Direct Evidence for the Formation of Diastereoisomeric Benzylpenicilloyl Haptens from Benzylpenicillin and Benzylpenicillenic Acid in Patients

Abstract

Covalent binding to proteins to form neoantigens is thought to be central to 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, benzylpenicillenic acid (PA). Mass spectrometric analysis of human serum albumin exposed to BP and PA in vitro revealed that at low concentrations (drug protein molar ratio 0.001:1) and during short time incubations BP and PA selectively target different residues, Lys199 and Lys525, respectively. Molecular modeling showed that the selectivity was a function of noncovalent interaction before 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 diastereoisomeric hapten formation.

Characterization of the Antigen Specificity of T-Cell Clones from Piperacillin-Hypersensitive Patients with Cystic Fibrosis

“…␤-Lactam antibiotics are known to be targeted by specific Lys residues on protein (Levine and Ovary, 1961;Batchelor et al, 1965;Meng et al, 2011). Nucleophilic attack leads to ring opening, binding of the penicilloyl group, and ultimately formation of a stable adduct.…”

“…To identify the key piperacillin-modified lysine residues in albumin involved in the stimulation of T-cell clones, we used our recently described mass spectrometry methods (Meng et al, 2011;Whitaker et al, 2011b). Before mass spectrometry, all samples were incubated with dithiothreitol (10 mM) at room temperature for 15 min and iodoacetamide (166 mM) for another 15 min at room temperature before again being subjected to methanol precipitation.…”

“…It is well established that ␤-lactam antibiotics form covalent bonds with lysine residues on protein (Levine and Ovary, 1961;Batchelor et al, 1965;Jenkins et al, 2009;Meng et al, 2011), and drug-protein binding is thought to be an obligatory step in the instigation of an immune response (Brander et al, 1995;Padovan et al, 1997). Consequently, we have recently developed and used mass spectrometry methods to characterize the amino acid residues on albumin modified with piperacillin in patients (Whitaker et al, 2011a).…”

Amoxicillin in the Aquatic Environment, Its Fate and Environmental Risk

“…The haptenation process occurs through the nucleophilic opening of the β-lactam ring by the attack of free amino groups in proteins, particularly modelling molecular studies found that the most reactive residues towards to amoxicillin is the Lysine, favouring the amoxicilloyl-protein adduct formation, which is able to elicit an immune response [109][110][111][112].…”

Staphylococcal enterotoxin B-derived haptens promote sensitization

“…9,10 Such a process may result in upregulation of the antigenicity of the carriers, or both of the carriers and haptens. A number of haptens have been recognized relating the pathogenesis of allergic diseases including some plant derivatives (such as urushiol 11 ), some drugs (such as penicillin 12 ) and chemicals (such as trinitrobenzene sulfonic acid 13 ). The role of hapten in the animal model of allergic dermatitis has been well recognized, 14 in which haptens fulfill the role of adjuvants.…”