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

• Published in: Toxins Vol. 4; no. 7; pp. 554 - 567
• Main Authors: Chavanayarn, Charnwit, Thanongsaksrikul, Jeeraphong, Thueng-in, Kanyarat, Bangphoomi, Kunan, Sookrung, Nitat, Chaicumpa, Wanpen
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 01.07.2012; MDPI
• Subjects: Amino Acid Sequence; Animals; Antibodies, Monoclonal, Humanized - chemistry; Camelus; Cloning, Molecular; Complementarity Determining Regions - metabolism; E coli; Elapid Venoms - chemistry; Elapid Venoms - enzymology; Elapidae; Enzymatic activity; Escherichia coli - genetics; Escherichia coli - metabolism; Hemolysis - drug effects; Hemorrhage - chemically induced; Horses - immunology; Humans; Immunoglobulin Variable Region - metabolism; Immunotherapy; Molecular Sequence Data; Phospholipases A2 - metabolism; Phospholipids - metabolism; Polymorphism, Restriction Fragment Length; Protein Conformation; Single-Domain Antibodies - chemistry; homology modeling; molecular docking; phospholipase A2 (PLA2); snake bite; VH/VHH; snake venom; single domain antibody (SdAb)
• ISSN: 2072-6651; 2072-6651
• DOI: 10.3390/toxins4070554

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/VHH) that bound specifically to the P3 and P5 were selected from a humanized-camel VH/VHH phage display library. Two phagemid transfected E. coli clones (P3-1 and P3-3) produced humanized-VHH, while another clone (P3-7) produced humanized-VH. At the optimal venom:antibody ratio, the VH/VHH 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/VHH 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/VHH 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.