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A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity.

Ordóñez A, Pérez J, Tan L, Dickens JA, Motamedi-Shad N, Irving JA, Haq I, Ekeowa U, Marciniak SJ, Miranda E, Lomas DA - FASEB J. (2015)

Bottom Line: The expression of scFv4B12 within the ER (scFv4B12KDEL) and along the secretory pathway (scFv4B12) reduced the intracellular polymerization of Z α1-antitrypsin by 60%.MAb4B12 recognized a discontinuous epitope probably located in the region of helices A/C/G/H/I and seems to act by altering protein dynamics rather than binding preferentially to the native state.This novel approach could reveal new target sites for small-molecule intervention that may block the transition to aberrant polymers without compromising the inhibitory activity of Z α1-antitrypsin.

View Article: PubMed Central - PubMed

Affiliation: *Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, Cambridge, United Kingdom; Department of Cell Biology, Genetics and Physiology, University of Malaga, Malaga, Spain; Wolfson Institute for Biomedical Research, University College London, London, United Kingdom; and Department of Biology and Biotechnologies, "Charles Darwin," and Pasteur Institute, Cenci Bolognetti Foundation, Sapienza University, Rome, Italy.

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Production and characterization of polymerization-blocking mAbs to Z α1-antitrypsin. A) Typical curves obtained with purified Z α1-antitrypsin monomer (Zm) and polymer (Zp; 0.2 mg/ml at 60°C in PBS, pH 7.4, for 3 hours) in a sandwich ELISA (2C1-Ag-9C5-HRP). MAb2C1 recognizes only polymers and mAb9C5 recognizes all conformers of α1-antitrypsin. B) Conditions tested to establish an assay for identification of mAbs able to block heat-induced polymerization. C) Purified mAbs (50 μg/ml) assessed in the polymerization assay shown in B. D) Competitive ELISA against the detection antibody (mAb9C5-HRP).
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Figure 1: Production and characterization of polymerization-blocking mAbs to Z α1-antitrypsin. A) Typical curves obtained with purified Z α1-antitrypsin monomer (Zm) and polymer (Zp; 0.2 mg/ml at 60°C in PBS, pH 7.4, for 3 hours) in a sandwich ELISA (2C1-Ag-9C5-HRP). MAb2C1 recognizes only polymers and mAb9C5 recognizes all conformers of α1-antitrypsin. B) Conditions tested to establish an assay for identification of mAbs able to block heat-induced polymerization. C) Purified mAbs (50 μg/ml) assessed in the polymerization assay shown in B. D) Competitive ELISA against the detection antibody (mAb9C5-HRP).

Mentions: Three cell fusions were performed as described previously (19), and the resulting hybridoma cells (∼2500 wells) were screened for the presence of antibodies against Z α1-antitrypsin by antigen-mediated ELISA. Seventy positive wells were subjected to a secondary screening for mAbs able to modify the polymerization of Z α1-antitrypsin. Polymer formation was quantified by sandwich ELISA using our 2C1 polymer-specific mAb (20) (Fig. 1A). The ability of candidate mAbs to block heat-induced polymerization was assessed by incubating Z α1-antitrypsin at concentrations similar to that expected for antibodies in a hybridoma supernatant (10–200 µg/ml) and temperatures that could drive polymerization while allowing antibody-antigen interaction (40–50°C). The final assay was performed using 20 µg/ml Z α1-antitrypsin, heated at 45°C for 45 hours (Fig. 1B). The fetal bovine serum contained in the media supernatant interfered with our assay by reducing polymerization (data not shown), and therefore each candidate antibody was purified and tested as a pure IgG reagent (Fig. 1C). Most mAbs did not modify Z α1-antitrypsin polymerization to any relevant extent. One of them caused an increase in polymer signal (5E3), 3 of them caused a mild decrease in polymer signal (5E4, 1D6, and 8E2), and 2 of them caused a strong decrease in polymer signal (4B12 and 3C4). These 2 antibodies were assessed in a competitive ELISA against mAb9C5-HRP to ensure that the reduction in signal was not caused by interference of the candidate mAb with the detection antibody. MAb3C4 competed the binding of 9C5-HRP to preformed Z α1-antitrypsin polymers (Fig. 1D), so it was discarded from our studies, whereas mAb4B12 showed no interference with polymer detection.


A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity.

Ordóñez A, Pérez J, Tan L, Dickens JA, Motamedi-Shad N, Irving JA, Haq I, Ekeowa U, Marciniak SJ, Miranda E, Lomas DA - FASEB J. (2015)

Production and characterization of polymerization-blocking mAbs to Z α1-antitrypsin. A) Typical curves obtained with purified Z α1-antitrypsin monomer (Zm) and polymer (Zp; 0.2 mg/ml at 60°C in PBS, pH 7.4, for 3 hours) in a sandwich ELISA (2C1-Ag-9C5-HRP). MAb2C1 recognizes only polymers and mAb9C5 recognizes all conformers of α1-antitrypsin. B) Conditions tested to establish an assay for identification of mAbs able to block heat-induced polymerization. C) Purified mAbs (50 μg/ml) assessed in the polymerization assay shown in B. D) Competitive ELISA against the detection antibody (mAb9C5-HRP).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4548814&req=5

Figure 1: Production and characterization of polymerization-blocking mAbs to Z α1-antitrypsin. A) Typical curves obtained with purified Z α1-antitrypsin monomer (Zm) and polymer (Zp; 0.2 mg/ml at 60°C in PBS, pH 7.4, for 3 hours) in a sandwich ELISA (2C1-Ag-9C5-HRP). MAb2C1 recognizes only polymers and mAb9C5 recognizes all conformers of α1-antitrypsin. B) Conditions tested to establish an assay for identification of mAbs able to block heat-induced polymerization. C) Purified mAbs (50 μg/ml) assessed in the polymerization assay shown in B. D) Competitive ELISA against the detection antibody (mAb9C5-HRP).
Mentions: Three cell fusions were performed as described previously (19), and the resulting hybridoma cells (∼2500 wells) were screened for the presence of antibodies against Z α1-antitrypsin by antigen-mediated ELISA. Seventy positive wells were subjected to a secondary screening for mAbs able to modify the polymerization of Z α1-antitrypsin. Polymer formation was quantified by sandwich ELISA using our 2C1 polymer-specific mAb (20) (Fig. 1A). The ability of candidate mAbs to block heat-induced polymerization was assessed by incubating Z α1-antitrypsin at concentrations similar to that expected for antibodies in a hybridoma supernatant (10–200 µg/ml) and temperatures that could drive polymerization while allowing antibody-antigen interaction (40–50°C). The final assay was performed using 20 µg/ml Z α1-antitrypsin, heated at 45°C for 45 hours (Fig. 1B). The fetal bovine serum contained in the media supernatant interfered with our assay by reducing polymerization (data not shown), and therefore each candidate antibody was purified and tested as a pure IgG reagent (Fig. 1C). Most mAbs did not modify Z α1-antitrypsin polymerization to any relevant extent. One of them caused an increase in polymer signal (5E3), 3 of them caused a mild decrease in polymer signal (5E4, 1D6, and 8E2), and 2 of them caused a strong decrease in polymer signal (4B12 and 3C4). These 2 antibodies were assessed in a competitive ELISA against mAb9C5-HRP to ensure that the reduction in signal was not caused by interference of the candidate mAb with the detection antibody. MAb3C4 competed the binding of 9C5-HRP to preformed Z α1-antitrypsin polymers (Fig. 1D), so it was discarded from our studies, whereas mAb4B12 showed no interference with polymer detection.

Bottom Line: The expression of scFv4B12 within the ER (scFv4B12KDEL) and along the secretory pathway (scFv4B12) reduced the intracellular polymerization of Z α1-antitrypsin by 60%.MAb4B12 recognized a discontinuous epitope probably located in the region of helices A/C/G/H/I and seems to act by altering protein dynamics rather than binding preferentially to the native state.This novel approach could reveal new target sites for small-molecule intervention that may block the transition to aberrant polymers without compromising the inhibitory activity of Z α1-antitrypsin.

View Article: PubMed Central - PubMed

Affiliation: *Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, Cambridge, United Kingdom; Department of Cell Biology, Genetics and Physiology, University of Malaga, Malaga, Spain; Wolfson Institute for Biomedical Research, University College London, London, United Kingdom; and Department of Biology and Biotechnologies, "Charles Darwin," and Pasteur Institute, Cenci Bolognetti Foundation, Sapienza University, Rome, Italy.

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Related in: MedlinePlus