Selection of a human butyrylcholinesterase-like antibody single-chain variable fragment resistant to AChE inhibitors from a phage library expressed in E. coli

Figures & data

Figure 1. (A) Molecular formula of the agent (succinylcholine) used as substrate to select the HB2151 E. coli cells able to grow in the selective minimum medium; (B) petri dish containing colonies of HB2151 cells from the first round of selection able to growth in the selective minimum medium.

Figure 2. (A) SDS-PAGE of the eluate from HiTrap^TM column: a single ~24 kDa protein was detectable; (B) Primary structure of the catalytic scFv as confirmed by MALDI TOF analysis and deposited in GenBank database under the accession number KF914159.

Figure 3. General formula of the thio-substrates, used in the modified Ellman assay (37 °C, pH 7.4) and kinetic curves for the hydrolysis respectively of the three substrates, acetylthiocholine, propionylthiocholine and butyrylthiocholine, by WZ-14.2.1 (10⁻⁷ M) in the modified Ellman assay.

Table 1. Kinetic parameters obtained for the three substrates hydrolysis by WZ-14.2.1 (10⁻⁷ M) in the modified Ellman assay (37 °C; pH 7.4)

Substrate	Km mM	Vmax µM/min	Kcat s^−1	Kuncat s^−1x10^−7	Kcat/Kuncat X10^7	Kcat/Km M^−1 s^−1
Acetylthiocholine	72.40 ± 9.57	31.45 ± 4.82	4.99 ± 2.12	5.07 ± 1.99	1.00 ± 0.41	68.92 ± 13.23
Propionylthiocholine	31.57 ± 5.90	20.01 ± 3.03	3.17 ± 0.88	7.93 ± 2.53	0.40 ± 0.12	100.41 ± 20.21
Butyrylthiocholine	75.40 ± 10.28	33.22 ± 5.41	5.27 ± 1.90	1.60 ± 0.51	3.29 ± 0.85	69.93 ± 10.98

Data are means ± SEM from three separate experiments.

Figure 4. Molecular formula of the AChE inhibitors used in the study.

Figure 5. Concentration-dependent inhibition by the BChE inhibitor ethopropazine on the hydrolysis by WZ-14.2.1 (10⁻⁷ M) of the substrate acetylthiocholine verified through the Ellman modified assay (37 °C, pH 7.4). Data are means ± SEM from three separate experiments. *P < 0.05; **P < 0.01

Figure 6. Concentration-dependent inhibition by the Ser blocking agent phenylmethanesuphonyl fluoride (PMSF) on the hydrolysis by WZ-14.2.1 (10⁻⁷ M) of the substrate acetylthiocholine verified through the Ellman modified assay (37 °C, pH 7.4). Data are means ± SEM from three separate experiments. *P < 0.05; **P < 0.01

Figure 7. Kinetic profile obtained in the presence and in the absence (control) of WZ-14.2.1 (10⁻⁷ M) using the Amplex Red ® fluorimetric acetylcholinesterase/acetylcholine assay; *P < 0.05; **P < 0.01

Figure 8. (A) CLUSTALW alignment between WZ-14.2.1 (query) and its closest homologous (identified by BLAST) with the PDB codes 2GHW, 4CAU, 1F3R, 2GKI and 1DZB; (B) 3D model of WZ-14.2.1 obtained by homology building on 2GHW template. The CDR zones (L1, L2, L3, H1, H2, and H3) are colored (in yellow, pink, green, red, dark green, and gray, respectively), the putative binding site is circled; (C) The best conformation of acetylthiocholine in the catalytic antibody obtained with the molecular docking analysis.

Table 2. The measured quantities for each ligand after molecular docking calculation

Compounds	Energy (Kcal/mol)	Distance between C = O* and O atom of Ser37 (Å)	Distance between C = O* and O atom of Ser101 (Å)	π-cation or Stacking with Trp107	Salt bridge with Asp 105
Acetylcholine	-3.45	3.4	4.1	–	+
Acetylthiocholine	-3.43	3.6	3.7	+	+
Propionylthiocholine	-3.44	3.9	9.2	+	+
Butyrylthiocholine	-3.65	3.9	9.1	+	+
Ethopropazine	-6.63	-	-	+	+
Neostigmine	-4.37	7.1	9.1	–	+
		Distance between P = O and O atom of Ser 37	Distance between P = O and O atom of Ser 101
Chlorpyrifos oxon	-5.11	5.5	8.8	–	–
Dichlorvos	-4.71	3.5	4.3	–	–
Iso-OMPA	-3.00	3.9/4.1	9.0/6.8	–	–
Paraoxon ethyl	-3.68	5.7	9.4	–	–

Interaction energy (Kcal/mol) calculated by the Auto Dock program; distance (in Å) between the C atom of the esteric group of the ligands and the O atom of Ser37 (second column) or Ser101 (third column); chance (+ or –) of engaging π-cation interaction between Trp107 and the quaternary N atom of the ligand; finally, chance to engage salt bridges with Asp105 residue. *C = O, P = O: functional groups inside the compounds

Kurkova IN, Smirnov IV, Belogurov AA Jr., Ponomarenko NA, Gabibov AG. Creation of catalytic antibodies metabolizing organophosphate compounds. Biochemistry (Mosc) 2012; 77:1139 - 46; http://dx.doi.org/10.1134/S0006297912100069; PMID: 23157294

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