Targeting a Novel N-terminal Epitope of Death Receptor 5 Triggers Tumor Cell Death

Peng Zhang,Yaxi Zhang,Yong Zheng,Yanxin Liu,Juan Shi,Shilian Liu,Dexian Zheng

doi:10.1074/jbc.m109.070680

Abstract

Tumor necrosis factor-related apoptosis-inducing ligand receptors death receptor (DR) 4 and DR5 are potential targets for antibody-based cancer therapy. Activation of the proapoptotic DR5 in various cancer cells triggers the extrinsic and/or intrinsic pathway of apoptosis. It has been shown that there are several functional domains in the DR5 extracellular domain. The cysteine-rich domains of DR5 have a conservative role in tumor necrosis factor-related apoptosis-inducing ligand-DR5-mediated apoptosis, and the pre-ligand assembly domain within the N1-cap contributes to the ligand-independent formation of receptor complexes. However, the role of the N-terminal region (NTR) preceding the N1-cap of DR5 remains unclear. In this study, we demonstrate that NTR could mediate DR5 activation that transmits an apoptotic signal when bound to a specific agonistic monoclonal antibody. A novel epitope in the NTR of DR5 was identified by peptide array. Antibodies against the antigenic determinant showed high affinities for DR5 and triggered caspase activation in a time-dependent manner, suggesting the NTR of DR5 might function as a potential death-inducing region. Moreover, permutation analysis showed that Leu(6) was pivotal for the interaction of DR5 and the agonistic antibody. Synthetic wild-type epitopes eliminated the cytotoxicity of all three agonistic monoclonal antibodies, AD5-10, Adie-1, and Adie-2. These results indicate that the NTR of DR5 could be a potential target site for the development of new strategies for cancer immunotherapy. Also, our findings expand the current knowledge about DR5 extracellular functional domains and provide insights into the mechanism of DR5-mediated cell death.

Highlights

There are a number of agonistic monoclonal antibodies (mAbs) against human DR4 or DR5 reported in the literature [11,12,13], and they have demonstrated more specific cell death-inducing activities
Given the differences in the cell signaling induced by AD5-10 and Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), we speculate that changes in the conformational structure of DR5 may contribute to this mechanism
To map the epitope in DR5 recognized by AD5-10, we first identified the type of the antigenic determinant, which was considered to be either a sequential epitope or conformational one

Summary

EXPERIMENTAL PROCEDURES

Reagents and Antibodies—Recombinant human TRAIL was purchased from R & D Systems (Minneapolis, MN). The membrane was first blocked with 5% bovine serum albumin in TBST buffer (0.1 M Tris-HCl (pH 7.4), 150 nM NaCl, and 0.1% Tween 20) for 2 h at room temperature and followed by incubation with AD5-10 or an isotype control antibody of normal mouse IgG3 (0.2 ␮g/ml) for 4 h at room temperature. The array membrane was washed three times with TBST buffer and incubated with HRP-conjugated goat anti-mouse IgG for 2 h at room temperature. Hoechst 33258 (1 ␮g/ml) was added to the fixed cells, incubated for 15 min at room temperature, and washed with PBS. The plates were washed three times with PBS/Tween and incubated with 100 ␮l of HRP-conjugated secondary antibodies at 37 °C for 60 min. Output for the final interaction model and predictions was shown after ligand explorer modification

RESULTS

Pathway upon Stimulation with

Random peptide

DISCUSSION