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

Charnwit Chavanayarn,Nitat Sookrung,Wanpen Chaicumpa,Kanyarat Thueng-in,Jeeraphong Thanongsaksrikul,Kunan Bangphoomi

doi:10.3390/toxins4070554

Charnwit Chavanayarn, Nitat Sookrung + Show 4 more

Open Access

https://doi.org/10.3390/toxins4070554

Copy DOI

Journal: Toxins	Publication Date: Jul 19, 2012
Citations: 61	License type: CC BY 4.0

Affiliation: Siriraj Hospital, Mahidol University, Kasetsart University

Abstract

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.

Highlights

IntroductionVenoms of poisonous snakes contain several isoforms of secreted phospholipase A2 (sPLA2) [1,2,3]
Venoms of poisonous snakes contain several isoforms of secreted phospholipase A2 [1,2,3].The principal role of the snake venom Phospholipase A2 (PLA2) is for digesting the prey
The protein peaks 3 and 5 (P3 and P5, respectively) which had been shown by LC-MS/MS to be PLA2 of the N. kaouthia [22] were dialysed against distilled water, concentrated, and the protein contents were measured

Summary

Introduction

Venoms of poisonous snakes contain several isoforms of secreted phospholipase A2 (sPLA2) [1,2,3]. VHH specific to botulinum neurotoxin derived from this library neutralized readily the zinc metalloproteinase activity of the neurotoxin light chain by inserting the CDR3 domain directly into the toxin catalytic groove [19] This enzyme inhibitory mechanism cannot be achieved from the large sized antibody molecules such as intact IgG (150 kDa). In this communication, humanized-camel SdAb that bound with PLA2 of Naja kaouthia (monocled cobra) which is a predominant snake species causing high hospitalized cases and relatively high mortality among the bitten victims in Thailand, were produced and tested for neutralization of enzymatic activity of the PLA2

Materials and Methods

Results

Discussions

Conclusions