Butyrylcholinesterase as anti-organophosphate intoxication agent: Studies on tetramer assembly and aging mechanisms

Butyrylcholinesterase (BChE) catalytic subunits assemble into tetramers in the neuromuscular junctions and central nervous system through interaction with the conserved proline-rich attachment domain of acetylcholinesterase-associated collagen tail proteins and proline-rich membrane anchor proteins. 98% of the human plasma BChE is tetramer. Tetramerization is critical to the stability of BChE in the circulation, which in turn is an important factor in developing BChE into an anti-organophosphate intoxication therapeutic. We hypothesized that a poly-proline peptide is an intrinsic component of soluble plasma BChE tetramers. We found that a series of proline-rich peptides was released from denatured human and horse plasma BChE. Eight peptides, with masses from 2072 to 2878, were purified by HPLC and sequenced by mass spectrometry and Edman degradation. All peptides derived from the same proline-rich core sequence but varied in length at their N- and C-termini. The source of these peptides was identified through database searching as Ras-associated and pleckstrin homology domainscontaining protein 1 (lamellipodin, gi:82581557). We propose that the proline-rich peptides organize the 4 subunits of BChE into a 340 kDa tetramer, by interacting with the C-terminal BChE tetramerization domain.;Organophosphorus compounds (OP) are toxic because they inhibit acetylcholinesterase (ACNE) by phosphylation of the active site serine, forming a stable conjugate. The conjugate can undergo an aging process which leads to a permanently inhibited enzyme. Current knowledge on aging mechanisms was drawn mostly from studies on the OP inhibited AChE. Recently studies have shown that there may be alternative aging pathways for certain OP inhibited BChE. We systematically investigated the aging mechanism of human BChE inhibited by a variety of OP, including dichlorvos, echothiophate, DFP, soman, sarin, cyclohexyl sarin, VX, VR, isomalathion and tabun. Inhibited BChE was aged in the presence of H2O 18 to allow incorporation of O18 into the aged OP-enzyme conjugates. The aged products were analyzed subsequently using mass spectrometry. We found that aging of all of the alkoxy-adducts followed the classical pathway, i.e. the O-C bond scission, whereas tabun inhibited BChE aged through a novel pathway never before described: that is two N-C bond scissions, followed by a spontaneous (3-elimination reaction. The unique tabun-aged product is expected to be useful for diagnosing exposure.