Differential detection of PAS‐positive inclusions formed by the Z, Siiyama, and Mmalton variants of α 1 ‐antitrypsin

Several point mutations of α 1 ‐antitrypsin cause a perturbation in protein structure with consequent polymerization and intracellular accumulation. The retention of polymers of α 1 ‐antitrypsin within hepatocytes results in protein overload that in turn is associated with juvenile hepatitis, cirrhosis, and hepatocellular carcinoma. The detection of α 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 α 1 ‐antitrypsin to detect polymers within hepatocytes of individuals with α 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 α 1 ‐antitrypsin that result in hepatic inclusions and profound plasma deficiency. Immunohistological staining with a polyclonal anti‐human α 1 ‐antitrypsin antibody showed hepatic inclusions in all 3 cases, while ATZ11 reacted with hepatic inclusions formed by only Z α 1 ‐antitrypsin. Polymers of plasma M and Z α 1 ‐antitrypsin prepared under different conditions in vitro and polymers of recombinant mutants of α 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 α 1 ‐antitrypsin at the reactive loop, or C‐sheet polymers formed by heating α 1 ‐antitrypsin in citrate. In conclusion , the ATZ11 monoclonal antibody detects Z α 1 ‐antitrypsin in hepatic inclusions by detecting a neoepitope that is specific to the polymeric conformer and that is localized close to residue 342. (H EPATOLOGY 2004;40:1203–1210.)