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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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Polymerization blocking activity of the 4B12 monoclonal antibody. A) Nondenaturing PAGE followed by silver staining of 0.02 mg/ml monomeric Z α1-antitrypsin incubated with serial 1:2 dilutions of purified mAb4B12 starting from 1:1 molar ratio (0.063 mg/ml) at 45°C for 60 hours. Right) ELISA quantification of α1-antitrypsin polymers by sandwich ELISA (2C1-Ag-9C5-HRP) as means ± sem, n = 5, each performed in triplicate. B) Quantification of α1-antitrypsin polymers after incubation with purified Fab domain of mAb4B12 in the same conditions as A as means ± sem; n = 5, each performed in triplicate. C) Nondenaturing PAGE followed by silver staining of samples processed as in A but in the presence of nonspecific IgG1. Right) ELISA quantification for α1-antitrypsin polymers (n = 3). m, monomer; p, polymers.
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Figure 2: Polymerization blocking activity of the 4B12 monoclonal antibody. A) Nondenaturing PAGE followed by silver staining of 0.02 mg/ml monomeric Z α1-antitrypsin incubated with serial 1:2 dilutions of purified mAb4B12 starting from 1:1 molar ratio (0.063 mg/ml) at 45°C for 60 hours. Right) ELISA quantification of α1-antitrypsin polymers by sandwich ELISA (2C1-Ag-9C5-HRP) as means ± sem, n = 5, each performed in triplicate. B) Quantification of α1-antitrypsin polymers after incubation with purified Fab domain of mAb4B12 in the same conditions as A as means ± sem; n = 5, each performed in triplicate. C) Nondenaturing PAGE followed by silver staining of samples processed as in A but in the presence of nonspecific IgG1. Right) ELISA quantification for α1-antitrypsin polymers (n = 3). m, monomer; p, polymers.

Mentions: The ability of the purified full-length 4B12 IgG to block polymerization was further assessed by nondenaturing PAGE (Fig. 2A). mAb4B12 completely blocked heat-induced polymerization of Z α1-antitrypsin at a 1:1 molar ratio in vitro (lane 4), and the effect gradually decreased (polymerization increased, lanes 5–8) when the concentration of mAb4B12 was reduced. A specific 2C1 sandwich ELISA was used to quantify this effect with the same results (Fig. 2A, right). Similar data were obtained with the 4B12 Fab region (antigen-binding fragment), particularly at 1:1 molar ratio (Fig. 2B). An isotype-matching mouse IgG control (IgG1) had no effects on polymerization in the same conditions (Fig. 2C), supporting the specific blocking effect of mAb4B12. These results demonstrate that mAb4B12 is able to bind Z α1-antitrypsin and block its heat-induced polymerization at a 1:1 molar ratio in vitro.


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)

Polymerization blocking activity of the 4B12 monoclonal antibody. A) Nondenaturing PAGE followed by silver staining of 0.02 mg/ml monomeric Z α1-antitrypsin incubated with serial 1:2 dilutions of purified mAb4B12 starting from 1:1 molar ratio (0.063 mg/ml) at 45°C for 60 hours. Right) ELISA quantification of α1-antitrypsin polymers by sandwich ELISA (2C1-Ag-9C5-HRP) as means ± sem, n = 5, each performed in triplicate. B) Quantification of α1-antitrypsin polymers after incubation with purified Fab domain of mAb4B12 in the same conditions as A as means ± sem; n = 5, each performed in triplicate. C) Nondenaturing PAGE followed by silver staining of samples processed as in A but in the presence of nonspecific IgG1. Right) ELISA quantification for α1-antitrypsin polymers (n = 3). m, monomer; p, polymers.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Polymerization blocking activity of the 4B12 monoclonal antibody. A) Nondenaturing PAGE followed by silver staining of 0.02 mg/ml monomeric Z α1-antitrypsin incubated with serial 1:2 dilutions of purified mAb4B12 starting from 1:1 molar ratio (0.063 mg/ml) at 45°C for 60 hours. Right) ELISA quantification of α1-antitrypsin polymers by sandwich ELISA (2C1-Ag-9C5-HRP) as means ± sem, n = 5, each performed in triplicate. B) Quantification of α1-antitrypsin polymers after incubation with purified Fab domain of mAb4B12 in the same conditions as A as means ± sem; n = 5, each performed in triplicate. C) Nondenaturing PAGE followed by silver staining of samples processed as in A but in the presence of nonspecific IgG1. Right) ELISA quantification for α1-antitrypsin polymers (n = 3). m, monomer; p, polymers.
Mentions: The ability of the purified full-length 4B12 IgG to block polymerization was further assessed by nondenaturing PAGE (Fig. 2A). mAb4B12 completely blocked heat-induced polymerization of Z α1-antitrypsin at a 1:1 molar ratio in vitro (lane 4), and the effect gradually decreased (polymerization increased, lanes 5–8) when the concentration of mAb4B12 was reduced. A specific 2C1 sandwich ELISA was used to quantify this effect with the same results (Fig. 2A, right). Similar data were obtained with the 4B12 Fab region (antigen-binding fragment), particularly at 1:1 molar ratio (Fig. 2B). An isotype-matching mouse IgG control (IgG1) had no effects on polymerization in the same conditions (Fig. 2C), supporting the specific blocking effect of mAb4B12. These results demonstrate that mAb4B12 is able to bind Z α1-antitrypsin and block its heat-induced polymerization at a 1:1 molar ratio in vitro.

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