title: Characterising the association of latency with α(1)-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.
authors.affiliation: Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, Cambridge, UK; Eawag, Swiss Federal Institute of Aquatic Science and Technology, Dübendorf, Switzerland.
authors.forename: Lu
authors.initials: L
authors.lastname: Tan
authors.affiliation: Department of Cell Biology, Genetics and Physiology, University of Málaga, Málaga, Spain.
authors.forename: Juan
authors.initials: J
authors.lastname: Perez
authors.affiliation: Division of Gastroenterology & Hepatology, University Department of Medicine, Cambridge University Hospitals, Cambridge, UK.
authors.forename: Marianna
authors.initials: M
authors.lastname: Mela
authors.affiliation: Department of Biology and Biotechnologies Charles Darwin and Pasteur Institute-Cenci Bolognetti Foundation-University of Rome La Sapienza, Rome, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Miranda
authors.affiliation: Core Biochemical Assay Laboratory, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK.
authors.forename: Keith A
authors.initials: KA
authors.lastname: Burling
authors.affiliation: Department of Medicine, College of Medicine, University of Florida, Gainesville, FL, USA.
authors.forename: Farshid N
authors.initials: FN
authors.lastname: Rouhani
authors.affiliation: Channing Division of Network Medicine, Department of Medicine, Brigham and Women's Hospital, Boston, MA, USA.
authors.forename: Dawn L
authors.initials: DL
authors.lastname: DeMeo
authors.affiliation: Wolfson Institute for Biomedical Research, University College London, London, UK.
authors.forename: Imran
authors.initials: I
authors.lastname: Haq
authors.affiliation: Wolfson Institute for Biomedical Research, University College London, London, UK.
authors.forename: James A
authors.initials: JA
authors.lastname: Irving
authors.affiliation: Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, Cambridge, UK.
authors.forename: Adriana
authors.initials: A
authors.lastname: Ordóñez
authors.affiliation: Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, Cambridge, UK.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Dickens
authors.affiliation: Department of Medicine, College of Medicine, University of Florida, Gainesville, FL, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Brantly
authors.affiliation: Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, Cambridge, UK.
authors.forename: Stefan J
authors.initials: SJ
authors.lastname: Marciniak
authors.affiliation: Division of Gastroenterology & Hepatology, University Department of Medicine, Cambridge University Hospitals, Cambridge, UK.
authors.forename: Graeme J M
authors.initials: GJ
authors.lastname: Alexander
authors.affiliation: Division of Asthma, Allergy and Lung Biology, King's College London, Guy's Hospital, 5th Floor, Tower Wing, London, UK. Electronic address: bibek.gooptu@kcl.ac.uk.
authors.forename: Bibek
authors.initials: B
authors.lastname: Gooptu
authors.affiliation: Wolfson Institute for Biomedical Research, University College London, London, UK. Electronic address: d.lomas@ucl.ac.uk.
authors.forename: David A
authors.initials: DA
authors.lastname: Lomas
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000378:metabolism; D007700:Kinetics; D058105:Polymerization; D017433:Protein Structure, Secondary; D000515:alpha 1-Antitrypsin / Q000737:chemistry*
chemical_list: D000911:Antibodies, Monoclonal; D000515:alpha 1-Antitrypsin
keywords: Augmentation therapy; Latency; Monoclonal antibodies; Polymerisation; α1-Antitrypsin