Differential detection of PAS-positive inclusions formed by the Z, Siiyama, and Mmalton variants of alpha1-antitrypsin

Several point mutations of alpha(1)-antitrypsin cause a perturbation in protein structure with consequent polymerization and intracellular accumulation. The retention of polymers of alpha(1)-antitrypsin within hepatocytes results in protein overload that in turn is associated with juvenile hepatitis, cirrhosis, and hepatocellular carcinoma. The detection of alpha(1)-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 alpha(1)-antitrypsin to detect polymers within hepatocytes of individuals with alpha(1)-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 alpha(1)-antitrypsin that result in hepatic inclusions and profound plasma deficiency. Immunohistological staining with a polyclonal anti-human alpha(1)-antitrypsin antibody showed hepatic inclusions in all 3 cases, while ATZ11 reacted with hepatic inclusions formed by only Z alpha(1)-antitrypsin. Polymers of plasma M and Z alpha(1)-antitrypsin prepared under different conditions in vitro and polymers of recombinant mutants of alpha(1)-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 alpha(1)-antitrypsin at the reactive loop, or C-sheet polymers formed by heating alpha(1)-antitrypsin in citrate. In conclusion, the ATZ11 monoclonal antibody detects Z alpha(1)-antitrypsin in hepatic inclusions by detecting a neoepitope that is specific to the polymeric conformer and that is localized close to residue 342.