Expression of Membrane-Bound Human AminopeptidaseP as a Soluble Enzyme and an Investigation into Its Efficacy Towards Offering Protection Against the Toxicity of Chemical Warfare Nerve Agents
Abstract
In humans there are two forms of aminopeptidaseP (APP), a cytosolic and a membrane-bound (hmAPP). In this study, we produced an adenovirus containing the hmAPP gene without the membrane binding domain and containing a C-terminus 6 histidine tag (AdshmAPP);this virus was used for expression of soluble hmAPP in HEK293A cells in vitro and in mice in vivo. The enzyme fromHEK293A cells was purified and investigated for its ability to hydrolyze GD, GB, GF, GA, and VX. While unable to hydrolyze VX, shmAPP was capable of hydrolyzing each G agent, hydrolyzing GD more efficiently than any other G-type nerve agent with a catalytic efficiency of (5 1) 106 M-1 min-1. However, the stereo chemical preference of shmAPP favors the hydrolysis of the non-toxic P (+)isomer(s) of each agent. We then evaluated whether mice containing elevated blood levels of shmAPP would be protected from the lethality of GD, GF and GA. Mice were infected with Ad-shmAPP and 5 days later were challenged subcutaneously with 1 x LD50 doses of GA,GF and GD. The survival rates of Ad-shmAPP-treated mice were not significantly different from those of mice treated with a control virus. These results suggest that the overexpression of wild-type shmAPP in mice failed to afford protection against nerve agent toxicity.
Document Details
- Document Type
- Technical Report
- Publication Date
- Sep 01, 2016
- Accession Number
- AD1019022
Entities
People
- David G. Mata
- Nageswararao Chilukuri
- Peter Rezk
- Praveena Sabnekar
Organizations
- United States Army Medical Research Institute of Chemical Defense