NOX4 forms macromolecular complexes with calnexin, which are needed for the proper maturation, processing, and function of NOX4 in the endoplasmic reticulum
A potential explanation for missing oxidative stress but constitutive expressed NOX4 is that NOX4 produces predominantly H2O2 rather than O2. as do NOX1, NOX2, NOX3, and NOX5 [7,8,9] and that NOX4 might be located at a specific site in the cell

Summary

Experimental Procedures

Cell Culture—Human embryonic kidney 293 cells (HEK293) were obtained from ATCC (Manassas, VA) and cultured in modified Eagle’s medium (MEM, Gibco) supplemented with fetal calf serum (FCS; 8%), non-essential amino acids (0.1 mM), sodium pyruvate (1 mM), and gentamycin (50 ␮g/ml) in a humidified atmosphere (5% CO2, 37 °C). Pellets were resuspended in 1 ml of HM buffer containing protease inhibitors and used for the protein amount determination by Bradford assay and subsequently for Western blot analysis or membranes were spun down at 100,000 ϫ g for 15 min (4 °C) and used for blue native electrophoresis. For quantitative SILAC-based NOX4-Co-IP (qCo-IP) HEK293 cells were grown in normal or heavy labeled SILAC medium (100 mg/liter of [13C]arginine/[13C,15N]lysine or [12C,14N]arginine/[12C,14N]lysine), lysed with either Tris/HCl, pH 7.5, buffered 1% digitonin or for total membrane preparation the cells were harvested in HM buffer, nuclei were removed (3,000 ϫ g for 10 min), and 100,000 ϫ g total membrane (20 min, 4 °C) pellets were solubilized with digitonin (6 g/g of protein) in solubilization buffer A (50 mM NaCl, 50 mM imidazole/HCl, pH 7.0, 2 mM 6-aminohexanoic acid, 1 mM EDTA). Mass Spectrometry and Data Analysis of SILAC-based qCo-IP—Gel lanes of the combined light and heavy labeled samples were cut into 8 slices. Anti-calnexin (number MAB3126, 1:2,000) was obtained from Merck Millipore; Na/K-ATPase (number ␣6F, 1:2,000) from Hybridoma Bank, University of Iowa; Golgin (number A21270, 1:1,000) from Invitrogen; GAPDH

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Results

Discussion