Biosynthesis and secretion of M- and Z-type alpha 1-proteinase inhibitor by human monocytes. Effect of inhibitors of glycosylation and of oligosaccharide processing on secretion and function

The biosynthesis and secretion of M-type and Z-type alpha 1-antitrypsin was studied in human monocytes. In monocytes of PiMM individuals alpha 1-antitrypsin represented 0.08% of the newly synthesized proteins and 0.44% of the secreted proteins. Two molecular forms of alpha 1-antitrypsin could be identified: a 51-kDa intracellular form, susceptible to endoglucosaminidase H, thus representing the high-mannose type precursor form and a 56-kDa form resistant to endoglucosaminidase H which was secreted into the medium. Inhibition of de novo glycosylation by tunicamycin impaired the secretion of M-type alpha 1-antitrypsin by about 75% whereas inhibition of oligosaccharide processing by the mannosidase II inhibitor swainsonine did not alter the secretion of M-type alpha 1-antitrypsin. alpha 1-Antitrypsin secreted by human monocytes was functionally active as measured by complex formation with porcine pancreatic elastase. Even unglycosylated alpha 1-antitrypsin secreted by human monocytes treated with tunicamycin formed a complex with elastase. In monocytes of PiZZ individuals the secretion of alpha 1-antitrypsin was decreased. 72% of newly synthesized M-type alpha 1-antitrypsin, but only 35% of newly synthesized Z-type alpha 1-antitrypsin were secreted during a labeling period of 3 h with [35S]methionine. The 51-kDa form of Z-type alpha 1-antitrypsin accumulated intracellularly, whereas the 56-kDa form was secreted. Inhibition of oligosaccharide processing by swainsonine did not alter the decreased secretion of Z-type alpha 1-antitrypsin, whereas inhibition of de novo glycosylation by tunicamycin blocked the secretion of Z-type alpha 1-antitrypsin completely.     

The ability of serum from alpha 1-antitrypsin-deficient patients to inhibit PRT-201, a recombinant human type I pancreatic elastase

PRT-201 is a recombinant human pancreatic elastase under development as a treatment for blood vessels to promote hemodialysis access patency. Proteases such as elastase are normally inactivated by antiproteases such as alpha 1-antitrypsin. It is unknown if serum from patients with alpha 1-antitrypsin deficiency will inhibit PRT-201 elastase activity. An assay for PRT-201 elastase activity in the presence of serum was developed and validated. PRT-201 elastase activity inhibition curves were developed using serum and also using purified alpha 1-antitrypsin and alpha 2-macroglobulin. Serum from 15 patients with documented alpha 1-antitrypsin deficiency, some of whom were receiving alpha 1-antitrypsin augmentation therapy, and four normal volunteers was analyzed. Serum from normal volunteers and patients with alpha 1-antitrypsin deficiency completely inactivated PRT-201 elastase activity in vitro. In the alpha 1-antitrypsin-deficient patients, the volume of serum necessary to inhibit elastase activity was related to the serum concentration of alpha 1-antitrypsin and augmentation therapy. Purified alpha 1-antitrypsin and alpha 2-macroglobulin were each alone capable of completely inhibiting PRT-201 elastase activity. It is unlikely that the use of PRT-201 will be associated with negative outcomes in patients with alpha 1-antitrypsin deficiency.     

Effect of swainsonine on the processing of the asparagine-linked carbohydrate chains of alpha 1-antitrypsin in rat hepatocytes. Evidence for the formation of hybrid oligosaccharides

The biosynthesis of the proteinase inhibitor alpha 1-antitrypsin has been studied in rat hepatocyte primary cultures. Newly synthesized alpha 1-antitrypsin was found in hepatocytes as a glycoprotein of an apparent molecular weight of 49,000 carrying oligosaccharide side chains of the high mannose type. In the hepatocyte medium a secreted alpha 1-antitrypsin of an apparent molecular weight of 54,000 could be identified as a glycoprotein with carbohydrate chains of the complex type. Pulse-chase experiments revealed a precursor-product relationship for the two forms of alpha 1-antitrypsin. When the hepatocytes were treated with swainsonine, an intracellular form of alpha 1-antitrypsin with an apparent molecular weight of 49,000 indistinguishable from that of control cells was found. However, the alpha 1-antitrypsin secreted from swainsonine-treated hepatocytes was different from that present in control media. It was characterized by a lower apparent molecular weight (51,000), a higher amount of [3H]mannose incorporation, half as much incorporation of [3H]galactose, and the same amount of [3H]fucose incorporation compared to alpha 1-antitrypsin of control media. In contrast to the 54,000 complex type alpha 1-antitrypsin from control media the 51,000 alpha 1-antitrypsin from the medium of swainsonine-treated cells was found to be susceptible to the action of endoglucosaminidase H, even when fucose was attached to the proximal GlcNAc residue. alpha 1-Antitrypsin secreted from swainsonine-treated cells combines features usually associated with either high mannose or complex type oligosaccharides and therefore represents a hybrid structure. In spite of its effect on the carbohydrate part of alpha 1-antitrypsin swainsonine did not impair the secretion of the incompletely processed glycoprotein.     

Phenotype and gene distribution of alpha-1-antitrypsin in a North Italian population.

A group of 202 unrelated Italians were screened for alpha1-antitrypsin using agarose-acrylamide electrophoresis and isoelectric focusing. The S and F gene frequencies were comparable to those found among Greeks and North European populations but they differed considerably from the frequencies found among Spaniards and Portuguese. The other gene frequencies appeared to be comparable to other populations, studied.     

Investigation of the heterogeneity of rhesus monkey alpha 1-antitrypsin

Rhesus monkey alpha 1-antitrypsin (n = 144) was examined for heterogeneity by acid starch gel electrophoresis, isoelectric focusing in agarose and agarose gel electrophoresis. In contrast to other studies, no heterogeneity of Rhesus monkey alpha 1-antitrypsin could be documented using specific antisera. Rhesus monkey alpha 1-antitrypsin contained a reactive thiol. The pIs of the major isoforms of Rhesus monkey alpha 1-antitrypsin were 4.63, 4.69, 4.84 and 4.86 at 4 degrees C. No deficiency state of Rhesus monkey alpha 1-antitrypsin was detected. The six protease inhibitors in Rhesus monkey sera cross-reacted with antisera to the six human protease inhibitors.     

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.     

Genetic polymorphism of the alpha 1-antitrypsin system in the native population of the Kazakh SSR

Phenotypes of alpha 1-antitrypsin of 218 natives from three ethno-historical regions of the Kazakh SSR were detected by isoelectric focusing in ultrathin layer polyacrylamide gel. Gene frequencies of the PiM1, PiM2, PiM3 subtypes in the summary extract were as follows: 0.8477, 0.1372 and 0.0106, respectively; the total gene frequency of two rare variants (PiN and PiZ) was 0.0046. The observed distribution of Pi subtypes shows good agreement with the Hardi-Weinberg equation. The analysis of the interpopulation intraethnic variability of the alpha 1-antitrypsin phenotype and allele frequencies in Kazakhs revealed clear local diversity. The frequency of PiM1 in the natives from North-Central ethno-historical region was reliably lower and that of PiM2 higher than in populations from South-East and West regions. The extracts analysed did not differ in the PiM3 incidences. The results of these studies were compared with the literature data for alpha 1-antitrypsin polymorphism in populations of the Euro-Asia. It is shown that PiM1 and PiM2 frequencies in the Kazakhs differ from the corresponding mean values both in mongoloid and europeoid groups. At the same time, they do not correspond to the intermediate frequency estimations, which could be expected from the fact of mixed origin of the Kazakh people and their border-line geographical position between Europe and Asia. Possible reason for such discrepancy is discussed.     

Osmolytes as modulators of conformational changes in serpins.

Protein misfolding and aggregation play an integral role in many diseases. The misfolding of the serpin (SERine Proteinase INhibitor) alpha1-antitrypsin results in the accumulation of insoluble polymers within hepatocytes and alpha1-antitrypsin deficiency in plasma, predisposing patients to liver cirrhosis and emphysema. We have examined the effect of three naturally occurring osmolytes, sarcosine, glycine betaine and trimethylamine N-oxide, on conformational changes in alpha1-antitrypsin. All three solutes protected native alpha1-antitrypsin against thermally induced polymerisation and inactivation in a concentration-dependent manner. Further spectroscopic analysis showed that sarcosine stabilises the native conformation of alpha1-antitrypsin, thus hindering its conversion to an intermediate state and subsequent polymerisation. On refolding in the presence of sarcosine, alpha1-antitrypsin formed a heterogeneous population, with increasing proportions of molecules adopting an inactive conformation in higher concentrations of the osmolyte. These data show that sarcosine can be used to prevent abnormal structural changes in native alpha1-antitrypsin, but is ineffective in facilitating the correct folding of the protein. The implications of these results in the context of conformational changes and states adopted by alpha1-antitrypsin are discussed.     

Protein abnormalities in adult respiratory distress syndrome, tuberculosis, and cystic fibrosis sera

Crossed immunoelectrophoresis (X-IEP) revealed several abnormalities in serum proteins from patients with adult respiratory distress syndrome (ARDS), tuberculosis (TB), and cystic fibrosis (CF). The two quite different kinds of pulmonary disease, one acute (ARDS) and the other chronic (TB and CF) exhibited serum changes specific for each disease and abnormalities associated with inflammation and pathogenesis, in general. In ARDS sera, most proteins were extremely low, presumably due to leakage into the lungs through damaged tissue, while the acute-phase proteins, orosomucoid, alpha 1-antitrypsin, alpha 1-antichymotrypsin, and haptoglobin, were markedly high when compared to the overall protein pattern. The extremely high alpha 1-antichymotrypsin values were not seen in corresponding TB and CF sera. Numerous TB patients had elevated alpha 1-antitrypsin, alpha 1-antichymotrypsin, and haptoglobin, but only the alpha 1-antitrypsin population mean was significantly different from normal. Gc-globulin, ceruloplasmin, and beta-lipoprotein were higher and alpha 1-lipoprotein and inter-alpha-trypsin inhibitor lower than normal. All other quantitative serum changes were not statistically significant. Surprisingly, all TB patients belonged to the Gc-1-1 genotype in contrast to the Gc-1-1, Gc-1-2, Gc-2-2 polymorphisms of the other populations. CF homozygote sera revealed statistically significant increases in the acute-phase proteins, alpha 1-antitrypsin, alpha 1-antichymotrypsin, and haptoglobin, while orosomucoid, transferrin, IgA, and IgG tended to be higher than normal. The tendency for higher levels of transferrin indicated possible iron deficiency in some patients. In contrast, prealbumin, alpha 1-lipoprotein, and inter-alpha-trypsin inhibitor were significantly depressed in CF patients. CF heterozygotes shared the decrease of alpha 1-lipoprotein with the patients while exhibiting small but significant depressions of alpha 2-macroglobulin and IgG. Though not statistically significant, lowered concentrations of alpha 1-antitrypsin were evident for the heterozygotes.     

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.     

Interaction between human pancreatic elastase and plasma protease inhibitors

1 ml of human serum inhibits about 0.9 mg of purified human pancreatic elastase owing to complexation with alpha 1-antitrypsin and alpha 2-macroglobulin. On addition to serum, elastase is preferentially bound by alpha 2-macroglobulin. The complexes between elastase and alpha 1-antitrypsin and alpha 2-macroglobulin, respectively, migrate as alpha 2-globulin on agarose gel electrophoresis. Elastase bound by alpha 1-antitrypsin is precipitated by antibodies against enzyme as well as inhibitor, while the alpha 2-macroglobulin-bound elastase is only precipitated by antibodies against the inhibitor. The molar combining ratio for elastase/alpha 1-antitrypsin is 1:1 and for elastase/alpha 2-macroglobulin 2:1. The elastase bound by alpha 2-macroglobulin retains its activity against low molecular weight substrates, while that bound by alpha 1-antitrypsin is enzymologically inactive.     

6-mer peptide selectively anneals to a pathogenic serpin conformation and blocks polymerization. Implications for the prevention of Z alpha(1)-antitrypsin-related cirrhosis.

Conformational diseases such as amyloidosis, Alzheimer's disease, prion diseases, and the serpinopathies are all caused by structural rearrangements within a protein that transform it into a pathological species. These diseases are typified by the Z variant of alpha(1)-antitrypsin (E342K), which causes the retention of protein within hepatocytes as inclusion bodies that are associated with neonatal hepatitis and cirrhosis. The inclusion bodies result from the Z mutation perturbing the conformation of the protein, which facilitates a sequential interaction between the reactive center loop of one molecule and beta-sheet A of a second. Therapies to prevent liver disease must block this reactive loop-beta-sheet polymerization without interfering with other proteins of similar tertiary structure. We have used reactive loop peptides to explore the differences between the pathogenic Z and normal M alpha(1)-antitrypsin. The results show that the reactive loop is likely to be partially inserted into beta-sheet A in Z alpha(1)-antitrypsin. This conformational difference from M alpha(1)-antitrypsin was exploited with a 6-mer reactive loop peptide (FLEAIG) that selectively and stably bound Z alpha(1)-antitrypsin. The importance of this finding is that the peptide prevented the polymerization of Z alpha(1)-antitrypsin and did not significantly anneal to other proteins (such as antithrombin, alpha(1)-antichymotrypsin, and plasminogen activator inhibitor-1) with a similar tertiary structure. These findings provide a lead compound for the development of small molecule inhibitors that can be used to treat patients with Z alpha(1)-antitrypsin deficiency. Furthermore they demonstrate how a conformational disease process can be selectively inhibited with a small peptide.     

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)     

Group-specific component, alpha1-antitrypsin and esterase D in Canadian Eskimos.

Three genetic markers - group-specific component (Gc), alpha1-antitrypsin, and esterase D - were examined in a population of Eskimos from Igloolik in the eastern Canadian Arctic. Gc and esterase D were found to be polymorphic. In addition to the common Gc types, an anodal variant called Gc Igloolik was found, probably identical to previously reported Gc Eskimo. Gene frequencies were Gc1: 0.6524, Gc2: 0.3373, GcIgl: 0.0104, for 338 Eskimos. Genetic types of alpha1-antitrypsin (Pi types) were mostly M, with two MS sibs who were half Caucasian, in 170 Eskimos. Frequencies of the esterase D allele in 336 Eskimos were EsD1: 0.7083, EsD2: 0.2917. The frequencies of Gc2 and EsD2 are both higher than are found in Caucasian populations.     

Differential effects of flavonols on inactivation of alpha1-antitrypsin induced by hypohalous acids and the myeloperoxidase-hydrogen peroxide-halide system

Alpha1-antitrypsin is well known for its ability to inhibit human neutrophil elastase. Pretreatment of alpha1-antitrypsin with hypohalous acids HOCl and HOBr as well as with the myeloperoxidase-hydrogen peroxide-chloride (or bromide) system inactivated this proteinase. The flavonols rutin, quercetin, myricetin, and kaempferol inhibited the inactivation of alpha1-antitrypsin by HOCl and HOBr with rutin having the most pronounced effect. In contrast, these flavonols did not remove the proteinase inactivation by the myeloperoxidase-hydrogen peroxide-halide system. Taurine did not protect against the inactivation of alpha1-antitrypsin by HOCl, HOBr, or the myeloperoxidase-hydrogen peroxide-halide system, while methionine was efficient in all systems. A close association between myeloperoxidase and alpha1-antitrypsin was revealed by native gel electrophoresis and in-gel peroxidase staining. In addition, alpha1-antitrypsin binds to the myeloperoxidase components transferred after SDS-PAGE on a blotting membrane. With this complex formation, myeloperoxidase overcomes the natural antioxidative protective system of plasma and prevents the inactivation of alpha1-antitrypsin.     

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.     

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.     

Cleaved antitrypsin polymers at atomic resolution

Alpha1-antitrypsin deficiency, which can lead to both emphysema and liver disease, is a result of the accumulation of alpha1-antitrypsin polymers within the hepatocyte. A wealth of biochemical and biophysical data suggests that alpha1-antitrypsin polymers form via insertion of residues from the reactive center loop of one molecule into the beta-sheet of another. However, this long-standing hypothesis has not been confirmed by direct structural evidence. Here, we describe the first crystallographic evidence of a beta-strand linked polymer form of alpha1-antitrypsin: the crystal structure of a cleaved alpha1-antitrypsin polymer.     

The alpha 1-antitrypsin gene and its deficiency states

alpha 1-antitrypsin, a 52 kDa antiprotease, provides the major defense to the lower respiratory tract against the ravages of neutrophil elastase, a powerful serine protease. A variety of mutations in the coding exons of the alpha 1-antitrypsin gene result in 'alpha 1-antitrypsin deficiency', leading to emphysema at an early age. A subset of mutations cause liver disease and a rare mutation is associated with a bleeding diathesis. Preventive treatment for the emphysema associated with alpha 1-antitrypsin deficiency is available in the form of intermittent infusions with alpha 1-antitrypsin, and strategies have been developed to reverse the deficiency state with gene therapy.