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...
The oxidation of low-density lipoprotein (LDL) is thought to be a major contributor to the development of atherosclerosis and considerable evidence has accumulated showing that oxidized LDL (ox LDL) induces cell damage and pro-atherogenic events. However, evidence that oxidized LDL directly causes atherosclerosis is lacking. We studied whether native and enzymatically or chemically ox LDL at concentrations of 5 and 100 microg/mL is cytotoxic to or promotes pro-atherogenic activation of human primary monocytes in culture. Both types of ox LDL (100 microg/mL), but not native LDL added to monocytes for 24 h significantly diminish DNA synthesis and increase cell death. In addition, both preparations of ox LDL inhibit cytokine and metalloproteinase production, diminish cellular oxygen consumption and induce PPAR gamma expression. Enzymatically ox LDL, but not LDL oxidized by copper sulfate, also increases the monocyte metabolic rate and induces intracellular lipid accumulation. Low concentrations of either preparation of oxidized and native LDL did not show significant effects on all parameters measured. These data establish a direct link between ox LDL concentration and cytotoxicity and suggest that oxidation by copper of the lipid moiety in LDL and of the protein moiety by enzyme creates ox LDL, which can damage monocytes without release of pro-inflammatory molecular species. In contrast to native and enzymatically ox LDL, copper ox LDL does not induce intracellular lipid accumulation. Differently oxidized LDL molecules may exert distinct effects in lesion development in atherosclerosis.
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