Cresyl saligenin phosphate, an organophosphorus toxicant, makes covalent adducts with histidine, lysine, and tyrosine residues of human serum albumin

Abstract

CBDP [2-(2-cresyl)-4H-1-3-2-benzodioxaphosphorin-2-oxide] is a toxic organophosphorus compound. It is generated in vivo from tri-ortho-cresyl phosphate (TOCP), a component of jet engine oil and hydraulic fluids. Exposure to TOCP was proven to occur on board aircraft by finding CBDP-derived phospho-butyrylcholinesterase in the blood of passengers. Adducts on BChE, however, do not explain the toxicity of CBDP. Critical target proteins of CBDP are yet to be identified. Our goal was to facilitate the search for the critical targets of CBDP by determining the range of amino acid residues capable of reacting with CBDP and characterizing the types of adducts formed. We used human albumin as a model protein. Mass spectral analysis of the tryptic digest of CBDP-treated human albumin revealed adducts on His-67, His-146, His-242, His-247, His-338, Tyr-138, Tyr-140, Lys-199, Lys-351, Lys-414, Lys-432, and Lys-525. Adducts formed on tyrosine residues were different from those formed on histidines and lysines. Tyrosines were organophosphorylated by CBDP, while histidine and lysine residues were alkylated. This is the first report of an organophosphorus compound with both phosphorylating and alkylating properties. The o-hydroxybenzyl adduct on histidine is novel. The ability of CBDP to form stable adducts on histidine, tyrosine, and lysine allows one to consider new mechanisms of toxicity from TOCP exposure.

Figure 1

Identification of His-338 as the adduction site on human serum albumin after reaction with CBDP. Panel A: MALDI mass spectra of tryptic digests of control (upper panel) and CBDP-treated albumin (lower panel). Peptide HPDYSVVLLLR at m/z 1311.7 increased in mass by 106 amu when its histidine was modified by reaction with CBDP. MS spectra were acquired in positive mode at 4000 V with CHCA matrix. The values shown indicate monoisotopic masses. Panel B: LTQ-Orbitrap MS/MS spectrum of the HPDYSVVLLLR parent ion [M+3H]⁺³ at m/z 473.3 showing major y- and b-ions that identify histidine as the adduction site. Ions that retain o-hydroxybenzyl are labeled with an asterisk.

Figure 2

His-67 of human albumin forms concatenated-[2]-o-hydroxybenzyl adduct upon treatment with CBDP. MALDI MS/MS spectrum of parent ion (MH) at m/z 1229.6 corresponds to the peptide SLH*TLFGDK, where the asterisk indicates the position of concatenated-[2]-o-hydroxybenzyl adduct. The peak at m/z 1123.5 is consistent with the neutral loss of o-hydroxybenzyl moiety (MH-106). The peak at m/z 1017.5 is consistent with the neutral loss of concatenated-[2]-o-hydroxybenzyl moiety (MH-212).

Figure 3

Identification of Lys-199 of human albumin as a residue reactive towards CBDP. MS/MS fragmentation of parent ion [M+2H]⁺² at m/z 612.3 in the LTQ-Orbitrap mass spectrometer yielded y- and b-ion series consistent with the peptide LK*C(carbamidomethylated)ASLQK. The asterisk indicates fragments bearing the +276-amu mass due to formation of the ring-opened CBDP-adduct on Lys-199.

Figure 4

Identification of o-cresyl phosphotyrosine adduct formed on Tyr-138 of human serum albumin upon treatment with CBDP. LTQ-Orbitrap MS/MS spectrum of singly charged parent ion at m/z 1097.5 corresponds to peptide Y*LYEIAR with the adduct (+170 amu) on Tyr-138. The peak at m/z 989.5 is consistent with the neutral loss of o-cresol. Ions bearing the adduct are labeled with an asterisk. Neutral loss of the entire adduct from parent ion is shown by symbol Δ.

Figure 5

Solvent accessible amino acid residues of albumin react with CBDP. The crystal structure of human albumin (Protein Data Bank code 1bmo) shows in sticks the 5 histidines, 5 lysines, and 2 tyrosines (plus Tyr-411) that are reactive towards CBDP. The structure was viewed with PyMOL software (DeLano, W.L. The PyMOL Molecular Graphics System (2002) DeLano Scientific, Palo Alto, CA, USA. http://www.pymol.org).

Scheme 1

Reaction of tyrosine residues of human serum albumin with CBDP. The masses are for the neutral charge state.

Scheme 2

Reaction of histidine residues of human serum albumin with CBDP. The masses are for the neutral charge state.