Biosynthesis, processing, and secretion of M and Z variant human alpha 1-antitrypsin

The Z genetic variant of human alpha 1-antitrypsin (alpha 1AT) is associated with decreased serum alpha 1AT levels, hepatic inclusion bodies, and an increased risk of lung and liver disease. We studied the biosynthesis, processing, and secretion of normal and Z variant alpha 1AT in cell-free translation systems, reconstituted in vitro processing systems, and in the Xenopus oocyte secretory system. Human liver mRNA was prepared from normal subjects (PiMM) and from individuals homozygous for alpha 1AT deficiency (PiZZ). Cell-free translation resulted in the synthesis of 49,000-Da preproteins with a 23-amino acid signal sequence. The genetic variants were synthesized at comparable levels and could be distinguished on the basis of charge. The majority of the amino acids in the ZZ signal peptide were identified and found to be the same as those comprising the MM signal sequence. These proteins were co-translationally processed with similar efficiency by dog pancreas microsomes, producing 52,000-Da glycoproteins which were completely translocated across the endoplasmic reticulum membrane. When the human liver RNA preparations were injected into Xenopus oocytes, both of the alpha 1AT variants were synthesized intracellularly and alpha 1AT was detected in the medium of all oocytes injected with MM RNA. However, the Z variant accumulated within the microsomal vesicles of the cell and was undetectable or present at decreased levels in the medium. We conclude that the single amino acid substitution in the Z variant of alpha 1AT does not affect its synthesis or co-translational processing but that it strongly affects its transport from the rough endoplasmic reticulum through the secretory pathway.     

The effect of amino acid substitutions at position 342 on the secretion of human alpha 1-antitrypsin from Xenopus oocytes

A glutamic acid to lysine change in the Z variant of human alpha 1-antitrypsin is associated with a failure to secrete the protein from synthesising cells. The block in export of the protein may be caused either by the loss of an acidic residue or the introduction of a basic one at this point in the polypeptide chain. Site-directed mutagenesis has been used to construct novel alpha 1-antitrypsin mutants which show that the side chain interactions from Glu-342 are not obligatory for protein export and it is rather the introduction of a basic residue at this point which produces the intracellular accumulation of the protein.     

The purification and properties of human alpha1-antitrypsin (alpha1-antiprotease), variant Z.

After three stages of preliminary purification, variant Z was chromatographed on a DEAE-cellulose column. Upon elution with a linearly increasing concentration of NaCl, variant Z was recovered in two separate peaks, the first of which contained 81% and the second 19% of the total. The preparation corresponding to the first peak was homogeneous by various criteria. The trypsin and chymotrypsin inhibiting capacities and the specific antigenic activity of the preparation were nearly the same as those of an authentic sample of variant M. Variant Z contained 8 or 9 more gycine residues than variant M, but no appreciable difference was found between their carbohydrate contents. By analytical isoelectrofocusing the isoinhibitors of purified variant Z overlapped with those in the plasma of the donor and were cathodal to, but partially overlapped with purified variant M. After desialysation, the overlap between the different variants became complete, but variant Z contained a larger proportion of cathodal and smaller proportion of anodal components than variant M. Both variants formed five distinct isoinhibitor-protease complexes after incubation with trypsin and chymotrypsin and the corresponding complexes in the different variants completely coincided.     

The N-terminal amino acid sequence from alpha 1-antitrypsin isolated from liver inclusion bodies

The alpha 1-antitrypsin from the liver of a subject with alpha i-antitrypsin deficiency was purified and subjected to automated Edman degradation. The N-terminal amino acid sequence from position 1 to 12 was identical to that in plasma alpha 1-antitrypsin, type Z. This result precludes that the intrahepatic accumulation of Z alpha 1-antitrypsin is due to a defective removal of a signal peptide.     

Structural and functional characterization of the abnormal Z alpha 1-antitrypsin isolated from human liver

alpha 1-Antitrypsin has been isolated from liver inclusion bodies of a subject with a homozygous Z deficiency. The inhibitor was recovered in a fully active form by extraction in high salt at either pH 2.0 or pH 8.0. Carbohydrate analysis indicated a protein in the 'high mannose' form, and this was collaborated by its sensitivity to endo-beta N-glucosaminidase. These data suggest that the abnormal alpha 1-antitrypsin is blocked in the secretory pathway prior to its entrance into the Golgi, and that this blockage is not due to a gross misfolding of the polypeptide.     

Sequence homology between human alpha 1-antichymotrypsin, alpha 1-antitrypsin, and antithrombin III

alpha 1-Antichymotrypsin mRNA was isolated by specific polysome immunoprecipitation from turpentine-treated baboon liver. The highly enriched mRNA was used for synthesis and cloning of the corresponding cDNA. Baboon alpha 1-antichymotrypsin cDNA clones were identified by hybrid-selected translation, and the insert DNA fragment from one of the putative clones was used as a probe to screen a human liver cDNA library comprised of 40 000 independent transformants. One of the human cDNA clones was unambiguously identified to contain alpha 1-antichymotrypsin DNA sequences by comparison of its 5'-terminal nucleotide sequence with the N-terminal amino acid sequence of the protein. This cDNA clone, designated phACT235, contains 1524 base pairs of human DNA, which was sequenced in its entirety. The inserted DNA codes for a 25 amino acid signal peptide sequence and the entire mature alpha 1-antichymotrypsin of 408 amino acid residues. Comparison of the amino acid sequence of alpha 1-antichymotrypsin with that of the human alpha 1-antitrypsin has revealed a homology level similar to that between chymotrypsin and trypsin.     

Segregation distortion of the alpha 1-antitrypsin Pi Z allele.

alpha 1-antitrypsin (alpha 1AT) of the Pi type Z is associated with two diseases: pulmonary emphysema and cirrhosis of the liver. We report 23 families with both parents heterozygous for the PiZ allele, characterized from our own analysis and from world literature sources. All families were identified through members expressing disease. From the extended pedigrees, 18 backcross families (parents with Pi types MM and MZ) were identified. Analysis of the backcross families reveals a significant increase in Pi MZ offspring (.73) among families where the male is heterozygous. The distortion is not detected among families where the female is heterozygous. Among the matings where both parents are heterozygous, we found 0.43 Pi ZZ from families where one or more members expressed hepatic cirrhosis, and 0.40 Pi ZZ for total families studied. This contrasts to the 0.25 Pi ZZ expected, but is consistent with the distortion observed in backcross matings. The implications of various statistical approaches are discussed, and we point out why our findings differ from previous reports. We suggest a possible biological explanation residing in the fertilization process.     

Complete amino acid sequence of human alpha 1-microglobulin

Complete amino acid sequence of human α₁-microglobulin has been established. It is composed of 167 amino acid residues and contains three carbohydrate attachment sites. No amino acid sequence heterogeneity was found.     

Xenopus oocytes can synthesise but do not secrete the Z variant of human alpha 1-antitrypsin

Human liver mRNA was prepared from a patient homozygous for alpha 1-antitrypsin deficiency (PiZZ) and from a normal subject (PiMM). Both liver RNAs were microinjected into Xenopus oocytes and alpha 1-antitrypsin identified by immunoprecipitation. The normal M variant of alpha 1-antitrypsin is synthesised and secreted by Xenopus oocytes, the abnormal Z protein is not secreted and an intracellular form accumulates in the oocytes. In the presence of tunicamycin an unglycosylated form of M alpha 1-antitrypsin appears in the incubation medium but no corresponding unglycosylated version of the Z protein is secreted.     

Properties of isolated human alpha1-antitrypsins of Pi types M, S and Z.

1. alpha1-Antitrypsin contains a single thiol group partly blocked in native plasma and reactive after mild reduction. 2. Human alpha1-antitrypsins of Pi types F, M, S and Z have been isolated with native microheterogeneity using thiol-disulfide (SH-SS) interchange reactions utilizing the reactive thiol group. 3. The pI of the various microheterogeneous fractions are given for protein M. Stepwise desialylation of alpha1-antitrypsin indicates that the charge difference between the major fractions is one sialic acid residue between each. This is further supported by the pI changes obtained on substitution of the single thiol with positively or negatively charged compounds. 4. Desialyation of purified proteins from each Pi type converts the individual microheterogeneous fractions to one major fraction. The pI shift for the variants studied indicate a difference of plus or minus one or two charge units between protein M and the variants. 5. A difference of one sialic acid residue was obtained for proteins M and Z by the thiobarbituric assay, but stepwise removal of sialic acid with neuraminidase revealed almost identical stepwise change of pattern of both proteins indicating the same number of sialic acid residues. 6. Electrofocusing has been used to identify CNBr fragments from proteins M, S and Z. 7. An amino acid substitution has been found to be located in one of the eight CNBr fragments, glutamic acid in protein M is substituted by lysine in protein Z. 8. The average concentration of alpha1-antityprsin in plasma from healthy males was found to be 1.32 g/1.     

Circular dichroism and conformation of human alpha1-antitrypsin.

The solution conformation of alpha 1-antitrypsin from human blood plasma was studied by the circular dichroism (CD) probe. The CD spectra revealed in this glycoprotein approximately 16-20% of alpha-helix, the rest of the main polypeptide chain possessing the pleated sheet (beta) and the aperiodic structures. The conformation was stable between pH 4.7 and 8.8. Reversible change in conformation was observed at pH 10.3, and more dratic denaturation occurred at pH 11.6. The environment of the side chain chromophores was strongly affected by acid at pH 2.5, whereas the main chain conformation was changed slightly. A drastic change in the CD spectra, indicating denaturation, was observed in 3.5 M guanidine hydrochloride. Sodium dodecyl sulfate was effective in disorganizing the tertiary structure and in enhancing the helix content. The phenylalanine band fine structure was observed in the native protein and also after denaturation with acid, guanidine hydrochloride and sodium dodecyl sulfate.     

Characterization and suppression of dysfunctional human alpha1-antitrypsin variants

Human alpha1-antitrypsin-deficient variants may aggregate in the liver, with subsequent deficiency in the plasma, which can lead to emphysema. The structural and functional characteristics of 10 dysfunctional alpha1-antitrypsin variants (R39C, S53F, V55P, I92N, G115S, N158K, E264V, A336T, P369S, and P369L) were analyzed in detail. Most of them were unstable, as compared to the wild-type molecule, and many of the variants folded into an intermediate form. When five thermostable mutations (T68A, A70G, M374I, S381A, and K387R) were introduced into dysfunctional alpha1-antitrypsin variants, the stabilities and inhibitory activities of most of the variants were restored to levels comparable to those of the wild-type molecule. However, the extremely unstable S53F variant was not stabilized sufficiently by these mutations so as to exhibit function. N158K variant, which carries a mutation in the region critical for the reactive site loop insertion into beta-sheet A, exhibited a reduced level of inhibitory activity, despite conformational stabilization. These results show that aberrant folding caused by conformational destabilization due to mutations can be compensated for by increasing the overall stability of the alpha1-antitrypsin molecule, with exception of a mutation in the highly localized region critical for functional execution.     

The S variant of human alpha 1-antitrypsin, structure and implications for function and metabolism

The S variant of the human alpha 1-antitrypsin with E-264----V, is responsible for a mild alpha 1-antitrypsin deficiency quite common in the European population. S protein specifically cleaved at the susceptible peptide bond was crystallized and its crystal structure determined and refined to 3.1 A resolution. The S variant crystallizes isomorphous to the normal M variant. The difference Fourier electron density map shows the E----V change as outstanding residual density. In addition, small structural changes of the main polypeptide chain radiate from the site of mutation and affect parts far removed from it. By the mutation, internal hydrogen bonds and salt linkages of E-264 to Y-38 and K-487, respectively, are lost. They cause the far-reaching slight distortions and are probably related to the reduced thermal stability of the S mutant. They may also be responsible for slower folding of the polypeptide chain and the clinical symptoms of alpha 1-antitrypsin deficiency. In a theoretical study by molecular dynamics methods simulations of the M and S proteins were made and the results analysed with respect to structural and dynamic properties and compared with the experimental results. There is a significant correlation between experimental and theoretical results in some respects.     

Elevated synthesis of human alpha 1-antitrypsin hinders the secretion of murine alpha 1-antitrypsin from hepatocytes of transgenic mice

alpha 1-Antitrypsin (AAT) is a major hepatic secretory protein. The elevated synthesis of human AAT within hepatocytes of transgenic mice results in its accumulation within a subset of distended cisternae of the rough endoplasmic reticulum. The protein does not accumulate in large insoluble aggregates as is the case for the human PiZ AAT variant. Furthermore, the accumulated protein is not associated with immunoglobulin heavy chain binding protein. Transgenic animals exhibiting an elevated synthesis and subsequent intrahepatic accumulation of human AAT exhibit reduced serum levels of murine AAT as a result of its hindered secretion and accumulation within the rough endoplasmic reticulum. Interestingly, the secretion of murine transferrin and albumin which represent glycosylated and non-glycosylated hepatic secretory proteins, respectively, is unaffected. Overall, these results demonstrate that the elevated synthesis of human AAT can hinder the export of murine AAT from the hepatic rough endoplasmic reticulum in an apparently specific manner.     

Antiprotease targeting: altered specificity of alpha 1-antitrypsin by amino acid replacement at the reactive centre

Alpha-1 antitrypsin (alpha 1AT) is an efficient inhibitor of the human neutrophil proteases, elastase and cathepsin G. The reactive centre P1 residue (Met358) of alpha 1AT is important in defining the specificity of inhibition; furthermore, oxidation of this residue results in a loss of inhibitor activity. There is evidence that oxidative inactivation of alpha 1AT may be involved in the pathogenesis of pulmonary emphysema associated with cigarette smoking. We have studied the effect of a series of amino acid replacements at the active centre on the inhibition properties of alpha 1AT. The mutant proteins were produced in E. coli following in vitro mutagenesis of the alpha 1AT cDNA. Alpha-1-AT (Ile358), (Ala358) and (Val358) were efficient inhibitors of both neutrophil and pancreatic elastase, but not cathepsin G. Alpha-1-AT (Ala356, Val358) and alpha 1AT (Phe358) were specific for pancreatic elastase and cathepsin G respectively. Alpha-1-AT (Leu358) inhibited both neutrophil elastase and cathepsin G. These data show that, for effective inhibition, a potential cleavage site for the protease must be displayed at the alpha 1AT active centre. In each case, replacement of Met358 led to resistance to oxidative inactivation. Since alpha 1AT (Leu358) inhibits both neutrophil proteases and is resistant to oxidation, this variant may be of increased potential for the therapy of destructive lung disorders.     

Hormonal regulation of serum alpha 1-antitrypsin and hepatic alpha 1-antitrypsin mRNA in rats

We evaluated the effects of pituitary dependent hormones on alpha 1-antitrypsin in male rats. Hepatic alpha 1-antitrypsin mRNA was measured by in vitro translation and by specific hybridization with a mouse cDNA alpha 1-antitrypsin probe. Hypophysectomy caused a 50-75% decrease in serum elastase inhibitory capacity (measuring functional alpha 1-antitrypsin) and hepatic alpha 1-antitrypsin mRNA content. In hypophysectomized animals, no increase in elastase inhibitory capacity or alpha 1-antitrypsin mRNA levels by translation was found when met-human growth hormone alone or corticosterone, dihydrotestosterone and thyroxine were given together. Growth hormone increased alpha 1-antitrypsin mRNA by hybridization to a small extent. Addition of growth hormone to the combination of corticosterone, dihydrotestosterone, and thyroxine increased serum elastase inhibitory capacity and alpha 1-antitrypsin mRNA. We conclude that growth hormone acts synergistically with the other pituitary dependent hormones to regulate serum and hepatic mRNA levels of alpha 1-antitrypsin.