Identification of cell-surface molecular interactions under living conditions by using the enzyme-mediated activation of radical sources (EMARS) method

Abstract

Important biological events associated with plasma membranes, such as signal transduction, cell adhesion, and protein trafficking, are mediated through the membrane microdomains. We have developed a novel method termed enzyme-mediated activation of radical sources (EMARS) to identify coclustering molecules on the cell surface under living conditions, which features a radical formation from an aryl azide reagent by horseradish peroxidase (HRP). For identification of molecules labeled by the EMARS reaction, antibody array system and mass spectrometry-based proteomics approaches are available. Spatio- temporally-regulated interaction between b1 integrin and ErbB4 involved in fibronectin-dependent cell migration and therapeutic antibody-stimulated interaction between FGFR3 and CD20 were discovered using the EMARS method.

Figure 1.

Coclustered molecules with β1-integrin and ganglioside GM1 revealed by the EMARS reaction. An antibody array analysis of the coclustered molecules of β1-integrin (upper left panel) and ganglioside GM1 (upper right panel) in HeLa S3 cells. Antibodies against 42 kinds of receptor tyrosine kinases (RTKs) are spotted in duplicate on the array. Cell surface expression of RTKs was examined by labeling with N-hydroxysuccinimide biotin (lower panels). Adapted and used with permission from [8].

Figure 2.

Suppression of the nonspecific labeling by endogeneous enzyme(s) in EMARS reaction with fluorescein-tagged aryl azide. EMARS reaction was performed with fluorescein-tagged aryl azide (left panel) or biotin-tagged aryl azide (right panel) after setting HRP on β1 integrin in T98 cells and HeLa S3 cells. After reaction, each sample was subjected to Western blotting with an anti-fluorescein antibody (left panel) or streptavidin (right panel). The 800 g fraction contains nuclei, mitochondria and peroxisomes. The 20,000 g fraction contains microsomes. The robust nonspecific labeling in the 800 g fraction in the sample with biotin-tagged aryl azide (right panel) disappeared in that with fluorescein-tagged aryl azide (left panel). Adapted from [11] and used with permission.

Figure 3.

Interaction with β1 integrin and phosphorylation of RTKs. (A) Association between β1 integrin and RTKs in HeLa S3 cells. The EMARS reaction was performed at 15 min, 2 h and 1 day after seeding of HeLa S3 cells onto fibronectin using an HRP-conjugated anti-β1 integrin antibody as a probe and aryl azide-fluorescein as a labeling reagent. (B) Phosphorylation of RTKs. A part of the EMARS product was separately applied to antibody array and reacted with an anti-phosphotyrosine antibody. Adapted from [13] and used with permission.

Figure 4.

RTKs coclustered with rituximab-CD20 complex. The EMARS method was performed using HRP-conjugated rituximab or HRP-conjugated 2H7 antibody in Raji cells. The EMARS products from rituximab-HRP (Rituximab-HRP) and 2H7-HRP (2H7-HRP) treated Raji cells were applied to the RTKs antibody array. Adapted and used with permission from [14]. (This research was originally published in The Journal of Biological Chemistry. Kotani, N.; Ishiura, Y.; Yamashita, R.; Ohnishi, T.; Honke, K. FGFR3 associated with the CD20 antigen regulates the rituximab-induced proliferation inhibition in B-cell lymphoma cells. J. Biol. Chem. 2012, 287, 37109–37118. © the American Society for Biochemistry and Molecular Biology).

Scheme 1.

The enzyme-mediated activation of radical source (EMARS) reaction. The aryl azide group that is usually utilized for photoaffinity labeling is activated by horseradish peroxidase (HRP) to a nitrene radical. The aryl azide group can be conjugated with various labeling tags such as biotin and fluorescein.

Scheme 2.

In vivo EMARS analysis.

Scheme 3.

Identification of the EMARS product by proteomics technique. The fluorescein-labeled EMARS products are purified and concentrated by immunoaffinity chromatography with anti-fluorescein antibody-immobilized beads and identified by mass spectrometry (MS)-based proteomics analysis.