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.     

Expression of PiM-and PiZ-mutated forms of the human alpha 1-antitrypsin gene in transfected monkey COS1 cells

The PiZ mutation of the gene coding for alpha 1-antitrypsin results in a serum deficiency of this protein leading to early onset emphysema and liver disease. The PiZ gene has a Z-specific point mutation in exon V together with a point mutation in exon III which is also present in some normal (PiM) individuals. There has thus far been no system to study the effects of PiZ point mutations in tissue culture. We constructed plasmids containing alpha 1-antitrypsin cDNA synthetically altered at either exon III or exon V mutation sites and linked to simian virus 40 promoter sequences. Such constructs with the exon V mutation were transfected into monkey COS1 cells followed by analysis of expression of alpha 1-antitrypsin gene products. COS1 cells normally synthesize virtually no alpha 1-antitrypsin mRNA or protein. alpha 1-Antitrypsin mRNA is transcribed at high levels in cells transfected with either M or Z plasmids. Immunologic staining of COS1 cells within 48 h of transfection localizes alpha 1-antitrypsin protein to specific regions of the cytoplasm. This extranuclear localization is also observed with human HepG2 hepatoma cells, which synthesize alpha 1-antitrypsin at high levels, and with human SK-Hep1 hepatoma cells transfected with an M plasmid. The cloned synthetically altered alpha 1-antitrypsin genes provide a system for dissecting contributions of distinct point mutations to the pathological effects of the PiZ protein.     

Regulator of G Signaling 16 is a marker for the distinct endoplasmic reticulum stress state associated with aggregated mutant alpha1-antitrypsin Z in the classical form of alpha1-antitrypsin deficiency

In the classical form of alpha(1)-antitrypsin deficiency, a mutant protein accumulates in a polymerized form in the endoplasmic reticulum (ER) of liver cells causing liver damage and carcinogenesis by a gain-of-toxic function mechanism. Recent studies have indicated that the accumulation of mutant alpha(1)-antitrypsin Z in the ER specifically activates the autophagic response but not the unfolded protein response and that autophagy plays a critical role in disposal of insoluble alpha(1)-antitrypsin Z. In this study, we used genomic analysis of the liver in a novel transgenic mouse model with inducible expression to screen for changes in gene expression that would potentially define how the liver responds to accumulation of this mutant protein. There was no unfolded protein response. Of several distinct gene expression profiles, marked up-regulation of regulator of G signaling (RGS16) was particularly notable. RGS16 did not increase when model systems were exposed to classical inducers of ER stress, including tunicamycin and calcium ionophore, or when a nonpolymerogenic alpha(1)-antitrypsin mutant accumulated in the ER. RGS16 was up-regulated in livers from patients with alpha(1)-antitrypsin deficiency, and the degree of up-regulation correlated with the hepatic levels of insoluble alpha(1)-antitrypsin Z protein. Taken together, these results indicate that expression of RGS16 is an excellent marker for the distinct form of "ER stress" that occurs in alpha(1)-antitrypsin deficiency, presumably determined by the aggregation-prone properties of the mutant protein that characterizes the deficiency.     

Molecular cloning and primary structure of rat alpha 1-antitrypsin

A cDNA clone encoding rat alpha 1-antitrypsin has been isolated from a lambda gt-11 rat liver cDNA library using an antigen-overlay immunoscreening method. The nucleotide sequence of this cDNA clone is 1306 base pairs in length and has a coding region of 1224 base pairs which can be translated into an alpha 1-antitrypsin precursor protein consisting of 408 amino acid residues. The cDNA sequence contains a termination codon, TAA, at position 1162 and a polyadenylation signal sequence, AATAAT, at position 1212. The calculated molecular weight of the translated mature protein is 43,700 with 387 amino acid residues; this differs from purified rat alpha 1-antitrypsin's apparent molecular weight of 54,000 because of glycosylation. Five potential glycosylation sites were identified on the basis of the cDNA sequence. The translated mature protein sequence from the cDNA clone matches completely with the N-terminal 33 amino acids of purified rat alpha 1-antitrypsin, which has an N-terminal Glu. The cDNA encoding rat alpha 1-antitrypsin shares 70% and 80% sequence identity with its human and mouse counterparts, respectively. The reactive center sequence of rat alpha 1-antitrypsin is highly conserved with respect to human alpha 1-antitrypsin, both having Met-Ser at the P1 and P1' residues. Genomic Southern blot analysis yielded a simple banding pattern, suggesting that the rat alpha 1-antitrypsin gene is single-copy. Northern blot analysis using the cDNA probe showed that rat alpha 1-antitrypsin is expressed at high levels in the liver and at low levels in the submandibular gland and the lung.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assignment of the alpha 1-antitrypsin gene and a sequence-related gene to human chromosome 14 by molecular hybridization

alpha 1-Antitrypsin is a major plasma protease inhibitor synthesized in the liver. Genetic deficiency of this protein predisposes the affected individuals to development of infantile liver cirrhosis or chronic obstructive pulmonary emphysema. The human chromosomal alpha 1-antitrypsin gene has been cloned and shown to contain three introns in the peptide-coding region. When the cloned alpha 1-antitrypsin gene was used as a hybridization probe to analyze Eco RI-digested genomic DNA from different individuals, two distinct bands of 9.6 kilobases (kb) and 8.5 kb in length were observed in every case. Further analysis using only labeled intronic DNA as the hybridization probe has indicated that the authentic alpha 1-antitrypsin gene resides within the 9.6-kb fragment. Thus the 8.5-kb fragment must contain another gene that is closely related in sequence to the alpha 1-antitrypsin gene. Using a series of human-Chinese hamster somatic cell hybrids containing unique combinations of human chromosomes, the alpha 1-antitrypsin gene as well as the sequence-related gene have been assigned to human chromosome 14 by Southern hybridization and synteny analysis.     

Retroviral mediated transfer and expression of human alpha 1-antitrypsin in cultured cells

Genetic deficiency of alpha 1-antitrypsin in man is a predisposing factor to emphysema and a disorder potentially correctable by somatic gene therapy. A full-length human alpha 1-antitrypsin cDNA was cloned into a retroviral vector and introduced into cells which package the recombinant gene in a retroviral capsule. Cells infected with the recombinant retrovirus express human alpha 1-antitrypsin mRNA and protein. The recombinant protein is glycosylated, secreted and exhibits anti-protease activity against human neutrophil elastase.     

Developmental expression, cellular localization, and testosterone regulation of alpha 1-antitrypsin in Mus caroli kidney

alpha 1-Antitrypsin (alpha 1-protease inhibitor), an essential plasma protein, is synthesized predominantly in the liver of all mammals. We have previously shown that Mus caroli, a Southeast Asian mouse species is exceptional in that it expresses abundantly alpha 1-antitrypsin mRNA and polypeptide, in the kidney as well as the liver (Berger, F.G., and Baumann, H. (1985) J. Biol. Chem. 260, 1160-1165) providing a unique model for examination of the evolution of genetic determinants of tissue-specific gene expression. In the present paper, we have further characterized alpha 1-antitrypsin expression in M. caroli. The extrahepatic expression of alpha 1-antitrypsin is limited to the kidney, specifically within a subset of the proximal tubule cells. The developmental pattern of alpha 1-antitrypsin mRNA expression in the kidney differs from that in the liver. In the kidney, alpha 1-antitrypsin mRNA is present at only 2-4% adult level at birth and increases very rapidly to adult level during puberty between 26 and 36 days of age. There are no significant changes in liver alpha 1-antitrypsin mRNA levels during this period. Testosterone, while having only modest affects on alpha 1-antitrypsin mRNA accumulation in the adult kidney, causes a 20-fold induction of the mRNA in the pre-pubertal kidney. This suggests that the increase in alpha 1-antitrypsin mRNA expression during puberty is testosterone mediated. Southern blot analyses of Mus domesticus and M. caroli genomic DNA and a cloned M. caroli alpha 1-antitrypsin genomic sequence, indicate that a single alpha 1-antitrypsin gene exists in M. caroli, whereas multiple copies exist in M. domesticus. These data show that the alteration in tissue specificity of alpha 1-antitrypsin mRNA accumulation that has occurred during Mus evolution is associated with distinctive developmental and hormonally regulated expression patterns.     

Aggregation of plasma Z type alpha 1-antitrypsin suggests basic defect for the deficiency

The abnormal type of alpha 1-antitrypsin, PI (protease inhibitor) type Z, is associated with inclusion bodies in the liver, which contain non-secreted alpha 1-antitrypsin. Our studies show that Z protein has an inherent tendency to aggregate, even in plasma. Depending upon conditions, from 15 to 70% of the Z protein in plasma was in a high-Mr form, compared with 1.5% of M type alpha 1-antitrypsin. The high-Mr complex in plasma cannot be disaggregated using Triton X detergent or reducing conditions. This increased tendency to aggregate can be explained by the mutation affecting, tertiary structure and salt bridge formation in Z protein. We have observed this same tendency to aggregate for Mmalton alpha 1-antitrypsin, a rarer variant also associated with a plasma deficiency.     

Divergent expression of alpha1-protease inhibitor genes in mouse and human.

The alpha1-protease inhibitor proteins of laboratory mice are homologous in sequence and function to human alpha1-antitrypsin and are encoded by a highly conserved multigene family comprised of five members. In humans, the inhibitor is expressed in liver and in macrophages and decreased expression or inhibitory activity is associated with a deficiency syndrome which can result in emphysema and liver disease in affected individuals. It has been proposed that macrophage expression may be an important component of the function of human alpha1-antitrypsin. Clearly, it is desirable to develop a mouse model of this deficiency syndrome, however, efforts to do this have been largely unsuccessful. In this paper, we report that aside from the issues of potentially redundant gene function, the mouse may not be a suitable animal for such studies, because there is no significant expression of murine alpha1-protease inhibitor in the macrophages of mice. This difference between the species appears to result from an absence of a functional macrophage-specific promoter in mice.     

Photoreduction and incorporation of iron into ferritins.

The characteristics of a new kallikrein-binding protein in human serum and its activities were studied. Both the kallikrein-binding protein and alpha 1-antitrypsin form 92 kDa SDS-stable and heat-stable complexes with human tissue kallikrein. In non-SDS/PAGE, the mobility of these complexes differ. Complex-formation between kallikrein and the binding protein is inhibited by heparin, whereas that between kallikrein and alpha 1-antitrypsin is heparin-resistant. In normal or alpha 1-antitrypsin-deficient-serum, the amount of 92 kDa SDS-stable complex formed upon addition of kallikrein is not related to serum alpha 1-antitrypsin levels. The rate of complex-formation between kallikrein and the binding protein is 12 times higher than that between kallikrein and alpha 1-antitrypsin. Purified alpha 1-antitrypsin, which exhibits normal elastase binding, has a kallikrein-binding activity less than 5% of that of serum. Binding of tissue kallikrein in serum is not inhibited by increasing elastase concentrations, and elastase binding in serum is not inhibited by excess tissue kallikrein. A specific monoclonal antibody to human alpha 1-antitrypsin does not bind to either 92 kDa endogenous or exogenous kallikrein complexes isolated from human serum. The studies demonstrate a new tissue kallikrein-binding protein, distinct from alpha 1-antitrypsin, is present in human serum.     

Retarded protein folding of deficient human alpha 1-antitrypsin D256V and L41P variants

alpha(1)-Antitrypsin is the most abundant protease inhibitor in plasma and is the archetype of the serine protease inhibitor superfamily. Genetic variants of human alpha(1)-antitrypsin are associated with early-onset emphysema and liver cirrhosis. However, the detailed molecular mechanism for the pathogenicity of most variant alpha(1)-antitrypsin molecules is not known. Here we examined the structural basis of a dozen deficient alpha(1)-antitrypsin variants. Unlike most alpha(1)-antitrypsin variants, which were unstable, D256V and L41P variants exhibited extremely retarded protein folding as compared with the wild-type molecule. Once folded, however, the stability and inhibitory activity of these variant proteins were comparable to those of the wild-type molecule. Retarded protein folding may promote protein aggregation by allowing the accumulation of aggregation-prone folding intermediates. Repeated observations of retarded protein folding indicate that it is an important mechanism causing alpha(1)-antitrypsin deficiency by variant molecules, which have to fold into the metastable native form to be functional.     

Physiologic inhibition of human activated protein C by alpha 1-antitrypsin

The plasma antithrombotic enzyme activated protein C (APC) has two major plasma inhibitors. One is heparin-dependent, has been characterized, and is known as protein C inhibitor. The second inhibitor was isolated based on its heparin-independent ability to inhibit and complex with APC. The purified inhibitor had the amino acid composition and NH2 terminus of alpha 1-antitrypsin and reacted with monoclonal antibodies to alpha 1-antitrypsin. The inhibitor was greater than 95% pure alpha 1-antitrypsin as judged by electroimmunoassay, inactivation of trypsin, and electrophoresis in two gel systems. To identify the second major plasma inhibitor of APC, immunoblot studies of enzyme-inhibitor complexes were made to compare APC addition to normal plasma and to plasma deficient in protein C inhibitor or alpha 1-antitrypsin. The results showed that alpha 1-antitrypsin is the second major plasma APC inhibitor. Given the association rate constant of alpha 1-antitrypsin for APC of 10 M-1 s-1 and its plasma concentration of approximately 40 microM, it accounts for approximately half of the heparin-independent APC inhibitory activity of plasma. Based on immunoblot analysis plasmas of 15 patients with intravascular coagulation contained APC-alpha 1-antitrypsin complexes suggesting that this inhibition reaction occurs in vivo. Thus, alpha 1-antitrypsin is a major physiologic inhibitor of APC.     

Organ cultures of human fetal hepatocytes in the study of extra-and intracellular alpha1-antitrypsin.

The rate of synthesis of alpha 1-antitrypsin has been studied in organ cultures of fetal human liver. By de novo synthesis, alpha 1-antitrypsin of the same electrophoretic mobility and molecular size as plasma alpha 1-antitrypsin was produced. Synthetic rate was comparable to in vivo conditions and was suppressed by cycloheximide, colchicine and neuraminidase. By increasing alpha 1-antitrypsin levels in cultre medium, suppression of alpha 1-antitrypsin release from the intra-to the extracellular site was achieved, i.e., synthesis does not proceed autonomously. This suppression was preceded by a temporary enhancement of synthesis. Both effects were found to be independent of degree of sialylation of add-d alpha 1-antitrypsin. In contrast to alpha 1-antitrypsin released in tissue culture, the intracellular protein, as analyzed by crossed immunoelectrophoresis of Triton X-100 extracts from fetal liver, was found to occur partly as slowly moving peaks. Whether these peaks represent proforms or incompletely glycosylated precursors of export alpha 1-antitrypsin or complexes with proteases remains unsettled. A variety of other plasma proteins are released in organ cultures making the system suitable for study of factors regulating plasma protein synthesis.     

Probing the unfolding pathway of alpha1-antitrypsin

Protein misfolding plays a role in the pathogenesis of many diseases. alpha1-Antitrypsin misfolding leads to the accumulation of long chain polymers within the hepatocyte, reducing its plasma concentration and predisposing the patient to emphysema and liver disease. In order to understand the misfolding process, it is necessary to examine the folding of alpha1-antitrypsin through the different structures involved in this process. In this study we have used a novel technique in which unique cysteine residues were introduced at various positions into alpha1-antitrypsin and fluorescently labeled with N, N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)ethylenediamine. The fluorescence properties of each protein were studied in the native state and as a function of guanidine hydrochloride-mediated unfolding. The studies found that alpha1-antitrypsin unfolded through a series of intermediate structures. From the position of the fluorescence probes, the fluorescence quenching data, and the molecular modeling, we show that unfolding of alpha1-antitrypsin occurs via disruption of the A and C beta-sheets followed by the B beta-sheet. The implications of these data on both alpha1-antitrypsin function and polymerization are discussed.     

Molecular evolution of serpins: homologous structure of the human alpha 1-antichymotrypsin and alpha 1-antitrypsin genes

alpha 1-Antichymotrypsin belongs to a supergene family that includes alpha 1-antitrypsin, antithrombin III, ovalbumin, and angiotensinogen. The human chromosomal alpha 1-antichymotrypsin gene has been cloned and its molecular structure established. The gene is approximately 12 kb in length and contains five exons and four introns. The locations of the introns within the alpha 1-antichymotrypsin gene are identical with those of the human alpha 1-antitrypsin and angiotensinogen genes. Other members of this supergene family contain introns located at nonhomologous positions of the genes. The homologous organization of the alpha 1-antichymotrypsin and alpha 1-antitrypsin genes corresponds with the high degree of homology between their protein sequences and suggests that these loci arose by recent gene duplication. A model is presented for the evolution of both the genomic structure and the protein sequences of the serine protease inhibitor superfamily.     

Alpha-1-antitrypsin stimulates fibroblast proliferation and procollagen production and activates classical MAP kinase signalling pathways

Connective tissue formation at sites of tissue repair is regulated by matrix protein synthesis and degradation, which in turn is controlled by the balance between proteases and antiproteases. Recent evidence has suggested that antiproteases may also exert direct effects on cell function, including influencing cell migration and proliferation. The antiprotease, alpha1-antitrypsin, is the major circulating serine protease inhibitor which protects tissues from neutrophil elastase attack. Its deficiency is associated with the destruction of connective tissue in the lung and the development of emphysema, whereas accumulation of mutant alpha1-antitrypsin within hepatocytes often leads to liver fibrosis. In this study, we report that alpha1antitrypsin, at physiologically relevant concentrations, promotes fibroblast proliferation, with maximal stimulatory effects of 118 +/- 2% (n=6, P < 0.02) above media controls for cells exposed to 60 microM. We further show that alpha1antitrypsin also stimulates fibroblast procollagen production, independently of its effects on cell proliferation, with values maximally increased by 34 +/- 3% (n = 6, P < 0.01) above media controls at 30 microM. Finally, mechanistic studies to examine the mechanism by which alpha1-antitrypsin acts, showed that alpha1-antitrypsin induced the rapid activation of p42MAPK and p44MAPK (also known as ERK1/2) and that the specific MEK1 inhibitor PD98059 totally blocked alpha1-antitrypsin's mitogenic effects. These results support the hypothesis that alpha1-antitrypsin may play a role in influencing tissue repair in vivo by directly stimulating fibroblast proliferation and extracellular matrix production via classical mitogen-activated signalling pathways.     

Intracellular inclusions containing mutant alpha1-antitrypsin Z are propagated in the absence of autophagic activity

Mutant alpha(1)-antitrypsin Z (alpha(1)-ATZ) protein, which has a tendency to form aggregated polymers as it accumulates within the endoplasmic reticulum of the liver cells, is associated with the development of chronic liver injury and hepatocellular carcinoma in hereditary alpha(1)-antitrypsin (alpha(1)-AT) deficiency. Previous studies have suggested that efficient intracellular degradation of alpha(1)-ATZ is correlated with protection from liver disease in alpha(1)-AT deficiency and that the ubiquitin-proteasome system accounts for a major route, but not the sole route, of alpha(1)-ATZ disposal. Yet another intracellular degradation system, autophagy, has also been implicated in the pathophysiology of alpha(1)-AT deficiency. To provide genetic evidence for autophagy-mediated disposal of alpha(1)-ATZ, here we used cell lines deleted for the Atg5 gene that is necessary for initiation of autophagy. In the absence of autophagy, the degradation of alpha(1)-ATZ was retarded, and the characteristic cellular inclusions of alpha(1)-ATZ accumulated. In wild-type cells, colocalization of the autophagosomal membrane marker GFP-LC3 and alpha(1)-ATZ was observed, and this colocalization was enhanced when clearance of autophagosomes was prevented by inhibiting fusion between autophagosome and lysosome. By using a transgenic mouse with liver-specific inducible expression of alpha(1)-ATZ mated to the GFP-LC3 mouse, we also found that expression of alpha(1)-ATZ in the liver in vivo is sufficient to induce autophagy. These data provide definitive evidence that autophagy can participate in the quality control/degradative pathway for alpha(1)-ATZ and suggest that autophagic degradation plays a fundamental role in preventing toxic accumulation of alpha(1)-ATZ.