Interaction with the 5D3 monoclonal antibody is regulated by intramolecular rearrangements but not by covalent dimer formation of the human ABCG2 multidrug transporter

Csilla Özvegy-Laczka; Rozália Laczkó; Csilla Hegedűs; Thomas Litman; György Várady; Katalin Goda; Tamás Hegedűs; Nikolay V. Dokholyan; Brian P Sorrentino; András Váradi; Balázs Sarkadi

doi:10.1074/jbc.M803230200

Interaction with the 5D3 monoclonal antibody is regulated by intramolecular rearrangements but not by covalent dimer formation of the human ABCG2 multidrug transporter

Csilla Özvegy-Laczka, Rozália Laczkó, Csilla Hegedűs, Thomas Litman, György Várady, Katalin Goda, Tamás Hegedűs, Nikolay V. Dokholyan, Brian P Sorrentino, András Váradi, Balázs Sarkadi

Cell Biology and Physiology

33 Citations (Scopus)

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.

Original language	English
Journal	The Journal of Biological Chemistry
Volume	283
Issue number	38
Pages (from-to)	26059-26070
Number of pages	12
ISSN	0021-9258
DOIs	https://doi.org/10.1074/jbc.M803230200
Publication status	Published - 2008

Keywords

ATP-Binding Cassette Transporters
Antibodies, Monoclonal
Cross-Linking Reagents
Cysteine
Dimerization
Dithiothreitol
Epitopes
Formaldehyde
Humans
Membrane Transport Proteins
Models, Biological
Mutation
Neoplasm Proteins
Polymers
Protein Binding
Protein Conformation
Protein Folding

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1074/jbc.M803230200

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Özvegy-Laczka, C., Laczkó, R., Hegedűs, C., Litman, T., Várady, G., Goda, K., Hegedűs, T., Dokholyan, N. V., Sorrentino, B. P., Váradi, A., & Sarkadi, B. (2008). Interaction with the 5D3 monoclonal antibody is regulated by intramolecular rearrangements but not by covalent dimer formation of the human ABCG2 multidrug transporter. The Journal of Biological Chemistry, 283(38), 26059-26070. https://doi.org/10.1074/jbc.M803230200

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 et al.
In: The Journal of Biological Chemistry, Vol. 283, No. 38, 2008, p. 26059-26070.

Research output: Contribution to journal › Journal article › Research › peer-review

Özvegy-Laczka, C, Laczkó, R, Hegedűs, C, Litman, T, Várady, G, Goda, K, Hegedűs, T, Dokholyan, NV, Sorrentino, BP, Váradi, A & Sarkadi, B 2008, 'Interaction with the 5D3 monoclonal antibody is regulated by intramolecular rearrangements but not by covalent dimer formation of the human ABCG2 multidrug transporter', The Journal of Biological Chemistry, vol. 283, no. 38, pp. 26059-26070. https://doi.org/10.1074/jbc.M803230200

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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.",

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author = "Csilla {\"O}zvegy-Laczka and Roz{\'a}lia Laczk{\'o} and Csilla Heged{\H u}s and Thomas Litman and Gy{\"o}rgy V{\'a}rady and Katalin Goda and Tam{\'a}s Heged{\H u}s and Dokholyan, {Nikolay V.} and Sorrentino, {Brian P} and Andr{\'a}s V{\'a}radi and Bal{\'a}zs Sarkadi",

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doi = "10.1074/jbc.M803230200",

language = "English",

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pages = "26059--26070",

journal = "The Journal of Biological Chemistry",

issn = "0021-9258",

publisher = "American Society for Biochemistry and Molecular Biology, Inc.",

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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

SN - 0021-9258

VL - 283

SP - 26059

EP - 26070

JO - The Journal of Biological Chemistry

JF - The Journal of Biological Chemistry

IS - 38

ER -

Interaction with the 5D3 monoclonal antibody is regulated by intramolecular rearrangements but not by covalent dimer formation of the human ABCG2 multidrug transporter

Abstract

Keywords

UN SDGs

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