Abstract
BackgroundAs a member of the TNF superfamily, TRAIL could induce human tumor cell apoptosis through its cognate death receptors DR4 or DR5, which can induce formation of the death inducing signaling complex (DISC) and activation of the membrane proximal caspases (caspase-8 or caspase-10) and mitochondrial pathway. Some monoclonal antibodies against DR4 or DR5 have been reported to have anti-tumor activity.ResultsIn this study, we reported a novel mouse anti-human DR5 monoclonal antibody, named as LaDR5, which could compete with TRAIL to bind DR5 and induce the apoptosis of Jurkat cells in the absence of second cross-linking in vitro. Using computer-guided molecular modeling method, the 3-D structure of LaDR5 Fv fragment was constructed. According to the crystal structure of DR5, the 3-D complex structure of DR5 and LaDR5 was modeled using molecular docking method. Based on distance geometry method and intermolecular hydrogen bonding analysis, the key functional domain in DR5 was predicted and the DR5 mutants were designed. And then, three mutants of DR5 was expressed in prokaryotic system and purified by affinity chromatograph to determine the epitope of DR5 identified by LaDR5, which was consistent with the theoretical results of computer-aided analysis.ConclusionsOur results demonstrated the specific epitope located in DR5 that plays a crucial role in antibody binding and even antineoplastic bioactivity. Meanwhile, revealed structural features of DR5 may be important to design or screen novel drugs agonist DR5.
Highlights
As a member of the TNF superfamily, TRAIL could induce human tumor cell apoptosis through its cognate death receptors DR4 or DR5, which can induce formation of the death inducing signaling complex (DISC) and activation of the membrane proximal caspases and mitochondrial pathway
Some positive monoclonal antibody clones against DR5 were screened out by enzyme-linked immunosorbent assay (ELISA), in which LaDR5, an IgG1 subtype antibody identified by double immunodiffusion method, was screened out
TRAIL could compete with LaDR5 to bind DR5 in a dose-dependent manner, which suggested the epitope overlapping of TRAIL and LaDR5 (Figure 1C)
Summary
As a member of the TNF superfamily, TRAIL could induce human tumor cell apoptosis through its cognate death receptors DR4 or DR5, which can induce formation of the death inducing signaling complex (DISC) and activation of the membrane proximal caspases (caspase-8 or caspase-10) and mitochondrial pathway. DR4 and DR5 recruit FAS associated protein with death domain (FADD) [5,6,7] and caspase-8 to form the deathinducing signaling complex (DISC), which activates caspase-8, subsequently leading to the activation of receptors [18]. It may have a more prominent role in bone and myeloid cell development than in regulating TRAIL-induced apoptosis
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