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The increased risk of developing emphysema among individuals who smoke cigarettes and who have normal levels of alpha 1-antitrypsin (alpha 1AT) is hypothesized to result from a decrease in the antineutrophil elastase capacity of the lower respiratory tract alpha 1AT of smokers compared with nonsmokers. To evaluate this hypothesis we compared the time-dependent kinetics of the inhibition of neutrophil elastase by lung alpha 1AT from healthy, young cigarette smokers (n = 8) and nonsmokers (n = 12). alpha 1-antitrypsin was purified from lavage fluid using affinity and molecular sieve chromatography, and the association rate constant (k assoc) for neutrophil elastase quantified. The k assoc of smoker plasma alpha 1AT (9.5 +/- 0.5 X 10(6) M-1s-1) was similar to that of nonsmoker plasma (9.3 +/- 0.7 X 10(6) M-1s-1, P greater than 0.5). In marked contrast, the k assoc of smoker lower respiratory tract alpha 1AT was significantly lower than that of nonsmoker alpha 1AT (6.5 +/- 0.4 X 10(6) M-1s-1 vs. 8.1 +/- 0.5 X 10(6) M-1s-1, P less than 0.01). Furthermore, the smoker lower respiratory tract alpha 1AT k assoc was significantly less than that of autologous plasma (P less than 0.01). When considered in the context of the concentration of alpha 1AT in the lower respiratory tract epithelial lining fluid, the inhibition time for neutrophil elastase of smoker lung alpha 1AT was twofold greater than that of nonsmoker lung alpha 1AT (smoker: 0.34 +/- 0.05 s vs. nonsmoker: 0.17 +/- 0.05 s, P less than 0.01). Consequently, for concentrations of alpha 1AT in the lower respiratory tract it takes twice as long for an equivalent amount of neutrophil elastase to be inhibited in the smoker's lung compared with the nonsmoker's lung. These observations support the concept that cigarette smoking is associated with a decrease in the lower respiratory tract neutrophil elastase inhibitory capacity, thus increasing the vulnerability of the lung to elastolytic destruction and thereby increasing the risk for the development of emphysema.

S-type alpha 1-antitrypsin (alpha 1AT) is a deficiency haplotype that differs from the common normal M1 (val213) alpha 1AT haplotype by a single amino acid (glu264 to val264). To evaluate the adequacy of the antineutrophil elastase protection associated with the S homozygous state, alpha 1AT plasma and lung epithelial lining fluid (ELF) levels and antineutrophil elastase function were analyzed in 9 PISS subjects. The plasma alpha 1AT levels of SS subjects were intermediate between that of M1M1 and ZZ subjects (p less than 0.001, all comparisons) and the plasma neutrophil elastase inhibitory capacity paralleled the differences in alpha 1AT concentration (p less than 0.001, all comparisons). The association rate constant for neutrophil elastase of the purified S protein was less than that of the normal molecule (S-type, 7.1 +/- 0.1 X 10(6) M-1 s-1; M1-type, 9.6 +/- 0.2 X 10(6) M-1 s-1; p less than 0.001), but much greater than that for the Z molecule (p less than 0.001). Exposure of the purified S protein to increasing oxidant burdens resulted in a dose-dependent reduction in the ability of the molecule to inhibit neutrophil elastase in a fashion parallel to that of the M1 and Z proteins. Quantification of ELF alpha 1AT levels and antineutrophil elastase capacity demonstrated that the SS ELF parameters were, as in plasma, intermediate between M1 homozygotes and Z homozygotes. Using the association rate constant together with the quantification of ELF alpha 1AT levels, the "in vivo lung inhibition time" was estimated, yielding an assessment of the relative antineutrophil elastase screen of the PISS lower respiratory tract.(ABSTRACT TRUNCATED AT 250 WORDS)

The normal M2 variant of alpha 1-antitrypsin (alpha 1AT) was cloned from a genomic DNA library of an individual homozygous for this allele. Sequencing of all coding exons of the M2 gene revealed it was identical to the common M1(Val213) gene except for two bases (M1(Val213) CGT Arg101, M2 CAT His101; M1(Val213) GAA Glu376 M2 GAC Asp376). Analysis of the sequence of the M1(Val213) and M2 genes around residue 101 revealed the M1 Arg101----M2 His101 caused a loss of the cutting site for the restriction endonuclease RsaI. Using this enzyme, as well as 19-mer oligonucleotides probes centered at residues 101 and 376, evaluation of genomic DNA from 22 M1 alleles and 14 M2 alleles revealed that residue 101 was Arg in all M1 alleles and His in all M2 alleles, while residue 376 was Glu in all M1 alleles and Asp in all M2 alleles. Despite the differences in sequence at two amino acids, the M1(Val213) and M2 proteins function similarly as assessed by quantification of the association rate constant of each for their natural substrate neutrophil elastase. In the context that there are two mutations separating the M1(Val213) and M2 alleles, it is likely that there is another alpha 1AT variant that was an intermediate in the evolution of these genes.

This study reports the entire nucleotide sequence of the protein coding region sequence of the alpha 1-antitrypsin (alpha 1AT) Z gene, a common form of the alpha 1AT gene associated with serum alpha 1AT deficiency. In addition to Glu342 to Lys342 mutation in exon V which has been previously identified by peptide analysis, another point mutation (GTG to GCG in exon III) in the gene sequence predicts a second amino acid substitution (Val213 to Ala213) in the Z protein. This Val213 to Ala213 mutation was confirmed to be a general finding in Z type alpha 1AT gene by evaluating genomic DNA from 40 Z haplotypes using synthetic oligonucleotide gene probes directed toward the mutated exon III sequences in the Z gene. Furthermore, the exon III Val213 to Ala213 mutation eliminates a BstEII restriction endonuclease site in the alpha 1AT Z gene, allowing rapid identification of this Val213 to Ala213 substitution at the genomic DNA level. Surprisingly, when genomic DNA samples from individuals thought to be homozygous for the M1 gene (the most common alpha 1AT normal haplotype) were evaluated with BstEII, 23% of the M1 haplotypes were BstEII site negative, thus identifying a new form of M1 (i.e. M1(Ala213], likely identical to M1 but with an isoelectric focusing "silent" amino acid substitution (Val213 to Ala213). Although the relative importance of the newly identified exon III Val213 to Ala213 mutation to the pathogenesis of the abnormalities associated with the Z gene is not known, it is likely that M1(Ala213) gene represents a common "normal" polymorphism of the alpha 1AT gene that served as an evolutionary intermediate between the M1(Val213) and Z genes.

Current concepts relating to the pathogenesis of emphysema associated with cigarette smoking is that an imbalance exists within the lower respiratory tract between neutrophil elastase and the local anti-neutrophil elastase screen, enabling uninhibited neutrophil elastase to destroy the alveolar structures over time. The possible role of alveolar macrophages in contributing to this imbalance was investigated by evaluating the ability of cigarette smokers' alveolar macrophages to inactivate alpha 1-antitrypsin (alpha 1AT), the major anti-neutrophil elastase of the human lower respiratory tract. In vitro, alveolar macrophages of smokers spontaneously released 2.5-fold more superoxide anion and eightfold more H2O2 than macrophages of nonsmokers (P less than 0.01, both comparisons). Using a model system that reproduced the relative amounts of alveolar macrophages and alpha 1AT found in the epithelial lining fluid of the lower respiratory tract, we observed that smokers' macrophages caused a 60 +/- 5% reduction in the ability of alpha 1AT to inhibit neutrophil elastase. In marked contrast, under the same conditions, nonsmokers' macrophages had no effect upon the anti-neutrophil elastase function of alpha 1AT. Addition of superoxide dismutase, catalase, mannitol, and methionine prevented inactivation of alpha 1AT by smokers' macrophages, implying that the release of oxidants mediated the inactivation of alpha 1AT. In addition, by utilizing a recombinant DNA produced modified form of alpha 1AT containing an active site substitution (met358----val), the inactivation of alpha 1AT by smokers' alveolar macrophages was prevented, suggesting that the smokers' macrophages inactivate alpha 1AT by oxidizing the active site of the alpha 1AT molecule. These results suggest that in cigarette smokers, the alveolar macrophage can modulate the activity of alpha 1AT as an inhibitor of neutrophil elastase and thus play a role in the pathogenesis of emphysema associated with cigarette smoking.

alpha 1-Antitrypsin (alpha 1AT) is a highly pleomorphic 52-kDa serum glycoprotein that functions as the major inhibitor of neutrophil elastase. Of these, the most common normal alpha 1AT haplotypes identified by isoelectric focusing (IEF) of serum are those of the M family, including M1, M2, and M3. In the course of studying the alpha 1AT type Z gene, we identified a restriction endonuclease BstEII polymorphism in the M1 gene that predicted the existence of a previously unidentified, but relatively common, haplotype of M, referred to as M1(Ala213) [Nukiwa, T., Satoh, K., Brantly, M. L., Ogushi, F., Fells, G. A., Courtney, M., & Crystal, R. G. (1986) J. Biol. Chem. 261, 15989-15994]. In this study we have cloned both alpha 1AT genes from an individual heterozygous for the M1(Ala213) and M1(Val213) haplotypes. Sequencing of the coding exons of both demonstrated that they are identical except for the Ala-Val difference at residue 213. The codominant transmission of the M1(Ala213) gene was demonstrated in a family study. Evaluation of 39 genomic samples of Caucasians with the IEF haplotype M1 demonstrated haplotype frequencies of 68% for M1(Val213) and 32% for M1(Ala213). alpha 1AT serum levels of individuals inheriting the M1(Ala213) gene in a homozygous fashion were in the same range as those for homozygous M1(Val213) as was the rate of association of the M1(Ala213) protein with neutrophil elastase.(ABSTRACT TRUNCATED AT 250 WORDS)

Seven active site variants of human alpha 1-antitrypsin (alpha 1AT) were produced in Escherichia coli following site-specific mutagenesis of the alpha 1AT complementary DNA. alpha 1AT (Ala358), alpha 1AT (Ile358) and alpha 1AT (Val358) were efficient inhibitors of both neutrophil and pancreatic elastases, but not of cathepsin G. alpha 1AT (Ala356, Val358) and alpha 1AT (Phe358) specifically inhibited pancreatic elastase and cathepsin G respectively. The most potent inhibitor of neutrophil elastase was alpha 1AT (Leu358), which also proved to be effective against cathepsin G. The alpha 1AT (Arg358) variant inactivated thrombin with kinetics similar to antithrombin III in the presence of heparin. Electrophoretic analysis showed that SDS-stable high mol. wt complexes were formed between the mutant inhibitors and the cognate proteases in each case. These data indicate that effective inhibition occurs when the alpha 1AT P1 residue (position 358) corresponds to the primary specificity of the target protease. Moreover, alteration of the P3 residue (position 356) can further modify the reactivity of the inhibitor. Two of the variants have therapeutic potential: alpha 1AT (Leu358) may be more useful than plasma alpha 1AT in the treatment of destructive lung disorders and alpha 1AT (Arg358) could be effective in the control of thrombosis.

To evaluate the potential use of recombinant DNA-produced alpha-1-antitrypsin (alpha-1-AT) to augment the lung antineutrophil elastase defenses in alpha-1-AT deficiency, we compared the kinetics of intravenously administered recombinant produced alpha-1-AT (r alpha-1-AT) and purified normal human plasma alpha-1-AT (p alpha-1-AT) in the blood and lung of rhesus monkeys. The r alpha-1-AT was produced in yeast transformed with an expressing plasmid containing a full-length human alpha-1-AT complementary deoxyribonucleic acid and purified to greater than 99% homogeneity. The r alpha-1-AT has a molecular weight of 45,000, no carbohydrates, and is identical in sequence to normal plasma alpha-1-AT except for an additional N-terminal acetylmethionine. Despite its lack of carbohydrates, the r alpha-1-AT inhibited human neutrophil elastase with an association rate constant similar to that of p alpha-1-AT. Rhesus monkeys were infused intravenously with 120 mg/kg of r alpha-1-AT (n = 13) or p alpha-1-AT (n = 12) and the serum, urine, and lung epithelial lining fluid (ELF) concentrations of these molecules quantified at various intervals.(ABSTRACT TRUNCATED AT 250 WORDS)