A novel monoclonal antibody to characterize pathogenic polymers in liver disease associated with α₁-antitrypsin deficiency

Abstract

Alpha 1 -antitrypsin is the most abundant circulating protease inhibitor. The severe Z deficiency allele (Glu342Lys) causes the protein to undergo a conformational transition and form ordered polymers that are retained within hepatocytes. This causes neonatal hepatitis, cirrhosis, and hepatocellular carcinoma. We have developed a conformation-specific monoclonal antibody (2C1) that recognizes the pathological polymers formed by a 1 -antitrypsin. This antibody was used to characterize the Z variant and a novel shutter domain mutant (His334Asp; a 1 -antitrypsin King's) identified in a 6-week-old boy who presented with prolonged jaundice. His334Asp a 1 -antitrypsin rapidly forms polymers that accumulate within the endoplasmic reticulum and show delayed secretion when compared to the wild-type M a 1 -antitrypsin. The 2C1 antibody recognizes polymers formed by Z and His334Asp a 1 -antitrypsin despite the mutations directing their effects on different parts of the protein. This antibody also recognized polymers formed by the Siiyama (Ser53Phe) and Brescia (Gly225Arg) mutants, which also mediate their effects on the shutter region of a 1 -antitrypsin. Conclusion: Z and shutter domain mutants of a 1 -antitrypsin form polymers with a shared epitope and so are likely to have a similar structure. (HEPATOLOGY 2010;52:1078-1088 T he serpinopathies are conformational diseases characterized by the polymerization and intracellular retention of members of the serine protease inhibitor or serpin superfamily of proteins.1 The best known is a 1 -antitrypsin deficiency, with the most common severe deficiency allele being the Z mutation (Glu342Lys). This mutation results in the retention of ordered polymers of a 1 -antitrypsin as periodic acid Schiff positive inclusion bodies within the endoplasmic reticulum (ER) of hepatocytes.2 These inclusions predispose the individual homozygous for the Z variant of the a 1 -antitrypsin protease inhibitor (PI*Z) to neonatal hepatitis, cirrhosis, and rarely, hepatocellular carcinoma.3 Deficiency of circulating a 1 -antitrypsin results in early onset panlobular emphysema.

Update on alpha-1 antitrypsin deficiency: New therapies

“…The cause of the disparate findings became clear with our development of the 2C1 monoclonal antibody that recognises polymers from the livers of individuals with α 1 -antitrypsin deficiency [7]. This antibody binds polymers formed by heating monomeric α 1 -antitrypsin, but not those formed by refolding from guanidine and urea [15].…”

“…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. …”

“…Alpha 1 -antitrypsin is the major circulating antiprotease but its key function is regulation of the proteolytic effects of neutrophil elastase within the lung. We showed that the severe Z deficiency mutant of α 1 -antitrypsin is retained within the endoplasmic reticulum (ER) of hepatocytes as ordered polymers that become sequestered in Periodic Acid Schiff-positive, diastase-resistant inclusions [2,7]. We have used biophysical and crystallographic techniques to dissect the pathway of α 1 -antitrypsin polymerisation [8][9][10][11][12].…”

“…Indeed, polymerisation is blocked by peptides that mimic the reactive loop sequence and so compete for binding to the insertion site in β-sheet A [2,16]. We subsequently showed that the same process explains the profound plasma deficiency and hepatic inclusions of 3 other mutants of α 1 -antitrypsin: Siiyama (Ser53Phe) [17], Mmalton (∆Phe52) [18] and King's (His334Asp) [7]. Polymerisation also underlies the deficiency of the mild S (Glu264Val), I (Arg39Cys), Queen's (Lys154Asn) and Baghdad (Ala336Pro) alleles of α 1 -antitrypsin [12,14,19,20] but the rate of polymer formation is much slower in keeping with the absence of liver disease and only mild plasma deficiency.…”

Reactive centre loop mutants of α-1-antitrypsin reveal position-specific effects on intermediate formation along the polymerization pathway

“…While denaturant can induce polymerization at concentrations that favour population of an unfolding intermediate state (I denat ) [6,20], observations indicate that there are multiple pathways favoured depending on the manner in which polymers are formed, and as a result intermediate ensembles represented by I pol and I denat could be structurally distinct [19]. In support of this, it has recently been shown that polymers produced in the presence of denaturant lack an epitope that is expressed on polymer obtained from patient samples [18,21]. Given this diversity it is unsurprising that there are currently three main models for the terminal polymer that differ from one another in fundamental respects ( Figure 1B).…”