Differential detection of PAS-positive inclusions formed by the Z, Siiyama, and Mmalton variants of α1-antitrypsin

Abstract

Several point mutations of α1-antitrypsin cause a perturbation in protein structure with consequent polymerization and intracellular accumulation. The retention of polymers of α1- antitrypsin within hepatocytes results in protein overload that in turn is associated with juvenile hepatitis, cirrhosis, and hepatocellular carcinoma. The detection of α1-antitrypsin polymers and understanding the molecular basis of polymer formation is of considerable clinical importance. We have used a monoclonal antibody (ATZ11) that specifically recognizes a conformation-dependent neoepitope on polymerized i-antitrypsin to detect polymers within hepatocytes of individuals with α1-antitrypsin deficiency. Paraffin-embedded liver tissue specimens were obtained from individuals who were homozygous for the Z (Glu342Lys), Mmalton (52Phe del), and Siiyama (Ser53Phe) alleles of α1-antitrypsin that result in hepatic inclusions and profound plasma deficiency. Immunohistological staining with a polyclonal anti-human α1-antitrypsin antibody showed hepatic inclusions in all 3 cases, while ATZ11 reacted with hepatic inclusions formed by only Z -antitrypsin. Polymers of plasma M and Z α1- antitrypsin prepared under different conditions in vitro and polymers of recombinant mutants of α1-antitrypsin demonstrated that the monoclonal antibody detected a neoepitope on the polymerized protein. It did not detect polymers formed by a recombinant shutter domain mutant (that mirrors the effects of the Siiyama and Mmalton variants), polymers formed by cleaving α1-antitrypsin at the reactive loop, or C-sheet polymers formed by heating α1-antitrypsin in citrate. In conclusion, the ATZ11 monoclonal antibody detects Z α1-antitrypsin in hepatic inclusions by detecting a neoepitope that is specific to the polymeric conformer and that is localized close to residue 342.