Characterising the association of latency with α1-antitrypsin polymerisation using a novel monoclonal antibody

Lu Tan; Juan Perez; Marianna Mela; Elena Miranda; Keith Burling; Farshid N Rouhani; Dawn L Demeo; Imran Haq; James Irving; Adriana Ordóñez; Jennifer Dickens; Mark L Brantly; Stefan J. Marciniak; Graeme Alexander; Bibekbrata Gooptu; David A. Lomas

doi:10.1016/j.biocel.2014.11.005

Characterising the association of latency with α1-antitrypsin polymerisation using a novel monoclonal antibody

Lu Tan, Juan Perez, Marianna Mela, Elena Miranda, Keith Burling, Farshid N Rouhani, Dawn L Demeo, Imran Haq, James Irving, Adriana Ordóñez, Jennifer Dickens, Mark L Brantly, Stefan J. Marciniak, Graeme Alexander, Bibekbrata Gooptu, David A. Lomas

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Abstract

α1-Antitrypsin is primarily synthesised in the liver, circulates to the lung and protects pulmonary tissues from proteolytic damage. The Z mutant (Glu342Lys) undergoes inactivating conformational change and polymerises. Polymers are retained within the hepatocyte endoplasmic reticulum (ER) in homozygous (PiZZ) individuals, predisposing the individuals to hepatic cirrhosis and emphysema. Latency is an analogous process of inactivating, intra-molecular conformational change and may co-occur with polymerisation. However, the relationship between latency and polymerisation remained unexplored in the absence of a suitable probe. We have developed a novel monoclonal antibody specific for latent α1-antitrypsin and used it in combination with a polymer-specific antibody, to assess the association of both conformers in vitro, in disease and during augmentation therapy. In vitro kinetics analysis showed polymerisation dominated the pathway but latency could be promoted by stabilising monomeric α1-antitrypsin. Polymers were extensively produced in hepatocytes and a cell line expressing Z α1-antitrypsin but the latent protein was not detected despite manipulation of the secretory pathway. However, α1-antitrypsin augmentation therapy contains latent α1-antitrypsin, as did the plasma of 63/274 PiZZ individuals treated with augmentation therapy but 0/264 who were not receiving this medication (p < 10−14). We conclude that latent α1-antitrypsin is a by-product of the polymerisation pathway, that the intracellular folding environment is resistant to formation of the latent conformer but that augmentation therapy introduces latent α1-antitrypsin into the circulation. A suite of monoclonal antibodies and methodologies developed in this study can characterise α1-antitrypsin folding and conformational transitions, and screen methods to improve augmentation therapy.

Original language	English
Pages (from-to)	81-91
Number of pages	11
Journal	International Journal of Biochemistry and Cell Biology
Volume	58
DOIs	https://doi.org/10.1016/j.biocel.2014.11.005
Publication status	Published - 1 Jan 2015

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10.1016/j.biocel.2014.11.005

Lu latent+mAb Int J Bioch Cell Biol 2015Final published version, 3.74 MB

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Tan, L., Perez, J., Mela, M., Miranda, E., Burling, K., Rouhani, F. N., Demeo, D. L., Haq, I., Irving, J., Ordóñez, A., Dickens, J., Brantly, M. L., Marciniak, S. J., Alexander, G., Gooptu, B., & Lomas, D. A. (2015). Characterising the association of latency with α1-antitrypsin polymerisation using a novel monoclonal antibody. International Journal of Biochemistry and Cell Biology, 58, 81-91. https://doi.org/10.1016/j.biocel.2014.11.005

@article{8cdd74a319cf4b58a048668fb5901738,

title = "Characterising the association of latency with α1-antitrypsin polymerisation using a novel monoclonal antibody",

abstract = "α1-Antitrypsin is primarily synthesised in the liver, circulates to the lung and protects pulmonary tissues from proteolytic damage. The Z mutant (Glu342Lys) undergoes inactivating conformational change and polymerises. Polymers are retained within the hepatocyte endoplasmic reticulum (ER) in homozygous (PiZZ) individuals, predisposing the individuals to hepatic cirrhosis and emphysema. Latency is an analogous process of inactivating, intra-molecular conformational change and may co-occur with polymerisation. However, the relationship between latency and polymerisation remained unexplored in the absence of a suitable probe. We have developed a novel monoclonal antibody specific for latent α1-antitrypsin and used it in combination with a polymer-specific antibody, to assess the association of both conformers in vitro, in disease and during augmentation therapy. In vitro kinetics analysis showed polymerisation dominated the pathway but latency could be promoted by stabilising monomeric α1-antitrypsin. Polymers were extensively produced in hepatocytes and a cell line expressing Z α1-antitrypsin but the latent protein was not detected despite manipulation of the secretory pathway. However, α1-antitrypsin augmentation therapy contains latent α1-antitrypsin, as did the plasma of 63/274 PiZZ individuals treated with augmentation therapy but 0/264 who were not receiving this medication (p < 10−14). We conclude that latent α1-antitrypsin is a by-product of the polymerisation pathway, that the intracellular folding environment is resistant to formation of the latent conformer but that augmentation therapy introduces latent α1-antitrypsin into the circulation. A suite of monoclonal antibodies and methodologies developed in this study can characterise α1-antitrypsin folding and conformational transitions, and screen methods to improve augmentation therapy.",

author = "Lu Tan and Juan Perez and Marianna Mela and Elena Miranda and Keith Burling and Rouhani, {Farshid N} and Demeo, {Dawn L} and Imran Haq and James Irving and Adriana Ord{\'o}{\~n}ez and Jennifer Dickens and Brantly, {Mark L} and Marciniak, {Stefan J.} and Graeme Alexander and Bibekbrata Gooptu and Lomas, {David A.}",

year = "2015",

month = jan,

day = "1",

doi = "10.1016/j.biocel.2014.11.005",

language = "English",

volume = "58",

pages = "81--91",

journal = "International Journal of Biochemistry and Cell Biology",

issn = "1357-2725",

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Tan, L, Perez, J, Mela, M, Miranda, E, Burling, K, Rouhani, FN, Demeo, DL, Haq, I, Irving, J, Ordóñez, A, Dickens, J, Brantly, ML, Marciniak, SJ, Alexander, G, Gooptu, B & Lomas, DA 2015, 'Characterising the association of latency with α1-antitrypsin polymerisation using a novel monoclonal antibody', International Journal of Biochemistry and Cell Biology, vol. 58, pp. 81-91. https://doi.org/10.1016/j.biocel.2014.11.005

TY - JOUR

T1 - Characterising the association of latency with α1-antitrypsin polymerisation using a novel monoclonal antibody

AU - Tan, Lu

AU - Perez, Juan

AU - Mela, Marianna

AU - Miranda, Elena

AU - Burling, Keith

AU - Rouhani, Farshid N

AU - Demeo, Dawn L

AU - Haq, Imran

AU - Irving, James

AU - Ordóñez, Adriana

AU - Dickens, Jennifer

AU - Brantly, Mark L

AU - Marciniak, Stefan J.

AU - Alexander, Graeme

AU - Gooptu, Bibekbrata

AU - Lomas, David A.

PY - 2015/1/1

Y1 - 2015/1/1

N2 - α1-Antitrypsin is primarily synthesised in the liver, circulates to the lung and protects pulmonary tissues from proteolytic damage. The Z mutant (Glu342Lys) undergoes inactivating conformational change and polymerises. Polymers are retained within the hepatocyte endoplasmic reticulum (ER) in homozygous (PiZZ) individuals, predisposing the individuals to hepatic cirrhosis and emphysema. Latency is an analogous process of inactivating, intra-molecular conformational change and may co-occur with polymerisation. However, the relationship between latency and polymerisation remained unexplored in the absence of a suitable probe. We have developed a novel monoclonal antibody specific for latent α1-antitrypsin and used it in combination with a polymer-specific antibody, to assess the association of both conformers in vitro, in disease and during augmentation therapy. In vitro kinetics analysis showed polymerisation dominated the pathway but latency could be promoted by stabilising monomeric α1-antitrypsin. Polymers were extensively produced in hepatocytes and a cell line expressing Z α1-antitrypsin but the latent protein was not detected despite manipulation of the secretory pathway. However, α1-antitrypsin augmentation therapy contains latent α1-antitrypsin, as did the plasma of 63/274 PiZZ individuals treated with augmentation therapy but 0/264 who were not receiving this medication (p < 10−14). We conclude that latent α1-antitrypsin is a by-product of the polymerisation pathway, that the intracellular folding environment is resistant to formation of the latent conformer but that augmentation therapy introduces latent α1-antitrypsin into the circulation. A suite of monoclonal antibodies and methodologies developed in this study can characterise α1-antitrypsin folding and conformational transitions, and screen methods to improve augmentation therapy.

AB - α1-Antitrypsin is primarily synthesised in the liver, circulates to the lung and protects pulmonary tissues from proteolytic damage. The Z mutant (Glu342Lys) undergoes inactivating conformational change and polymerises. Polymers are retained within the hepatocyte endoplasmic reticulum (ER) in homozygous (PiZZ) individuals, predisposing the individuals to hepatic cirrhosis and emphysema. Latency is an analogous process of inactivating, intra-molecular conformational change and may co-occur with polymerisation. However, the relationship between latency and polymerisation remained unexplored in the absence of a suitable probe. We have developed a novel monoclonal antibody specific for latent α1-antitrypsin and used it in combination with a polymer-specific antibody, to assess the association of both conformers in vitro, in disease and during augmentation therapy. In vitro kinetics analysis showed polymerisation dominated the pathway but latency could be promoted by stabilising monomeric α1-antitrypsin. Polymers were extensively produced in hepatocytes and a cell line expressing Z α1-antitrypsin but the latent protein was not detected despite manipulation of the secretory pathway. However, α1-antitrypsin augmentation therapy contains latent α1-antitrypsin, as did the plasma of 63/274 PiZZ individuals treated with augmentation therapy but 0/264 who were not receiving this medication (p < 10−14). We conclude that latent α1-antitrypsin is a by-product of the polymerisation pathway, that the intracellular folding environment is resistant to formation of the latent conformer but that augmentation therapy introduces latent α1-antitrypsin into the circulation. A suite of monoclonal antibodies and methodologies developed in this study can characterise α1-antitrypsin folding and conformational transitions, and screen methods to improve augmentation therapy.

U2 - 10.1016/j.biocel.2014.11.005

DO - 10.1016/j.biocel.2014.11.005

M3 - Article

SN - 1357-2725

VL - 58

SP - 81

EP - 91

JO - International Journal of Biochemistry and Cell Biology

JF - International Journal of Biochemistry and Cell Biology

ER -

Characterising the association of latency with α1-antitrypsin polymerisation using a novel monoclonal antibody

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