Interaction with the 5D3 monoclonal antibody is regulated by intramolecular rearrangements but not by covalent dimer formation of the human ABCG2 multidrug transporter
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Interaction with the 5D3 monoclonal antibody is regulated by intramolecular rearrangements but not by covalent dimer formation of the human ABCG2 multidrug transporter. / Özvegy-Laczka, Csilla; Laczkó, Rozália; Hegedűs, Csilla; Litman, Thomas; Várady, György; Goda, Katalin; Hegedűs, Tamás; Dokholyan, Nikolay V.; Sorrentino, Brian P; Váradi, András; Sarkadi, Balázs.
I: The Journal of Biological Chemistry, Bind 283, Nr. 38, 2008, s. 26059-26070.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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TY - JOUR
T1 - Interaction with the 5D3 monoclonal antibody is regulated by intramolecular rearrangements but not by covalent dimer formation of the human ABCG2 multidrug transporter
AU - Özvegy-Laczka, Csilla
AU - Laczkó, Rozália
AU - Hegedűs, Csilla
AU - Litman, Thomas
AU - Várady, György
AU - Goda, Katalin
AU - Hegedűs, Tamás
AU - Dokholyan, Nikolay V.
AU - Sorrentino, Brian P
AU - Váradi, András
AU - Sarkadi, Balázs
PY - 2008
Y1 - 2008
N2 - 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.
AB - 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.
KW - ATP-Binding Cassette Transporters
KW - Antibodies, Monoclonal
KW - Cross-Linking Reagents
KW - Cysteine
KW - Dimerization
KW - Dithiothreitol
KW - Epitopes
KW - Formaldehyde
KW - Humans
KW - Membrane Transport Proteins
KW - Models, Biological
KW - Mutation
KW - Neoplasm Proteins
KW - Polymers
KW - Protein Binding
KW - Protein Conformation
KW - Protein Folding
U2 - 10.1074/jbc.M803230200
DO - 10.1074/jbc.M803230200
M3 - Journal article
C2 - 18644784
VL - 283
SP - 26059
EP - 26070
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 38
ER -
ID: 119646095