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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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Intrabodies are efficiently expressed within the ER of COS-7 cells. A) Cells were transfected (Tx) with Z α1-antitrypsin or each intrabody: scFv4B12KDEL, scFv4B12, or scFv9C5KDEL for 24 hours and pulse-labeled with [35S]-Met/Cys for 20 minutes. Total α1-antitrypsin and intrabody proteins were immunoprecipitated from the cell lysates, separated by SDS-PAGE, and subjected to autoradiography. MAb2C1 was also used to quantify polymer formation. The asterisk indicates a nonspecific band. B) Protein expression levels represented as means ± sem; Mann-Whitney test, n = 3; ns, nonsignificant. C) Confocal microscopy of fixed cells transfected with scFv4B12KDEL or scFv9C5KDEL in combination with the KDEL-GFP plasmid as an ER marker and stained with an anti-myc antibody to visualize the intrabodies.
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Figure 4: Intrabodies are efficiently expressed within the ER of COS-7 cells. A) Cells were transfected (Tx) with Z α1-antitrypsin or each intrabody: scFv4B12KDEL, scFv4B12, or scFv9C5KDEL for 24 hours and pulse-labeled with [35S]-Met/Cys for 20 minutes. Total α1-antitrypsin and intrabody proteins were immunoprecipitated from the cell lysates, separated by SDS-PAGE, and subjected to autoradiography. MAb2C1 was also used to quantify polymer formation. The asterisk indicates a nonspecific band. B) Protein expression levels represented as means ± sem; Mann-Whitney test, n = 3; ns, nonsignificant. C) Confocal microscopy of fixed cells transfected with scFv4B12KDEL or scFv9C5KDEL in combination with the KDEL-GFP plasmid as an ER marker and stained with an anti-myc antibody to visualize the intrabodies.

Mentions: We have previously shown that polymers of Z α1-antitrypsin accumulate within the ER of COS-7 cells, reproducing the secretory defects seen in patients (20). Hence, we decided to use this model system to evaluate the correct expression of the different intrabodies. COS-7 cells were transfected with Z α1-antitrypsin or each intrabody and after 24 hours were pulse-labeled with [35S]-Met/Cyst. Protein expression levels were determined by immunoprecipitation with either an antibody against all conformers of α1-antitrypsin or against the myc-tag for the intrabodies (Fig. 4A). As expected, a 52 kDa band was detected for α1-antitrypsin (lane 2), and bands of ∼32 kDa were detected in cells expressing scFv4B12KDEL, scFv4B12, and scFv9C5KDEL (lanes 3–5). The slightly faster migration of scFv4B12 and scFv9C5KDEL compared with scFv4B12KDEL (Fig. 4A; lanes 4 and 5 vs. lane 3) was in agreement with their predicted molecular weights. To ensure correct quantification, cell lysates were subjected to a second round of immunoprecipitation. There were similar expression levels for all proteins when total radioactivity was corrected for the number of Cys/Met residues contained in Z α1-antitrypsin (13 Met/Cys) and in the intrabodies (all of them with 9 Met/Cys; Fig. 4B). As a control, we evaluated the presence of polymers in the cell lysates; no polymers were detected at the short time of the pulse (lane 1), as reported previously for polymerogenic mutants of the neuronal serpin neuroserpin (31). Immunofluorescence staining revealed a reticular distribution pattern for the intrabodies that colocalized with the ER as detected with an anti-KDEL antibody (Fig. 4C). These results demonstrate that all the intrabodies were efficiently expressed in COS-7 cells at protein levels comparable to α1-antitrypsin and that they were successfully driven into the ER.


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)

Intrabodies are efficiently expressed within the ER of COS-7 cells. A) Cells were transfected (Tx) with Z α1-antitrypsin or each intrabody: scFv4B12KDEL, scFv4B12, or scFv9C5KDEL for 24 hours and pulse-labeled with [35S]-Met/Cys for 20 minutes. Total α1-antitrypsin and intrabody proteins were immunoprecipitated from the cell lysates, separated by SDS-PAGE, and subjected to autoradiography. MAb2C1 was also used to quantify polymer formation. The asterisk indicates a nonspecific band. B) Protein expression levels represented as means ± sem; Mann-Whitney test, n = 3; ns, nonsignificant. C) Confocal microscopy of fixed cells transfected with scFv4B12KDEL or scFv9C5KDEL in combination with the KDEL-GFP plasmid as an ER marker and stained with an anti-myc antibody to visualize the intrabodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Intrabodies are efficiently expressed within the ER of COS-7 cells. A) Cells were transfected (Tx) with Z α1-antitrypsin or each intrabody: scFv4B12KDEL, scFv4B12, or scFv9C5KDEL for 24 hours and pulse-labeled with [35S]-Met/Cys for 20 minutes. Total α1-antitrypsin and intrabody proteins were immunoprecipitated from the cell lysates, separated by SDS-PAGE, and subjected to autoradiography. MAb2C1 was also used to quantify polymer formation. The asterisk indicates a nonspecific band. B) Protein expression levels represented as means ± sem; Mann-Whitney test, n = 3; ns, nonsignificant. C) Confocal microscopy of fixed cells transfected with scFv4B12KDEL or scFv9C5KDEL in combination with the KDEL-GFP plasmid as an ER marker and stained with an anti-myc antibody to visualize the intrabodies.
Mentions: We have previously shown that polymers of Z α1-antitrypsin accumulate within the ER of COS-7 cells, reproducing the secretory defects seen in patients (20). Hence, we decided to use this model system to evaluate the correct expression of the different intrabodies. COS-7 cells were transfected with Z α1-antitrypsin or each intrabody and after 24 hours were pulse-labeled with [35S]-Met/Cyst. Protein expression levels were determined by immunoprecipitation with either an antibody against all conformers of α1-antitrypsin or against the myc-tag for the intrabodies (Fig. 4A). As expected, a 52 kDa band was detected for α1-antitrypsin (lane 2), and bands of ∼32 kDa were detected in cells expressing scFv4B12KDEL, scFv4B12, and scFv9C5KDEL (lanes 3–5). The slightly faster migration of scFv4B12 and scFv9C5KDEL compared with scFv4B12KDEL (Fig. 4A; lanes 4 and 5 vs. lane 3) was in agreement with their predicted molecular weights. To ensure correct quantification, cell lysates were subjected to a second round of immunoprecipitation. There were similar expression levels for all proteins when total radioactivity was corrected for the number of Cys/Met residues contained in Z α1-antitrypsin (13 Met/Cys) and in the intrabodies (all of them with 9 Met/Cys; Fig. 4B). As a control, we evaluated the presence of polymers in the cell lysates; no polymers were detected at the short time of the pulse (lane 1), as reported previously for polymerogenic mutants of the neuronal serpin neuroserpin (31). Immunofluorescence staining revealed a reticular distribution pattern for the intrabodies that colocalized with the ER as detected with an anti-KDEL antibody (Fig. 4C). These results demonstrate that all the intrabodies were efficiently expressed in COS-7 cells at protein levels comparable to α1-antitrypsin and that they were successfully driven into the ER.

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