Detection of Circulating and Endothelial Cell Polymers of Z and Wild Type α1-Antitrypsin by a Monoclonal Antibody

Abstract

Globular inclusions of abnormal ␣1-antitrypsin (AAT)in the endoplasmic reticulum of hepatocytes are a characteristic feature of AAT deficiency of the PiZZ phenotype. Monoclonal antibodies, which contain constant specificity and affinity, are often used for the identification of Z-mutation carriers. A mouse monoclonal antibody (ATZ11) raised against PiZZ hepatocytic AAT was successfully used in enzyme-linked immunosorbent assays (ELISA) and in identification of Z-related AAT globular inclusions by immunohistochemical techniques. Using electrophoresis, Western blotting, and ELISA procedures, we have shown in the present study that this monoclonal antibody specifically detects a conformationdependent neoepitope on both polymerized and elastase-complexed molecular forms of AAT. The antibody has no apparent affinity for native, latent, or cleaved forms of AAT. The antibody ATZ11 illustrates the structural resemblance between the polymerized form of AAT and its complex with elastase and provides evidence that Z-homozygotes beyond the native form may have at least one more circulating molecular form of AAT, i.e. its polymerized form. In addition, staining of endothelial cells with ATZ11 antibody in both M-and Z-AAT individuals shows that AAT attached to endothelial cells is in a polymerized form. The antibody can be a powerful tool for the study of the molecular profile of AAT, not only in Z-deficiency cases but also in other (patho)physiological conditions.The capacity to undergo conformational changes is crucial for the physiological function of many proteins; the serine proteinase inhibitors (serpins) 1 are a clear case for such changes having been exploited during evolution as a means of modulating inhibitory activity (1). Serpins possess two structural elements that are conformationally labile and are essential for efficient proteinase inhibition: a reactive center loop and a ␤-sheet (␤-sheet A) that is able to open and accommodate the reactive center loop after its cleavage by the attacking proteases (2-5). The x-ray crystal structures of native, cleaved, cleaved in complex with protease, and polymerized forms of serpins have confirmed the abilities of these proteins to undergo profound conformational changes under certain environmental conditions (6 -8). The crystal structure of the ␣1-antitrypsin-trypsin complex showed complete insertion of the reactive site loop, which substantially increases thermal and conformational stability of the serpin and results in the irreversible inhibition and then the structural destruction of the proteinase (9).The conformational changes that occur in the serpin molecule during the formation of the enzyme-serpin complexes, interaction with other molecules, polymerization, cleavage, and oxidation lead to the generation of conformation-dependent neoepitopes (10). Immunochemical analysis by means of monoclonal antibodies is a useful approach for study of the formation of new epitopes that occur as a result of the conformational polymorphism of serpins (11,12). For example, the sugge...

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

“…Moreover, previous studies have shown that the antibody does not detect a linear peptide containing the Glu342Lys substitution. 6 Our previous work has shown that the Z mutant favors the formation of polymers by perturbing the relationship between strand 5 of ␤-sheet A and the reactive center loop. The mutation causes a partial insertion of the reactive center loop into ␤-sheet A (Fig.…”

“…Recent investigations have shown that the ATZ11 monoclonal antibody detects the polymeric and enzyme-complexed conformers of ␣ 1 -antitrypsin. 6 However, little is known about its ability to bind to polymers of ␣ 1 -antitrypsin formed by different linkages (i.e., via interaction with ␤-sheet A or C) or in association with different mutations. We therefore investigated the cause of the difference in immunostaining between inclusions formed by the Z, Siiyama, or Mmalton variants by characterizing the binding of the monoclonal antibody to polymers prepared under defined conditions in vitro.…”

“…Immunohistochemistry was performed according to the standard peroxidase-antiperoxidase method 25 with a commercial polyclonal anti-␣ 1 -antitrypsin antibody (1:5000; Dako, Denmark) or the monoclonal ATZ11 (1:100) antibody. 6 Sample Preparation. Human plasma M and Z ␣ 1 -antitrypsin were purified as detailed previously 26 or purchased from Calbiochem (Boston, MA), .…”

“…3 The reactive loop/␤-sheet A interaction is crucial for ␣ 1 -antitrypsin to function as an antiproteinase, but this conformational flexibility can also be detrimental because point mutations facilitate the sequential insertion of the reactive site loop into a ␤-sheet of another molecule, thereby leading to formation of polymers. 1,4,5 It is this polymeric ␣ 1 -antitrypsin that is retained within hepatocytes in association with the severe Z (Glu342Lys), 4,6 Siiyama (Ser53Phe), 7 and Mmalton (52Phe del) 8 deficiency variants. The accumulation of ␣ 1 -antitrypsin as polymers within the endoplasmic reticulum of hepatocytes causes protein overload that is associated with juvenile hepatitis, 9 cirrhosis, and hepatocellular carcinoma.…”

“…22,23 Polymers linked by ␤-sheet C or as strand 7A have been reported in crystal structures, but these were not stable when dissolved in aqueous solution. 17,18,22,23 We have used the monoclonal antibody ATZ11, which specifically recognizes an epitope on polymerized ␣ 1 -antitrypsin, 6 to explore the conformation of polymers of different mutants of ␣ 1 -antitrypsin within hepatocytes. The specificity of the antibody has been characterized using polymers of plasma ␣ 1 -antitrypsin formed under different conditions and polymers of recombinant ␣ 1 -antitrypsin induced by different mutations.…”