Covalent binding to proteins to form neoantigens is thought to be central in the pathogenesis of penicillin hypersensitivity reactions. We have undertaken detailed mass spectrometric studies to define the mechanism and protein chemistry of hapten formation from benzylpenicillin (BP) and its rearrangement product, benzyl penicillenic acid (PA). Mass spectrometric analysis of human serum albumin exposed to BP and its rearrangement product, PA in vitro revealed that at low concentrations (drug protein molar ratio 0.001:1) and short time incubation, BP and PA selectively target different residues, lys199 and lys525 respectively. Molecular modelling showed that the selectivity was a function of non-covalent interaction prior to covalent modification. With increased exposure to higher concentrations of BP and PA, multiple epitopes were detected on albumin, demonstrating that the multiplicity of hapten formation is a function of time and concentration. More importantly, we have demonstrated direct evidence that PA is a hapten accounting for the diastereoisomeric BP antigen formation in albumin isolated from the blood of patients receiving penicillin. Furthermore, PA was found to be more potent than BP with respect to stimulation of T cells from patients with penicillin hypersensitivity, illustrating the functional relevance of disastereoisomeric hapten formation.
- Received May 12, 2011.
- Revision received June 9, 2011.
- Accepted June 13, 2011.
- The American Society for Pharmacology and Experimental Therapeutics