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.

An antibody that prevents serpin polymerisation acts by inducing a novel allosteric behaviour

“…However, antibodies have been found that induce another serpin, PAI-1, to adopt an inactive yet monomeric latent conformation [33,34]. In the case of α 1 -AT, the latent conformation is a by-product of the polymerisation pathway that can be induced by heating [60] and augmented by the presence of buffer components that disfavour intermolecular interactions [61]. If the antibody was exerting an analogous effect — permitting conformational change while sterically preventing the association of single subunits — a comparable loss of activity might be expected to occur.…”

“…These polymers have been shown to consist of hyperstable, folded protein in an unbranched arrangement [73] and form through self-association of a polymerisation-prone monomeric species [21,22]. However, there is debate as to whether polymers arise from a near-native conformation in vivo or whether the polymerisation-prone species is a substantially unfolded intermediate on the folding pathway [29,30,44,60]. In this regard, it is notable that a single-chain variant of a monoclonal antibody that prevents polymerisation from native material in vitro is also effective during co-expression of a polymerisation-prone variant of α 1 -AT in a cell model of disease [44].…”

“…Many lines of evidence favour the former: there is a well described absence of the UPR, indicating in turn an absence of an unfolded protein load [7,8,32]; expression in the absence of ER chaperones results in the appearance of atypical small circular oligomeric species [81]; there is a lack of the misfolded latent conformation in ex vivo tissue [60] and the molten globule-like unfolding intermediate [27] results in a polymer that is distinct immunologically [23], and most likely therefore structurally [25]. It is noteworthy that co-expression of a single-chain variant (scFv) of mAb 4B12 results in a reduction in the intracellular polymerisation of Z α 1 -AT by 60% [44].…”

Targeting the site encoded by SERPINA1*E342K for treating alpha‐1 antitrypsin deficiency‐associated liver diseases

“…We measured effects of the top 40 scoring compounds using a hepatocyte cell line derived from PiZ mice, a transgenic line that stably expresses Z‐hAAT. The PiZ hepatocytes were positively stained by PASD (a hallmark of alpha‐1 antitrypsin deficiency) and an antibody raised against Z‐AAT polymer . In addition, the IC Z‐hAAT protein levels were much higher than EC Z‐hAAT protein levels, which further suggest the protein is misfolded and trapped in the cells.…”

Update on alpha-1 antitrypsin deficiency: New therapies

“…This has proved to be powerful technology that has allowed us to identify antibodies that specifically detect the polymeric [7] and latent [92] conformers of α 1 -antitrypsin and antibodies that can accelerate [93] and block [94] Reproduced from [17] with permission. …”