Plant-derived human butyrylcholinesterase, but not an organophosphorous-compound hydrolyzing variant thereof, protects rodents against nerve agents

Abstract

The concept of using cholinesterase bioscavengers for prophylaxis against organophosphorous nerve agents and pesticides has progressed from the bench to clinical trial. However, the supply of the native human proteins is either limited (e.g., plasma-derived butyrylcholinesterase and erythrocytic acetylcholinesterase) or nonexisting (synaptic acetylcholinesterase). Here we identify a unique form of recombinant human butyrylcholinesterase that mimics the native enzyme assembly into tetramers; this form provides extended effective pharmacokinetics that is significantly enhanced by polyethylene glycol conjugation. We further demonstrate that this enzyme (but not a G117H/E197Q organophosphorus acid anhydride hydrolase catalytic variant) can prevent morbidity and mortality associated with organophosphorous nerve agent and pesticide exposure of animal subjects of two model species.

Fig. 1.

Highly purified preparation of pBChE. Water-soluble proteins were extracted from leaves of WT plants (lane 1) or transgenic plants that express pBChE (lane 2). The recombinant protein was purified from the extract by ammonium sulfate fractionation (30%–70% fraction, lane 3), and in tandem affinity chromatography steps using Con A-sepharose (lane 4) and procainamide (lane 5). Protein samples were resolved by SDS-PAGE and either (A) subjected to silver staining or (B) transferred to a PVDF membrane, immunodecorated by an anti-human BChE antibody followed by HRP-conjugated secondary antibody, and visualized by chemiluminescence. Gels were loaded based on equal BChE activity at 24 mU (A) or 2.4 mU (B). Crude extract samples contained equivalent amounts of total soluble proteins. One unit of enzyme will hydrolyze 1.0 µmol of butyrylthiocholine to thiocholine and butyrate per minute.

Fig. 2.

Tetramerization of pBChE. (A) SEC-HPLC analysis of preparations of pBChE and hBChE. Monomers—1°, dimers—2°, tetramers—4°. β-Amylase (200 kDa) and BSA (66 kDa) were used as size standards. (B) Fractions collected during SEC-HPLC analysis were assayed for BChE activity. (C) Sucrose density gradient sedimentation analysis of pBChE and hBChE preparations. Fractions were assayed for BChE activity.

Fig. 3.

BTCh hydrolysis and its inhibition by POX. (A) Steady-state kinetics of BTCh hydrolysis by pBChE and hBChE. (B) POX IC₅₀ curves for pBChE and hBChE. Error bars indicate ± SEM.

Fig. 4.

Pharmacokinetic profiles of pBChE and its PEGylated derivatives. (A) Unmodified pBChE was administered intravenously to mice at 2,000 units/kg (N = 10) or through a carotid catheter to GPs at 7,000 units/kg (low, N = 2) or at 14,000 units/kg (high, N = 2). (B) PEGylated BChEs were administered i.v. to mice: pBChE-5Kp (N = 10), pBChE-5Kc (N = 10), and pBChE-20K (N = 5). Plasma BChE levels were determined at the specified time points and converted to units per kilogram body weight assuming blood constitutes 7% of body weight in rodents. Control BChE levels were 100–130 units/kg in both mice and GPs. Data points represent mean ± SEM. (Insert) PEGylated pBChE was resolved by SDS-PAGE and protein bands were visualized by silver staining. Unconjugated pBChE (lane 1), or pBChE following conjugation to 5K PEG [2 h partial conjugation (lane 2), and 4 h complete conjugation (lane 3)], or 20K PEG (lane 4).

Fig. 5.

Protection from acute symptoms of OP toxicity with pBChE. Mice (23–30 g) were i.v. injected with pBChE in saline (0–100 mg/kg). Actual BChE concentrations in blood were determined at 5 min after injection and ranged 0–58 mg/kg. At 10 min after BChE administration, mice were injected with POX (50–68 nmol per animal). Molar ratios ([BChE]/[POX]) were calculated and ranged between 0 and 0.51. Symptom score was 0, asymptomatic; 1, decreased motor activity; 2, tremors or fasciculation; 3, convulsions; 4, death.