Chlorpyrifos oxon promotes tubulin aggregation via isopeptide cross-linking between diethoxyphospho-Lys and Glu or Asp: Implications for neurotoxicity.

Exposure to organophosphorus toxicants (OP) can have chronic adverse effects that are not explained by inhibition of acetylcholinesterase, the cause of acute OP toxicity. We therefore hypothesized that OP-induced chronic illness is initiated by the formation of organophosphorus adducts on lysine residues in proteins, followed by protein cross-linking and aggregation. Here, Western blots revealed that exposure to the OP chlorpyrifos oxon converted porcine tubulin from its original 55-kDa mass to high-molecular-weight aggregates. Liquid chromatography-tandem MS analysis of trypsin-digested samples identified several diethoxyphospho-lysine residues in the OP-treated tubulin. Using a search approach based on the Batch Tag program, we identified cross-linked peptides and found that these chemically activated lysines reacted with acidic amino acid residues creating γ-glutamyl-ϵ-lysine or aspartyl-ϵ-lysine isopeptide bonds between β- and α-tubulin. Of note, these cross-linked tubulin molecules accounted for the high-molecular-weight aggregates. To the best of our knowledge, this is the first report indicating that chlorpyrifos oxon-exposed tubulin protein forms intermolecular cross-links with other tubulin molecules, resulting in high-molecular-weight protein aggregates. It is tempting to speculate that chronic illness from OP exposure may be explained by a mechanism that starts with OP adduct formation on protein lysines followed by protein cross-linking. We further speculate that OP-modified or cross-linked tubulin can impair axonal transport, reduce neuron connections, and result in neurotoxicity.