Plant-expressed cocaine hydrolase variants of butyrylcholinesterase exhibit altered allosteric effects of cholinesterase activity and increased inhibitor sensitivity

Abstract

Butyrylcholinesterase (BChE) is an enzyme with broad substrate and ligand specificities and may function as a generalized bioscavenger by binding and/or hydrolyzing various xenobiotic agents and toxicants, many of which target the central and peripheral nervous systems. Variants of BChE were rationally designed to increase the enzyme's ability to hydrolyze the psychoactive enantiomer of cocaine. These variants were cloned, and then expressed using the magnICON transient expression system in plants and their enzymatic properties were investigated. In particular, we explored the effects that these site-directed mutations have over the enzyme kinetics with various substrates of BChE. We further compared the affinity of various anticholinesterases including organophosphorous nerve agents and pesticides toward these BChE variants relative to the wild type enzyme. In addition to serving as a therapy for cocaine addiction-related diseases, enhanced bioscavenging against other harmful agents could add to the practicality and versatility of the plant-derived recombinant enzyme as a multivalent therapeutic.

Figure 1

Plant production and biochemical characterization of a cocaine hydrolase variant of BChE. (a) Plant-based strategy for the production of BChE. (i) Plant-expression optimized synthetic genes encoding human BChE and variants thereof were cloned into the TMV-based MagnICON vector system, which recombines in vivo to yield a cell-to-cell-spreading replicon. (ii) WT Nicotiana benthamiana plants were infiltrated with agrobacteria harboring the MagnICON vectors (iii) and on peak accumulation day of the transiently-expressed recombinant enzymes, leaf material was harvested, homogenized and the enzymes were purified. Transient expression replicon: RpRd, RNA-dependent RNA polymerase; MP, movement protein gene; α, barley alpha-amylase signal peptide. Wavy lines represent the translation products of the replicon genes. (b) Purification of pBChE_V4. Leaf extract from pBChE_V4 –expressing plants was clarified by 70% (NH₄)₂SO₄ precipitation then subject to ConA purification and eluted with stepwise increasing concentrations of methyl-α-D-mannopyranoside ([E1]-[E5]). Samples from these purification steps, protein size markers (M) and an un-infiltrated WT N. benthamiana extract control (C) were subject to SDS-PAGE followed by silver-staining (top) or BChE-specific immunoblotting (bottom). Lanes in respective gels were loaded based on equal enzymatic activity. (c) Oligomerization of pBChE_V4. Purified preparation of pBChE_V4 was analyzed by SEC-HPLC; fractions were monitored for total protein content (top) and pooled fractions (0.5 mL every 1 min) for enzymatic activity (bottom). Inset: fractionation pattern of WT pBChE. Molecular mass standards are indicated with arrows. (d) Enzymatic hydrolysis of (−)-cocaine by WT pBChE and pBChE_V4. Purified samples of WT pBChE (green, 1.21 × 10⁻¹ µM, upper and lower panel) and pBChE_V4 (pink, 6.06 × 10⁻⁴ µM, upper panel). Curves represent nonlinear regression fitted to the Michaelis-Menten model (Equation 1). Fitting the data to the Radić model (substrate inhibition, Equation 2) does not result in a significantly better fit (based on the extra sum-of-squares F test; p > 0.12 and p > 0.78 for the mutant and WT enzymes, respectively).

Figure 2

BTC hydrolysis by WT hBChE, WT pBChE, and pBChE_V2-5. (a) Reaction rates are plotted against substrate concentration (mean ± SEM). Plots in (b) zoom in on the low range of substrate concentrations. The 100% values and the goodness of fit values are as follows: WT hBChE, 100% = 1.57 ± 0.04 nmol/min, Equation (2), R² = 0.98; WT pBChE, 100% = 1.21 ± 0.07 nmol/min, Equation (2), R² = 0.99; pBChE_V2, 100% = 0.79 ± 0.10 nmol/min, Equation (3), R² = 0.83; pBChE_V3, 100% = 0.96 ± 0.01 nmol/min, Equation (3), R² = 0.95; pBChE_V4, 100% = 8.9 ± 0.6 nmol/min, Equation (3), R² = 0.95; pBChE_V5, 100% = 8.1 ± 0.3 nmol/min, Equation (3), R² = 0.95.

Figure 3

ATC hydrolysis by WT hBChE, WT pBChE, and pBChE_V2-5. (a) Reaction rates are plotted against substrate concentration (mean ± SEM). Plots in (b) zoom in on the low range of substrate concentrations. The 100% values and the goodness of fit values are as follows: WT hBChE, 100% = 0.97 ± 0.04 nmol/min, Equation (2), R² = 0.99; WT pBChE, 100% = 3.0 ± 0.1 nmol/min, Equation (2), R² = 1.00; pBChE_V2, 100% = 4.7 ± 0.1 nmol/min, Equation (2), R² = 0.99; pBChE_V3, 100% = 1.39 ± 0.00 nmol/min, Equation (3), R² = 0.98; pBChE_V4, 100% = 2.6 ± 0.1 nmol/min, Equation (1), R² = 0.94; pBChE_V5, 100% = 12.0 ± 0.7 nmol/min, Equation (3), R² = 0.99.

Figure 4

Inhibition profiles of WT hBChE, WT pBChE and pBChE_V2-5. Residual BTC hydrolytic activity (mean ± SEM) with the indicated concentrations of paraoxon and Iso-OMPA (OP inhibitors), neostigmine bromide (a carbamate inhibitor) and BW (an AChE-specific bis-quaternary inhibitor). The legends in each panel list the traces in order of decreasing IC50. Plots of variants are compared to the human plasma-derived enzyme. ns, no statistical difference; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 5

% DCI profile of WT hBChE. The % DCI profiles for hBChE are color-coded in a cartoon diagram from a spectrum of red-white-blue (red -highest, blue -lowest coupling to perturbation locations). (a) Upon perturbation of catalytic residues (S198, E325, and H438 shown as grey sticks) the five mutation positions (A199, F227, S287, A328, and Y332 shown as red sticks) shows high coupling (high % DCI values). (b) Upon perturbation of five mutation positions (A199, F227, S287, A328, and Y332 shown as grey sticks) the catalytic residues (S198, G325, and H438 shown as red sticks) shows high coupling (high % DCI values).

Figure 6

% DFI profile of WT hBChE and pentavalent mutant. (a) The % DFI profiles of WT BChE (blue) and BChE_V4 (x-axis – residue numbers, y-axis – % DFI values at each position). (b) Color-coded structure diagrams depicting the % DFI values at each position. The circled regions are part of the monomer-monomer contact region (V377, D378, T457, K458, A459, I462, Y500, R509, M511, T512, K513, R514, L515). (c) The % DFI profiles of monomeric (blue) and dimeric (green) WT BChE. (d) The % DFI profiles of monomeric (red) and dimeric (purple) BChE_V4. (e) Color-coded structure diagrams depicting the values of % DFI differences between the dimeric forms of WT BChE and BChE_V4 at each position. The red-white-blue code reveals loci with increased flexibility (shades of red), decreased flexibility (shades of blue) or no change (white) in the mutant dimer vs. the WT dimer.