The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is a novel cytotoxic ligand belonging to the TNF superfamily which is currently being developed as a cancer therapeutic drug. Here, we observed the different functions of recombinant TRAIL protein with a foreign protein label and non-labeled TRAIL. We used a prokaryotic expression system to prepare two different versions of the extracellular TRAIL 114–281aa protein: TRAIL-HS, a protein modified with 6xHis-Tag and S-Tag; and TRAIL-FT, which had no foreign protein. The proteins were purified using Ni-NTA chromatography (TRAIL-HS) and cation ion-exchange column chromatography (TRAIL-FT) and identified by SDS-PAGE and western blot analysis. We compared the abilities of the proteins to bind to death receptor 5 (DR5) by ELISA and to induce apoptosis in a normal liver cell line (Chang liver) and a human T-lymphocyte leukemia cell line (Jurkat) by MTT assay, GR staining and FACS. The results indicate that the biological functions of TRAIL-FT were superior to those of TRAIL-HS in binding and the induction of apoptosis, and may be useful to further the development and applications of TRAIL.

Background

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. Some monoclonal antibodies against DR4 or DR5 have been reported to have anti-tumor activity.

Results

In 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.

Conclusions

Our 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.