MOLECULAR CHARACTERISTICS AND CLINICAL ASPECTS OF ALPHA 1-ANTITRYPSIN DEFICIENCY

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(a)Alpha1-Antitrypsin (alpha1-AT) deficiency is an inherited disorder associated with a profound reduction in the serum alpha1-AT, a major antiprotease of the lower respiratory tract. Oxidants in the lower respiratory tract reduce the function of alpha1-AT by oxidizing methionine residue(s) at its active site. Interaction of alpha1-AT and its major target neutrophil elastase (NE) is dependent on the conformation of the reactive-site loop (RSL) of alpha1-AT. We hypothesized that oxidation of the RSL might alter its stressed conformation and ability to dock and lock with NE. To evaluate this hypothesis, we compared the conformation of wild-type (M) and Zalpha1-AT, and their oxidized counterparts, before and after interaction with NE, using transverse urea gradient gel and SDS-PAGE. (b)Following intravenous administration of alpha1-antitrypsin, there was a significant correlation between the concentrations of functional and antigenic alpha1-AT in both the epithelial lining fluid (ELF) and serum. The predicted maximum concentrations of alpha1-AT in ELF was 2.5 muM. It was calculated that a serum concentration of 11.5 muM alpha1-AT would result in an ELF alpha1-AT concentration of 1.25 muM. The results indicate that serum alpha1-AT concentrations above 20 muM are not associated with further linear increases in ELF alpha1-AT concentrations. (c)Individuals with alpha1-AT deficiency develop severe destructive lung disease much earlier and undergo more rapid decline in lung function than do the general population of individuals with chronic obstructive lung disease. We quantified factors associated with early lung destruction in individuals with alpha1-AT deficiency. (d)The mutant Z form of alpha1-AT accounts for more than 95% of all individuals with alpha1-AT deficiency, an important inherited cause of emphysema and liver disease. The Zalpha1-AT gene encodes a misfolded form of alpha1-AT, which polymerizes, accumulates intracellularly, and is degraded in the rough endoplasmic reticulum (RER) or the pre-Golgi compartment. Using a Chinese Hamster Ovary (CHO) cell line permanently transfected with the Zalpha1-AT gene, we compared the intracellular fate of newly synthesized Zalpha1-AT, radiolabeled with [S35]-methionine in a pulse-chase fashion, in the presence of several inhibitors of intracellular degradation. Cycloheximide and lactacystin prevented intracellular degradation of Zalpha1-AT. N-acetyl-leucinyl-leucinyl-norleucinal (ALLN), which inhibits calpains, proteasomes, and lysosomal cysteine protease, prevented degradation of Zalpha1-AT but did not significantly alter its secretion. Inhibitors of lysosomal proteolysis, including ammonium chloride, chloroquine, leupeptin and colchicine were without effect. In the presence of brefeldin A, cycloheximide and lactacystin did not prevent Zalpha1-AT degradation, however, leupeptin and ALLN increased T1/2 of Zalpha1-AT by 3-and 5-fold, respectively. These observations are consistent with the existence of at least two intracellular pathways for Zalpha1-AT degradation. (e)An Italian individual was identified with asthma, emphysema, and a very low level of serum alpha1-AT. DNA sequencing demonstrated the Mprocida deficiency allele and a novel null allele QOtrastevere (c654 G A, W194Z), a nonsense mutation near the intron 2 (IVS2) splice acceptor site. A Chinese hamster ovary cell line was permanently transfected with QOtrastevere or normal Malpha1-AT with and without IVS2. The nonsense mutation was associated with moderately reduced alpha1-AT mRNA regardless of IVS2, supporting a translational-translocation model as the cause of reduced alpha1-AT mRNA rather than a nuclear scanning model. Pulse-chase studies followed by immunoprecipitation demonstrated an endoplasmic reticulum-retained 31-kDa QOtrastevere alpha1-AT, which was rapidly degraded; retention and degradation appeared to be the major reasons for the absence of secreted alpha1-AT. (f)In smokers, unopposed protease activity may lead to emphysema by unrestricted activity of neutrophil elastase (NE). Alpha1-Antitrypsin inhibitor (alpha1-AT), the main inhibitor of NE, contains critical methionines which, if oxidized, block its anti-NE activity. Hydrogen peroxide is a critical component of cigarette smoke that inactivates alpha1-AT. To establish the molecular basis of oxidative inactivation, we studied the sites oxidized by hydrogen peroxide in alpha1-AT. Using simultaneous Edman sequencing of peptides produced by treatment with cyanogen bromide to identify all methionines in alpha1-AT, it was observed that two of the nine methionines were particularly susceptible to oxidation. One was methionine 358, whose oxidation is known to cause loss of anti-NE activity. The other methionine, 351, was as susceptible to oxidation as methionine 358. Its oxidation also resulted in loss of anti-NE activity. Methionine 226 was less readily oxidized and none of the other six methionines was susceptible to attack by hydrogen peroxide. Under experimental conditions formation of an alpha1-AT-NE complex protected both methionines from oxidation, while methionine 226 was not protected, consistent with methionines 351 and 358 being located at or near the site of inhibitor interaction. The susceptibility to oxidation of all nine methionines correlates well with the extent of their exposure on the surface of the molecule. Oxidative inactivation of alpha1-AT provides a mechanism for regulation of its activity at sites of inflammation.