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Longlong Luo,
Qun Luo, Leiming Guo,
Ming Lv,
Zhou Lin,
Jing Geng,
Xinying Li,
Yan Li,
Beifen Shen,
Chunxia Qiao,
Jiannan Feng
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ABSTRACT: Ricin is a highly lethal toxin. Anti-ricin chimeric monoclonal antibody (mAb) C4C13 was prepared in our lab; however, its binding affinity was much weaker than that of the parent antibody 4C13. In this study, based on the computer-guided homology modeling and conformational optimization methods, the 3-D structure of C4C13 variable regions Fv was constructed and optimized. Using molecular docking and dynamics simulation methods, the 3-D complex structure of ricin and C4C13 Fv was obtained. Considering the orientation property, surface electrostatic distribution, residues chemical andphysical character and intermolecular hydrogen bond, the binding mode and key residues were predicted. According to C4C13 Fv fragment and ricin complementary binding surface, electrostatic attraction periphery and van der Waals interaction interface, three mutants (i.e., M1 (N(H102)F, W(H103)Y); M2 (W(H103)Y) and M3 (R(L90)G)) were designed, in which M1 and M2 were predicted to possess higher antigen-binding activity than C4C13, while M3 was weaker. The relative affinity assays by ELISA showed that M1 and M2 mutations had higher affinity (9.6 and 18.3 nmol/L) than C4C13 (130 nmol/L) and M3 had weaker affinity (234.5 nmol/L) than C4C13. The results showed that the modeling complex structure of the antigen (ricin) and antibody (C4C13) is reasonable. Our work offered affinity maturated antibodies by site mutations, which were beneficial for valuable anti-ricin antibody design and preparation in future.
Journal of biomolecular structure & dynamics 03/2013; · 4.99 Impact Factor
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Chunxia Qiao,
Meiyun Hu, Leiming Guo,
Ming Lv,
Zhou Lin,
Jing Geng,
Xiaoling Lang,
Xinying Li,
Yan Li,
Yuanfang Ma,
Jiannan Feng,
Beifen Shen
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ABSTRACT: 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.
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.
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.
BMC Immunology 07/2012; 13:40. · 2.53 Impact Factor
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ABSTRACT: Evidence showed that the extracellular part of Fc(epsilon)RIalpha (FCR) with its own transmembrane domain (TMF) cannot be expressed as a transmembrane form in CHO cell line. However, FCR could be displayed on cell surface with the transmembrane domain (TM) of human IL2Ralpha (TMI). Theoretical analysis of TMF and TMI using TM prediction methods showed that TMI possessed strong orientation tendency to form "outside to inside" transmembrane mode from N-terminal to C-terminal, while TMF was prone to form "inside to outside" mode. Based on the analyzing results, the TM of Her2 (TMH) was studied and showed similar transmembrane mode as that of TMI, which implied that TMH might be a novel TM to obtain the surface display of FCR. Then, DNA sequences encoding TMH and TMF were fused to 3'-end of FCR gene, respectively. Fluorescent microscope observation indicated that FCR_TMH seemed to be located mainly on cell surface, while FCR_TMF appeared in endochylema. Flow cytometry analysis and Western blot also showed that the surface expression of FCR was enhanced significantly by TMH, while FCR_TMF could not be surface displayed in 293T cell. The experimental results were consistent with the theoretical predictions and demonstrated that the orientation tendency of TM may be very important in subcellular location of proteins.
Journal of Biotechnology 01/2009; 139(3):195-202. · 3.05 Impact Factor
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ABSTRACT: So far, no specific therapeutic agent is available for the treatment of ricin intoxication. Here, V(H) and V(L) genes were cloned from a hybridoma cell line secreting anti-ricin mAb 4C13, which could neutralize the toxicity of ricin. A chimeric antibody, c4C13, containing 4C13 mAb variable region genes fused to human constant region genes (gamma 1, kappa), was constructed. C4C13 retained the binding activity and recognized the same, or a closely related, epitope as the original mouse antibody. Furthermore, c4C13 blocked ricin-induced cytotoxicity to SP2/0 cells. Compared with its parental mouse antibody, c4C13 will be safer when used in human body to reverse clinical ricin intoxication.
Biotechnology Letters 01/2008; 29(12):1811-6. · 1.68 Impact Factor
