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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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Construction of the scFv9C5 and scFv4B12 intrabodies. A) Representation of a whole antibody. Antigen specificity is defined by the Fab, composed of one constant (C) and one variable (V) domain of each of the heavy (H) and light (L) chains. The shortest variable-region fragment is called Fv. Schema of a single-chain variable fragment (scFv). CDR1–3 denotes complementarity-determining regions. B) Schema of the intrabody-encoding plasmids: 2 based on the mAb9C5 or mAb4B12 sequences with a myc epitope tag to facilitate detection and an ER retention signal (KDEL) (scFv9C5KDEL and scFv4B12KDEL), and a third construct based on mAb4B12 but without the ER retention signal (scFv4B12). The GFPKDEL vector expressed a nonrelated protein targeted to the ER. ERsp denotes ER signal peptide. C) Representative alignment of the DNA sequences of several clones for identifying the light chain sequence of mAb4B12. The frameworks regions (FR) and complementary regions (CDR) are indicated.
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Figure 3: Construction of the scFv9C5 and scFv4B12 intrabodies. A) Representation of a whole antibody. Antigen specificity is defined by the Fab, composed of one constant (C) and one variable (V) domain of each of the heavy (H) and light (L) chains. The shortest variable-region fragment is called Fv. Schema of a single-chain variable fragment (scFv). CDR1–3 denotes complementarity-determining regions. B) Schema of the intrabody-encoding plasmids: 2 based on the mAb9C5 or mAb4B12 sequences with a myc epitope tag to facilitate detection and an ER retention signal (KDEL) (scFv9C5KDEL and scFv4B12KDEL), and a third construct based on mAb4B12 but without the ER retention signal (scFv4B12). The GFPKDEL vector expressed a nonrelated protein targeted to the ER. ERsp denotes ER signal peptide. C) Representative alignment of the DNA sequences of several clones for identifying the light chain sequence of mAb4B12. The frameworks regions (FR) and complementary regions (CDR) are indicated.

Mentions: The striking polymer-blocking properties of mAb4B12 in vitro encouraged us to evaluate the effect of this antibody in a cell model of disease. To this end, we generated the scFv, composed of the VH and VL domains joined by a flexible linker (Gly4Ser)3 (Fig. 3A, B). Two hybridoma cell lines were used as the source of RNA for scFv design: cells producing mAb4B12 and mAb9C5, an antibody against Z α1-antitrypsin polymers that recognizes all conformers of α1-antitrypsin (20) but that does not block polymer formation, used here as a negative control. The unique cDNA sequence for the variable domains (VH and VL) containing the complementarity-determining regions (hypervariable domains) responsible for antigen binding were identified by comparison of multiple sequenced clones to the mouse IG set from the ImMunoGeneTics information system for V-QUEry and STandardization (IMGT/V-QUEST) reference directory (28) (Fig. 3C). The resulting scFv4B12 and scFv9C5 constructs were sequenced, revealing a 750 bp open reading frame full-length cDNA, encoding a 244 amino acid protein with an estimated molecular weight of 27 kDa (Supplemental Fig. S1).


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)

Construction of the scFv9C5 and scFv4B12 intrabodies. A) Representation of a whole antibody. Antigen specificity is defined by the Fab, composed of one constant (C) and one variable (V) domain of each of the heavy (H) and light (L) chains. The shortest variable-region fragment is called Fv. Schema of a single-chain variable fragment (scFv). CDR1–3 denotes complementarity-determining regions. B) Schema of the intrabody-encoding plasmids: 2 based on the mAb9C5 or mAb4B12 sequences with a myc epitope tag to facilitate detection and an ER retention signal (KDEL) (scFv9C5KDEL and scFv4B12KDEL), and a third construct based on mAb4B12 but without the ER retention signal (scFv4B12). The GFPKDEL vector expressed a nonrelated protein targeted to the ER. ERsp denotes ER signal peptide. C) Representative alignment of the DNA sequences of several clones for identifying the light chain sequence of mAb4B12. The frameworks regions (FR) and complementary regions (CDR) are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Construction of the scFv9C5 and scFv4B12 intrabodies. A) Representation of a whole antibody. Antigen specificity is defined by the Fab, composed of one constant (C) and one variable (V) domain of each of the heavy (H) and light (L) chains. The shortest variable-region fragment is called Fv. Schema of a single-chain variable fragment (scFv). CDR1–3 denotes complementarity-determining regions. B) Schema of the intrabody-encoding plasmids: 2 based on the mAb9C5 or mAb4B12 sequences with a myc epitope tag to facilitate detection and an ER retention signal (KDEL) (scFv9C5KDEL and scFv4B12KDEL), and a third construct based on mAb4B12 but without the ER retention signal (scFv4B12). The GFPKDEL vector expressed a nonrelated protein targeted to the ER. ERsp denotes ER signal peptide. C) Representative alignment of the DNA sequences of several clones for identifying the light chain sequence of mAb4B12. The frameworks regions (FR) and complementary regions (CDR) are indicated.
Mentions: The striking polymer-blocking properties of mAb4B12 in vitro encouraged us to evaluate the effect of this antibody in a cell model of disease. To this end, we generated the scFv, composed of the VH and VL domains joined by a flexible linker (Gly4Ser)3 (Fig. 3A, B). Two hybridoma cell lines were used as the source of RNA for scFv design: cells producing mAb4B12 and mAb9C5, an antibody against Z α1-antitrypsin polymers that recognizes all conformers of α1-antitrypsin (20) but that does not block polymer formation, used here as a negative control. The unique cDNA sequence for the variable domains (VH and VL) containing the complementarity-determining regions (hypervariable domains) responsible for antigen binding were identified by comparison of multiple sequenced clones to the mouse IG set from the ImMunoGeneTics information system for V-QUEry and STandardization (IMGT/V-QUEST) reference directory (28) (Fig. 3C). The resulting scFv4B12 and scFv9C5 constructs were sequenced, revealing a 750 bp open reading frame full-length cDNA, encoding a 244 amino acid protein with an estimated molecular weight of 27 kDa (Supplemental Fig. S1).

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