Characterization of butyrylcholinesterase in bovine serum

Abstract

Human butyrylcholinesterase (HuBChE) protects from nerve agent toxicity. Our goal was to determine whether bovine serum could be used as a source of BChE. Bovine BChE (BoBChE) was immunopurified from 100 mL fetal bovine serum (FBS) or 380 mL adult bovine serum by binding to immobilized monoclonal mAb2. Bound proteins were digested with trypsin and analyzed by liquid chromatography-tandem mass spectrometry. The results proved that FBS and adult bovine serum contain BoBChE. The concentration of BoBChE was estimated to be 0.04 μg/mL in FBS, and 0.03 μg/mL in adult bovine serum, values lower than the 4 μg/mL BChE in human serum. Nondenaturing gel electrophoresis showed that monoclonal mAb2 bound BoBChE but not bovine acetylcholinesterase (BoAChE) and confirmed that FBS contains BoBChE and BoAChE. Recombinant bovine BChE (rBoBChE) expressed in serum-free culture medium spontaneously reactivated from inhibition by chlorpyrifos oxon at a rate of 0.0023 min^-1 (t_1/2 = 301 min^-1) and aged at a rate of 0.0138 min^-1 (t_1/2 = 50 min^-1). Both BoBChE and HuBChE have 574 amino acids per subunit and 90% sequence identity. However, the apparent size of serum BoBChE and rBoBChE tetramers was much greater than the 340,000 Da of HuBChE tetramers. Whereas HuBChE tetramers include short polyproline rich peptides derived from lamellipodin, no polyproline peptides have been identified in BoBChE. We hypothesize that BoBChE tetramers use a large polyproline-rich protein to organize subunits into a tetramer and that the low concentration of BoBChE in serum is explained by limited quantities of an unidentified polyproline-rich protein.

Keywords: Bovine butyrylcholinesterase; Immunopurification; Mass spectrometry; Molecular dynamics; Serum.

Figure 1

Comparison of amino acid sequences of human and bovine BChE. Residues in the catalytic triad are Ser 198, Glu 325, and His 438. Boxed residues Ser 117, Leu 285 and Ile 398 are implicated in spontaneous reactivation of BoBChE inhibited by chlorpyrifos oxon.

Figure 2

Nondenaturing gradient gel stained with butyrylthiocholine (BTC) or acetylthiocholine (ATC). Lane 1, mouse ascites fluid containing mAb2; lane 2, mAb2 purified from ascites fluid; lane 3, recombinant mAb2 purified from HEK293 culture medium containing 10% (v/v) FBS; lanes 4 and 5, FBS; lanes 6 and 7, FBS after treatment with mAb2 from ascites; lanes 8 and 9, FBS; lanes 10 and 11, FBS after treatment with mAb2 from ascites.

Figure 3

Mass spectrometry identified BoBChE (P32749) in FBS. Peptides colored green were identified with ≥95% confidence. Peptides colored yellow were identified with 50–95% confidence.

Figure 4

Nondenaturing gradient gels stained for cholinesterase activity with ATC or BTC. Human plasma (lanes 1, 2, 8, 9) has an intense band of BChE activity for the C4 tetramer. Minor HuBChE components C1, C2, and C3 are more easily visualized by staining with BTC. FBS has an intense band for BoAChE tetramers in the gel stained with ATC (lanes 3, 4) and a weak AChE band in the gel stained with BTC (lanes 10, 11). BoBChE in FBS (lanes 3, 4, 10, 11) is the band at the top of the gel. The most intense band for rBoBChE is at the top of the 19 gel (lanes 5, 6, 12, 13). Pure HuBChE tetramers (lanes 7, 14) migrate to the same position as BChE tetramers in human plasma.

Figure 5

Spontaneous reactivation of CPO-inhibited rBoBChE.

Figure 6

Fraction of snapshots from the 50 ns MD trajectory with a water molecule found within 3.5A of the phosphorus atom from the diethoxyphosphate adduct of S198, for different mutants of HuBChE. The triple mutants allow water to have greater access to the phosphorus atom.

Figure 7

Model to explain spontaneous reactivation of BoBChE inhibited by chlorpyrifos oxon. The model is based on the crystal structure of diethoxyphosphorylated HuBChE (1XLW). Carbon atoms in the mutated residues G117S/P285L/F398I are light blue. Carbon atoms in native HuBChE residues are green. The CPO (diethoxyphosphate) bound serine 198 and a water molecule are shown as balls and sticks. Residue F398 in HuBChE is shown as a semi-transparent surface. Residue I398 in BoBChE creates space for a water molecule near the phosphorus molecule (orange). Mutation P285L increases water access to the phosphorus atom because L285 in BoBChE, but not P285 in HuBChE, points away from the active site Ser 198. The orientation of L285 is stabilized by interaction with Y332 and F357. Residues Y332 and F357 are the same in HuBChE and BoBChE. The structure was drawn with PyMol.

Figure 8

Models of the human and bovine BChE tetramers. Panels A and B are top and side views of the human BChE tetramer, showing the location of a short polyproline peptide within the C-terminal tetramerization domain (Reproduced from Pan Y, Muzyka JL, Zhan CG. J Phys Chem 2009; 113, 6543-52). Panels C and D are top and side views of the bovine BChE tetramer showing the location of a large polyproline-rich protein within the tetramerization domain and extending beyond the plane of the 4 subunits. This model was developed for the collagen-tailed AChE tetramer by Dvir et al. (Reproduced from Dvir H, Harel M, Bon S, Liu WQ, Vidal M, Garbay C, Sussman JL, Massoulie J, Silman I. EMBO J 2004; 23, 4394–405).