Humanized-Single Domain Antibodies (VH/VHH) that Bound Specifically to Naja kaouthia Phospholipase A2 and Neutralized the Enzymatic Activity

Toxins

Charnwit Chavanayarn³  Jeeraphong Thanongsaksrikul¹  Kanyarat Thueng-in¹  Kunan Bangphoomi²  Nitat Sookrung⁴ 

[1] Laboratory for Research and Technology Development, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand;Department of Biochemistry, Kasetsart University, Bangkok 10900, Thailand;Graduate Program in Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand;Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand;

关键词: snake bite;    snake venom;    phospholipase A2 (PLA₂);    single domain antibody (SdAb);    VH/V_HH;    homology modeling;    molecular docking;   

DOI  :  10.3390/toxins4070554

来源: mdpi

【 摘 要 】

Naja kaouthia (monocled cobra) venom contains many isoforms of secreted phospholipase A2 (sPLA₂). The PLA₂ exerts several pharmacologic and toxic effects in the snake bitten subject, dependent or independent on the enzymatic activity. N. kaouthia venom appeared in two protein profiles, P3 and P5, after fractionating the venom by ion exchange column chromatography. In this study, phage clones displaying humanized-camel single domain antibodies (VH/V_HH) that bound specifically to the P3 and P5 were selected from a humanized-camel VH/V_HH phage display library. Two phagemid transfected E. coli clones (P3-1 and P3-3) produced humanized-V_HH, while another clone (P3-7) produced humanized-VH. At the optimal venom:antibody ratio, the VH/V_HH purified from the E. coli homogenates neutralized PLA₂ enzyme activity comparable to the horse immune serum against the N. kaouthia holo-venom. Homology modeling and molecular docking revealed that the VH/V_HH covered the areas around the PLA₂ catalytic groove and inserted their Complementarity Determining Regions (CDRs) into the enzymatic cleft. It is envisaged that the VH/V_HH would ameliorate/abrogate the principal toxicity of the venom PLA₂ (membrane phospholipid catabolism leading to cellular and subcellular membrane damage which consequently causes hemolysis, hemorrhage, and dermo-/myo-necrosis), if they were used for passive immunotherapy of the cobra bitten victim. The speculation needs further investigations.

【 授权许可】

CC BY   
© 2012 by the authors; licensee MDPI, Basel, Switzerland.