title: Interaction with the 5D3 monoclonal antibody is regulated by intramolecular rearrangements but not by covalent dimer formation of the human ABCG2 multidrug transporter.
abstract: Human ABCG2 is a plasma membrane glycoprotein working as a homodimer or homo-oligomer. The protein plays an important role in the protection/detoxification of various tissues and may also be responsible for the multidrug-resistant phenotype of cancer cells. In our previous study we found that the 5D3 monoclonal antibody shows a function-dependent reactivity to an extracellular epitope of the ABCG2 transporter. In the current experiments we have further characterized the 5D3-ABCG2 interaction. The effect of chemical cross-linking and the modulation of extracellular S-S bridges on the transporter function and 5D3 reactivity of ABCG2 were investigated in depth. We found that several protein cross-linkers greatly increased 5D3 labeling in ABCG2 expressing HEK cells; however, there was no correlation between covalent dimer formation, the inhibition of transport activity, and the increase in 5D3 binding. Dithiothreitol treatment, which reduced the extracellular S-S bridge-forming cysteines of ABCG2, had no effect on transport function but caused a significant decrease in 5D3 binding. When analyzing ABCG2 mutants carrying Cys-to-Ala changes in the extracellular loop, we found that the mutant C603A (lacking the intermolecular S-S bond) showed comparable transport activity and 5D3 reactivity to the wild-type ABCG2. However, disruption of the intramolecular S-S bridge (in C592A, C608A, or C592A/C608A mutants) in this loop abolished 5D3 binding, whereas the function of the protein was preserved. Based on these results and ab initio folding simulations, we propose a model for the large extracellular loop of the ABCG2 protein.
authors.affiliation: Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University and National Blood Center, 1113 Budapest, Hungary.
authors.forename: Csilla
authors.initials: C
authors.lastname: Ozvegy-Laczka
authors.forename: Rozália
authors.initials: R
authors.lastname: Laczkó
authors.forename: Csilla
authors.initials: C
authors.lastname: Hegedus
authors.forename: Thomas
authors.initials: T
authors.lastname: Litman
authors.forename: György
authors.initials: G
authors.lastname: Várady
authors.forename: Katalin
authors.initials: K
authors.lastname: Goda
authors.forename: Tamás
authors.initials: T
authors.lastname: Hegedus
authors.forename: Nikolay V
authors.initials: NV
authors.lastname: Dokholyan
authors.forename: Brian P
authors.initials: BP
authors.lastname: Sorrentino
authors.forename: András
authors.initials: A
authors.lastname: Váradi
authors.forename: Balázs
authors.initials: B
authors.lastname: Sarkadi
mesh_terms: D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004229:Dithiothreitol / Q000737:chemistry; D000939:Epitopes / Q000737:chemistry; D005557:Formaldehyde / Q000494:pharmacology; D006801:Humans; D026901:Membrane Transport Proteins / Q000737:chemistry; D008954:Models, Biological; D009154:Mutation; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000378:metabolism*; D011108:Polymers / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding
chemical_list: C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D000939:Epitopes; D026901:Membrane Transport Proteins; D009363:Neoplasm Proteins; D011108:Polymers; D005557:Formaldehyde; D003545:Cysteine; D004229:Dithiothreitol; C003043:paraform