Protein Haptenation and Its Role in Allergy

Abstract

Humans are exposed to numerous electrophilic chemicals either as medicines, in the workplace, in nature, or through use of many common cosmetic and household products. Covalent modification of human proteins by such chemicals, or protein haptenation, is a common occurrence in cells and may result in generation of antigenic species, leading to development of hypersensitivity reactions. Ranging in severity of symptoms from local cutaneous reactions and rhinitis to potentially life-threatening anaphylaxis and severe hypersensitivity reactions such as Stephen-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN), all these reactions have the same Molecular Initiating Event (MIE), i.e. haptenation. However, not all individuals who are exposed to electrophilic chemicals develop symptoms of hypersensitivity. In the present review, we examine common chemistry behind the haptenation reactions leading to formation of neoantigens. We explore simple reactions involving single molecule additions to a nucleophilic side chain of proteins and complex reactions involving multiple electrophilic centers on a single molecule or involving more than one electrophilic molecule as well as the generation of reactive molecules from the interaction with cellular detoxification mechanisms. Besides generation of antigenic species and enabling activation of the immune system, we explore additional events which result directly from the presence of electrophilic chemicals in cells, including activation of key defense mechanisms and immediate consequences of those reactions, and explore their potential effects. We discuss the factors that work in concert with haptenation leading to the development of hypersensitivity reactions and those that may act to prevent it from developing. We also review the potential harnessing of the specificity of haptenation in the design of potent covalent therapeutic inhibitors.

Figure 1

Simple protein haptenation results in a single electrophilic molecule covalently attached to a single nucleophilic amino acid in a protein. (A) DNCB covalently binds to proteins through an S_NAr reaction. (B) Haptenation of select HaCaT cell line proteins by DNCB. Image Nexu Science Communication.

Figure 2

Mechanistic classification of skin sensitizers.

Figure 3

Examples of complex adducts formed via multistep haptenation reactions. (A) Complex reactions of formaldehyde with nucleophilic side chains of amino acids (reproduced from Metz, B.; Kersten, G. F.; Baart, G. J.; de Jong, A.; Meiring, H.; ten Hove, J.; van Steenbergen, M. J.; Hennink, W. E.; Crommelin, D. J.; Jiskoot, W. Identification of formaldehyde-induced modifications in proteins: reactions with insulin. Bioconjug Chem 2006, 17 (3), 815–822. DOI: 10.1021/bc050340f. Copyright [2006] American Chemical Society). (B) Clavulanic acid forms multiple adducts through direct nucleophilic addition to the beta-lactam ring, its degradation products formed under hydrolysis, and cross-linking through multiple reactive intermediates (reproduced from Meng, X.; Earnshaw, C. J.; Tailor, A.; Jenkins, R. E.; Waddington, J. C.; Whitaker, P.; French, N. S.; Naisbitt, D. J.; Park, B. K. Amoxicillin and Clavulanate Form Chemically and Immunologically Distinct Multiple Haptenic Structures in Patients. Chem Res Toxicol 2016, 29 (10), 1762–1772. DOI: 10.1021/acs.chemrestox.6b00253. Copyright [2016] American Chemical Society). (C) Sulfamethoxazole nitroso metabolite forms an arylazoalkane adduct with lysine residues on proteins, which are not stable and can undergo hydrolysis under acidic conditions or high temperatures, generating an allysine intermediate. Further reaction between sulfamethoxazole and allysine resulted in a Schiff bas adduct (reproduced from Tailor, A.; Waddington, J. C.; Hamlett, J.; Maggs, J.; Kafu, L.; Farrell, J.; Dear, G. J.; Whitaker, P.; Naisbitt, D. J.; Park, K.; et al. Definition of Haptens Derived from Sulfamethoxazole: In Vitro and in Vivo. Chem Res Toxicol 2019, 32 (10), 2095–2106. DOI: 10.1021/acs.chemrestox.9b00282. Copyright [2019] American Chemical Society).

Figure 4

Examples of complex cross-link adducts formed via multistep haptenation reactions. (A) CAD MS/MS spectrum of the [M+3H]³⁺ 514.3 ion, derived from a tryptic digest of the 21-mer (VLSPADKTNWGHEYRMFCQIG) peptide incubated with cinnamaldehyde (*-modified residues). Inset - structures of cinnamaldehyde adducts derived from CAD MS/MS data of [M+3H]³⁺ 514.3, indicating that cinnamaldehyde can react initially via Schiff base formation on one N-terminus followed by Michael addition with the second N-terminus, or first via Michael addition with one N-terminus followed by Schiff base formation with the second N-terminus. It is equally possible that the Cys thiol of peptide fragment MFCQIG may be involved in the peptide fragments cross-linking. (B) KHK sequence on cytochrome c (reproduced from Person, M. D.; Monks, T. J.; Lau, S. S. An Integrated Approach To Identifying Chemically Induced Posttranslational Modifications Using Comparative MALDI-MS and Targeted HPLC-ESI-MS/MS. Chem Res Toxicol 2003, 16, 598–608. DOI: 10.1021/tx020109f. Copyright [2003] American Chemical Society). (C) Reaction of (glutathione-S-yl)-benzoquinone adjacent Lys residues on cytochrome c (reproduced from Person, M. D.; Mason, D. E.; Liebler, D. C.; Monks, T. J.; Lau, S. S. Alkylation of cytochrome C by (glutathion-S-yl)-1,4-benzoquinone and iodoacetamide demonstrates compound-dependent site specificity. Chem Res Toxicol 2005, 18 (1), 41–50. DOI: 10.1021/tx049873n. Copyright [2005] American Chemical Society).

Figure 5

Examples of generation of reactive species in interactions with GSH. (A) 5-Chloro-2-methylisothiazol-3-one (MCI) interacts with GSH to form additional amide and thioamide adducts with His and Lys residues on proteins (reproduced from Alvarez-Sanchez, R.; Divkovic, M.; Basketter, D. A.; Pease, C.; Panico, M.; Dell, A.; Morris, H.; Lepoittevin, J. P. Effect of Glutathione on the Covalent Binding of the (13)C-Labeled Skin Sensitizer 5-Chloro-2-methylisothiazol-3-one to Human Serum Albumin: Identification of Adducts by Nuclear Magnetic Resonance, Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry, and Nanoelectrospray Tandem Mass Spectrometry. Chem Res Toxicol 2004, 17 (9), 1280–1288. DOI: 10.1021/tx049935+. Copyright [2004] American Chemical Society). (B) Phenyl isothiacyanate (PITC) reacts with GSH to generate reactive intermediates which further conjugate with amine based nucleophiles of proteins (reproduced from Nakamura, T.; Kawai, Y.; Kitamoto, N.; Osawa, T.; Kato, Y. Covalent modification of lysine residues by allyl isothiocyanate in physiological conditions: plausible transformation of isothiocyanate from thiol to amine. Chem Res Toxicol 2009, 22 (3), 536–542. DOI: 10.1021/tx8003906. Copyright [2009] American Chemical Society). (C) Haptenation by 2-bromo-2-(bromomethyl)glutaronitrile (MDBGN) proceeds via an SN2 reaction as well as being mediated by a sulfhydryl intermediate of MDBGN (reproduced from Ndreu, L.; Erber, L. N.; Törnqvist, M.; Tretyakova, N. Y.; Karlsson, I. Characterizing Adduct Formation of Electrophilic Skin Allergens with Human Serum Albumin and Hemoglobin. Chem Res Toxicol 2020, 33 (10), 2623–2636. DOI: 10.1021/acs.chemrestox.0c00271. Copyright [2020] American Chemical Society). (D) Omeprazole-GSH conjugation plays a double role in both activation and detoxification through the formation of disulfides MDBGN (reproduced from Shin, J. M.; Cho, Y. M.; Sachs, G. Chemistry of covalent inhibition of the gastric (H+, K+)-ATPase by proton pump inhibitors. J Am Chem Soc 2004, 126 (25), 7800–7811. DOI: 10.1021/ja049607w. Copyright [2004] American Chemical Society).

Figure 6

Haptenation through reactive phase II metabolites. (A) Nevirapine haptenated proteins in skin through a reactive sulfate metabolite formed in skin. Proteins (reproduced from Sharma, A. M.; Novalen, M.; Tanino, T.; Uetrecht, J. P. 12-OH-nevirapine sulfate, formed in the skin, is responsible for nevirapine-induced skin rash. Chem Res Toxicol 2013, 26 (5), 817–827. DOI: 10.1021/tx400098z. Copyright [2013] American Chemical Society). (B) Acyl glucluronide metabolites can react with proteins through two distinct pathways: direct transacylation of lysine, cysteine, or arginine residues on proteins and a Schiff base formation where the glucuronide moiety is retained (reproduced from Kenny, J. R.; Maggs, J. L.; Meng, X.; Sinnott, D.; Clarke, S. E.; Park, B. K.; Stachulski, A. V. Syntheses and characterization of the acyl glucuronide and hydroxy metabolites of diclofenac. J Med Chem 2004, 47 (11), 2816–2825. DOI: 10.1021/jm030891w. Copyright [2013] American Chemical Society).

Figure 7

Assays for studying haptenation. (A)In chemico reactivity assays use a large molar excess of the chemical to model nucleophile to screen haptenation. (B) ¹H NMR and HR MAS NMR have been used for mechanistic studies on haptenation with model nucleophiles and complex biological samples. (C) LC-MS/MS has been used to identify protein targets and levels of hapenation.

Figure 8

The fate of haptenation. Haptenation occurs when reactive chemicals or drugs are not cleared by tier 1 detoxification (GSH conjugation and phase II metabolism). The haptenated proteins can be cleared through tier 2 detoxification (I). Permanent haptenation may lead to intrinsic toxicity through alteration of protein function, induction of ROS, or generation of DAMPs (II). Alternatively, haptenation can generate neoantigens presented as haptenated peptides by HLA molecules on the surface of antigen presenting cells, leading to activation of hapten-specific T cells. With imbalanced tolerance, activation of hapten-specific T cells can result in hapten-induced immunotoxicity (III). However, controlled and intentional haptenation may result in new modalities for targeting undruggable proteins (IV).