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Proteomic analysis of the cellular response to a potent sensitiser unveils the dynamics of haptenation in living cells

Proteomic analysis of the cellular response to a potent sensitiser unveils the dynamics of haptenation in living cells
Proteomic analysis of the cellular response to a potent sensitiser unveils the dynamics of haptenation in living cells

Haptenation of model nucleophiles, representing the key MIE in skin sensitisation, is routinely measured in chemico to provide data for skin allergy risk assessment. Better understanding of the dynamics of haptenation in human skin could provide the metrics required to improve determination of sensitiser potency for risk assessment of chemicals. We have previously demonstrated the applicability and sensitivity of the dual stable isotope labelling approach to detect low level haptenation in complex mixtures of proteins. In the present study, we investigated haptenation in a relevant living cell model over time at a subtoxic concentration. DNCB, an extremely potent sensitiser, caused minimal changes in overall protein differential expression in HaCaT cells and haptenated approximately 0.25 % of all available nucleophiles when applied at a subtoxic concentration (10μM) for 4 h. The data shows that the maximum level of haptenation occurs at 2 h and that DNCB, whilst being a promiscuous hapten, shows a preference for Cys residues, despite the considerably higher concentration of amine-based nucleophiles. Although a proportion of highly abundant proteins were haptenated, numerous haptenated sites were also detected on low abundant proteins. Certain proteins were modified at residues buried deep inside the protein structure which are less accessible to haptenation compared with surface exposed nucleophiles. The microenvironment of the buried residues may be a result of several factors influencing the reactivity of both the target nucleophile and the hapten.

Allergic contact dermatitis, Cellular response, DNCB, Haptenation, Proteomics, Sensitisation
0300-483X
152603
Parkinson, Erika
b7294dcc-43d3-46c4-bd19-7f6795b80fe6
Aleksic, Maja
152a8f09-84c2-4ebb-94c7-79c31edf53aa
Kukic, Predrag
17ed0bcc-ac30-4dc2-8eff-47f9e8cdaf0c
Bailey, Alistair
19a50642-79ab-4760-b367-fafd3fccdf01
Cubberley, Richard
e436b046-0d24-4503-bc78-8122f3bf14a8
Skipp, Paul
1ba7dcf6-9fe7-4b5c-a9d0-e32ed7f42aa5
Parkinson, Erika
b7294dcc-43d3-46c4-bd19-7f6795b80fe6
Aleksic, Maja
152a8f09-84c2-4ebb-94c7-79c31edf53aa
Kukic, Predrag
17ed0bcc-ac30-4dc2-8eff-47f9e8cdaf0c
Bailey, Alistair
19a50642-79ab-4760-b367-fafd3fccdf01
Cubberley, Richard
e436b046-0d24-4503-bc78-8122f3bf14a8
Skipp, Paul
1ba7dcf6-9fe7-4b5c-a9d0-e32ed7f42aa5

Parkinson, Erika, Aleksic, Maja, Kukic, Predrag, Bailey, Alistair, Cubberley, Richard and Skipp, Paul (2020) Proteomic analysis of the cellular response to a potent sensitiser unveils the dynamics of haptenation in living cells. Toxicology, 445, 152603, [152603]. (doi:10.1016/j.tox.2020.152603).

Record type: Article

Abstract

Haptenation of model nucleophiles, representing the key MIE in skin sensitisation, is routinely measured in chemico to provide data for skin allergy risk assessment. Better understanding of the dynamics of haptenation in human skin could provide the metrics required to improve determination of sensitiser potency for risk assessment of chemicals. We have previously demonstrated the applicability and sensitivity of the dual stable isotope labelling approach to detect low level haptenation in complex mixtures of proteins. In the present study, we investigated haptenation in a relevant living cell model over time at a subtoxic concentration. DNCB, an extremely potent sensitiser, caused minimal changes in overall protein differential expression in HaCaT cells and haptenated approximately 0.25 % of all available nucleophiles when applied at a subtoxic concentration (10μM) for 4 h. The data shows that the maximum level of haptenation occurs at 2 h and that DNCB, whilst being a promiscuous hapten, shows a preference for Cys residues, despite the considerably higher concentration of amine-based nucleophiles. Although a proportion of highly abundant proteins were haptenated, numerous haptenated sites were also detected on low abundant proteins. Certain proteins were modified at residues buried deep inside the protein structure which are less accessible to haptenation compared with surface exposed nucleophiles. The microenvironment of the buried residues may be a result of several factors influencing the reactivity of both the target nucleophile and the hapten.

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Accepted/In Press date: 23 September 2020
e-pub ahead of print date: 28 September 2020
Published date: 1 December 2020
Keywords: Allergic contact dermatitis, Cellular response, DNCB, Haptenation, Proteomics, Sensitisation

Identifiers

Local EPrints ID: 444672
URI: http://eprints.soton.ac.uk/id/eprint/444672
ISSN: 0300-483X
PURE UUID: b416749e-2f6c-459b-8f62-6c8be3f7d875
ORCID for Paul Skipp: ORCID iD orcid.org/0000-0002-2995-2959

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Date deposited: 29 Oct 2020 17:31
Last modified: 18 Feb 2021 16:39

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