TY - JOUR

T1 - The cysteine residue in beta-lactoglobulin reacts with oxidized tyrosine residues in beta-casein to give casein-lactoglobulin dimers

AU - Doblas, Laura

AU - Hägglund, Per M.

AU - Fuentes-Lemus, Eduardo

AU - Davies, Michael J.

N1 - Publisher Copyright: © 2022 The Authors

PY - 2023

Y1 - 2023

N2 - Proteins are modified during milk processing and storage, with sidechain oxidation and crosslinking being major consequences. Despite the prevalence and importance of proteins in milk, and particularly caseins (∼80% of total content), the nature of the cross-links formed by oxidation, and their mechanisms of formation, are poorly characterized. In this study, we investigated the formation and stability of cross-links generated by the nucleophilic addition of Cys residues to quinones generated on oxidation of Tyr residues. The mechanisms and stability of these adducts was explored using ubiquitin as a model protein, and β-casein. Ubiquitin and β-casein were oxidized using a rose Bengal/visible light/O2 system, or by the enzyme tyrosinase. The oxidized proteins were incubated with glutathione or β-lactoglobulin (non-oxidized, but unfolded by treatment at 70 °C), before analysis by SDS-PAGE, immunoblotting and LC-MS. Our data indicate that Cys-quinone adducts are readily-formed, and are stable for >48 h. Thus, oxidized β-casein reacts efficiently with the thermally unfolded β-lactoglobulin, likely via Michael addition of the exposed Cys to a Tyr-derived quinone. These data provide a novel, and possibly general, mechanism of protein cross-link formation, and provides information of the stability of these species that have potential as markers of protein quality.

AB - Proteins are modified during milk processing and storage, with sidechain oxidation and crosslinking being major consequences. Despite the prevalence and importance of proteins in milk, and particularly caseins (∼80% of total content), the nature of the cross-links formed by oxidation, and their mechanisms of formation, are poorly characterized. In this study, we investigated the formation and stability of cross-links generated by the nucleophilic addition of Cys residues to quinones generated on oxidation of Tyr residues. The mechanisms and stability of these adducts was explored using ubiquitin as a model protein, and β-casein. Ubiquitin and β-casein were oxidized using a rose Bengal/visible light/O2 system, or by the enzyme tyrosinase. The oxidized proteins were incubated with glutathione or β-lactoglobulin (non-oxidized, but unfolded by treatment at 70 °C), before analysis by SDS-PAGE, immunoblotting and LC-MS. Our data indicate that Cys-quinone adducts are readily-formed, and are stable for >48 h. Thus, oxidized β-casein reacts efficiently with the thermally unfolded β-lactoglobulin, likely via Michael addition of the exposed Cys to a Tyr-derived quinone. These data provide a novel, and possibly general, mechanism of protein cross-link formation, and provides information of the stability of these species that have potential as markers of protein quality.

KW - Beta-casein

KW - Beta-lactoglobulin

KW - Photosensitization

KW - Protein cross-linking

KW - Quinone

KW - Thiol

UR - http://www.scopus.com/inward/record.url?scp=85142823545&partnerID=8YFLogxK

U2 - 10.1016/j.abb.2022.109482

DO - 10.1016/j.abb.2022.109482

M3 - Journal article

C2 - 36457258

AN - SCOPUS:85142823545

VL - 733

JO - Archives of Biochemistry and Biophysics

JF - Archives of Biochemistry and Biophysics

SN - 0003-9861

M1 - 109482

ER -