The effect of alpha 1 antitrypsin on the proliferative response of human peripheral blood lymphocytes

In evaluating immune aberrations in patients with alpha 1 antitrypsin (alpha 1 AT) deficiency, we have previously shown that they exhibit enhanced lymphocyte responsiveness to PHA that is serum mediated. In this study, we demonstrate suppression of the PHA response by using purified alpha 1 AT, and also similar but less marked suppression of the Con A response. alpha 1 AT, however, has no effect whatsoever on PWM-induced proliferation. This effect is demonstrable provided alpha 1 AT is added within 4 hr of mitogen activation and is mediated by its action on adherent cells rather than on proliferating lymphocytes. Adherent cells still exhibit this effect if pulsed with alpha 1 AT, then thoroughly washed before their activation. This suggests that it may be inhibiting a membrane serine esterase already activated before the addition of PHA. Thus alpha 1 AT may modulate the activation of T cells through its effect on monocytes, leading to abnormalities in immunoregulation, and hence a predisposition to the development of a variety of immunologic disorders in alpha 1 AT-deficient subjects.     

Characterization of the gene and protein of the alpha 1-antitrypsin "deficiency" allele Mprocida

The "deficiency" group of alpha 1-antitrypsin (alpha 1AT) alleles is characterized by alpha 1AT genes that code for alpha 1AT present in serum but in amounts insufficient to protect the lower respiratory tract from progressive destruction by its burden of neutrophil elastase. Mprocida, a rare alpha 1AT allele associated with alpha 1AT serum levels less than 10 mg/dl (normal 150-350 mg/dl), codes for an alpha 1AT molecule that focuses on immobilized pH gradient isoelectric gels slightly cathodal to the common normal M1 (Val213) protein. On a per molecule basis, Mprocida has a mildly reduced function as an inhibitor, with an association rate constant for human neutrophil elastase of 7.0 +/- 0.1 x 10(6) M-1 s-1 (normal M1 (Val213) 9.3 +/- 0.8 x 10(6), p less than 0.01). The Mprocida molecule behaves normally in vivo with a half-life similar to normal M1 alpha 1AT molecules. Restriction endonuclease mapping demonstrates that the cloned Mprocida gene was grossly intact. Sequencing of all the exons, exon-intron junctions, and the major promoter region demonstrated Mprocida to be identical to the M1 (Val213) gene except for a single base substitution in exon II coding for amino acid 41 of the mature protein (M1 (Val213) Leu41 CTG----Mprocida Pro41 CCG). Usefully, the coding sequence of the alpha 1AT residues 40-41 is recognized by the restriction endonuclease PvuII so that using a probe corresponding to this region of exon II, the Mprocida mutation can be rapidly identified by Southern analysis. Evaluation of the crystallographic structure of alpha 1AT suggests the Leu41 to Pro41 mutation may disrupt alpha-helix A in the region of Pro21-Ser45, suggesting the possibility that the alpha 1AT Mprocida molecule is unstable and degraded intracellularly prior to secretion.     

Biosynthesis and processing of rat alpha 1-antitrypsin

Various biosynthetic forms of rat alpha 1-antitrypsin (alpha 1AT) have been isolated by immunoprecipitation of in vitro and in vivo synthesized products. Rat alpha 1AT is synthesized in a rabbit reticulocyte system as a 45,000-Da preprotein with a 23-amino acid signal sequence. The majority of the amino acids in the signal sequence have been identified and resemble the signal peptides of other secretory proteins with respect to the abundance and positions of hydrophobic amino acids. Evidence from the translation of rat liver RNA in the presence of dog pancreas microsomes, from the translation of rat liver polysomes, and from tunicamycin-treated rat hepatocytes established that cleavage of the signal peptide of pre-alpha 1AT results in the formation of a 42,000-Da protein, the polypeptide backbone of mature alpha 1AT. A 50,000-Da glycoprotein is immunoprecipitated from translations programmed with rat liver microsomes or with rat liver mRNA and dog pancreas microsomes. Cotranslational glycosylation of alpha 1AT appears to occur in a stepwise fashion since three glycosylated forms of alpha 1AT (approximately 45,000, 47,000, and 50,000 Da) can be detected in polysome translations. These proteins are susceptible to cleavage by endo-beta-N-acetylglucosaminidase H and are digested to the same product, indicating that they have identical polypeptide chains. Two intracellular forms of alpha 1AT were detected in cultured rat hepatocytes, a 50,000- and a 52,000-Da protein; only the larger protein was immunoprecipitated from the medium of these cells. Digestion with endo-beta-N-acetylglucosaminidase H indicated that the 50,000-Da protein is a core glycosylated processing intermediate, whereas the 52,000-Da protein, which comigrated with purified serum alpha 1AT, appears to contain complex carbohydrate sidechains. When glycosylation was inhibited by incubation of hepatocytes with tunicamycin, a nonglycosylated 42,000-Da protein was immunoprecipitated from the cells and the culture medium, indicating that glycosylation of alpha 1AT is not essential for its secretion.     

Alpha 1-antitrypsin Wbethesda: molecular basis of an unusual alpha 1-antitrypsin deficiency variant

Molecular analysis of alpha 1-antitrypsin (alpha 1AT) Wbethesda revealed that it differs from the normal M1 (Ala213) allele by a single base mutation causing an amino acid substitution Ala336 GCT----Thr ACT. Evaluation of alpha 1AT biosynthesis directed by the Wbethesda allele showed that although Wbethesda alpha 1AT mRNA was translated normally in vitro, transfection of the Wbethesda cDNA into COS-I cells was associated with human alpha 1AT secretion of 50% that of cells transfected with a normal alpha 1AT cDNA. The pattern of alpha 1AT biosynthesis was not intracellular accumulation as observed with the common Z alpha 1AT deficiency allele, but reduced intracellular alpha 1AT, suggesting intracellular degradation of the newly synthesized Wbethesda molecule. Together these observations suggest that in heterozygous combination with a Z or Null alpha 1AT allele, the Wbethesda variant causes "alpha 1AT deficiency", thus classifying it as an alpha 1AT "at risk" allele for emphysema.     

cis-dichlorodiammineplatinum (II) as a selective modifier of the oxidation-sensitive reactive-center methionine in alpha 1-antitrypsin

Methionine 358 in the plasma protein alpha 1-antitrypsin (alpha 1AT) is an oxidation-sensitive reactive-center residue critical for proteinase-inhibitory activity. Reaction of alpha 1AT with 20 microM to 1.67 mM cis-dichlorodiammineplatinum (II) (cis-DDP) or trans-DDP afforded concentration-dependent loss of trypsin-inhibitory activity. This effect, studied by gel electrophoresis and activity assays, is essentially independent of pH over the range 4.9-8.6. Binding assays showed covalent incorporation of 1 mol of cis-DDP into each mol of alpha 1AT. cis-DDP protected a single methionine residue from oxidation and made alpha 1AT resistant to degradation by papain, which cleaves alpha 1AT at Met358. These findings strongly suggest that cis-DDP inactivates alpha 1AT by binding exclusively to its reactive-center methionine. alpha 1AT bound twice as much platinum when reacted with trans-DDP. Because carboxamidomethylated alpha 1AT incorporated nearly 1 mol of both cis- and trans-DDP, the trans isomer apparently binds to both the reactive-center methionine and to the single cysteine residue of alpha 1AT. Because of its greater selectivity, cis-DDP is the superior reagent for modification of the alpha 1AT reactive-center methionine.     

Heterologous protein expression by transimmortalized differentiated liver cell lines derived from transgenic mice (hepatomas/alpha 1 antitrypsin/ONC mouse)

A number of therapeutic plasma proteins are synthesized by human hepatocytes. Since many of these proteins undergo liver-specific post-translational modifications which are required for full biological activity, it may therefore be necessary to develop hepatocyte-based expression systems for their production. Using transgenic mice we have developed a transimmortalisation technique for the isolation of differentiated hepatic cell lines, already engineered to secrete human alpha 1 antitrypsin (alpha 1 AT), a plasma protein which is produced mainly in liver cells. This was achieved by co-expression of the mouse c-myc proto-oncogene and a genomic copy of the human alpha 1 AT gene, both under the control of the human alpha 1 AT promoter. Transgenic mice carrying this construct developed hepatomas producing human alpha 1 AT. Under defined culture conditions, cell lines secreting active alpha 1 AT were derived from these tumours. These cells maintain a differentiated hepatic phenotype and continue to secrete human alpha 1 AT for at least 40 generations.     

Mechanisms of alpha1-antitrypsin inhibition of cellular serine proteases and HIV-1 protease that are essential for HIV-1 morphogenesis

Proprotein processing is essential for HIV infectivity. Cellular trans-Golgi network (TGN) serine proteases (e.g., furin) are required to cleave HIV envelope gp160 to gp120. In addition, HIV protease (PR), an aspartyl protease, cleaves p55(Gag) to p24, etc., in budding virions. alpha1-Antitrypsin (alpha(1)AT) is cleaved by serine proteases, causing a conformational change in alpha(1)AT that sequesters and so inactivates the protease. alpha(1)AT blocks both gp160 and p55 processing, and so is a powerful inhibitor of HIV replication. We hypothesized that alpha(1)AT inhibited gp160 and p55 processing via different mechanisms, and that in both cases, alpha(1)AT bound and was itself cleaved by the proteases whose activities were blocked. alpha(1)AT delivered by SV(AT), a recombinant, Tag-deleted SV40-derived vector, localized to the TGN, co-precipitated with furin, and depleted furin from the TGN. After SV(AT) transduction and HIV challenge, alpha(1)AT was detected in resulting nascent immature HIV-1 virions. alpha(1)AT also blocked incorporation of the enzymatically active dimeric form of PR into HIV virions. Western analysis using recombinant proteins showed that alpha(1)AT directly bound HIV PR, and was cleaved by it. The simultaneous inhibition of two different steps in HIV morphogenesis both increases alpha(1)AT antilentiviral activity and decreases the possibility that HIV mutations will allow escape from inhibition.     

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.     

Sheep plasma protease inhibitors influencing protease activity and growth of Lucilia cuprina larvae in vitro

The enzyme inhibitors alpha 2-macroglobulin (alpha 2M), anti-thrombin III (AT III) and alpha 1-proteinase inhibitor (alpha 1PI) were isolated from sheep plasma and tested for their ability to affect L. cuprina larval proteases and larval growth in vitro. Casein radial diffusion gels indicated that both alpha 2M and alpha 1PI completely inhibited the protease activity of a larval excretory-secretory preparation, while AT III had a partial effect. Casein zymograms revealed that alpha 2M inhibited all of the larval proteases, while AT III was able to modify the normal plaque pattern; alpha 1PI inhibited all plaques except a doublet present at pI 8.5. Larval growth in vitro was significantly inhibited by alpha 2M and AT III (P less than 0.05) when compared to albumin controls but was not affected by alpha 1PI. The levels of alpha 2M in sheep serum were monitored over the course of a larval fly infection. A significant increase in alpha 2M (P less than 0.05) was recorded in the serum of flystruck sheep. It is suggested that, under certain circumstances, these inhibitors may be involved in influencing flystrike through reducing the activity of larval proteases necessary for wound formation and larval nutrition.     

Immuno-biochemical findings in groups of individuals occupationally and non-occupationally exposed to emissions containing nickel and cobalt

Immune reactions elicited in the sera of individuals exposed to nickel and cobalt were assessed by changes in the concentration of serum immunoglobulins IgG, IgA and IgM and serum proteins alpha 2 macroglobulin (A2M), transferrin (TRF), alpha 1-antitrypsin (A1AT), ceruloplasmin (CPL) and lysozyme (LYS). Examinations were carried out in workers occupationally exposed to Ni (38 individuals) or Co (35 individuals) and in groups of non-occupationally exposed children living in areas with a different degree of air pollution from a nearby source of Ni and Co emissions (one group was made up of 54 exposed children, the other one of 64 "less exposed" children of the same age). Groups of non-exposed controls were represented by a group of 42 male adults matched by age and by a group of 48 children from a non-polluted area. Significantly increased average values were obtained for IgG, IgA and IgM in group of workers exposed to Ni, for IgA in workers exposed to Co and for A1AT, A2M, CPL and LYS in both groups of occupationally exposed adults (p less than 0.001 - p less than 0.005). Among non-occupationally exposed children the group of the most exposed had significantly elevated average values for A2M and A1AT which were higher than those recorded in groups of "less exposed" and control children (p less than 0.02 and p less than 0.05, respectively). The biomedical importance of these findings is discussed in detail.     

Evaluation of recombinant DNA-directed E.coli produced alpha 1-antitrypsin as an anti-neutrophil elastase for potential use as replacement therapy of alpha 1-antitrypsin deficiency

alpha 1-antitrypsin (alpha 1AT) deficiency is an inherited disorder almost always associated with the development of panacinar emphysema in the fourth to fifth decades. One source of alpha 1AT for chronic replacement therapy of such individuals is that produced by E.coli directed by a cDNA coding for the human alpha 1AT molecule. Using TG1(E.coli), an alpha 1AT molecule produced by E.coli transformed with the plasmid-expressing vector pTG922, the present study shows that recombinant DNA-directed E.coli-produced alpha 1AT is as an effective inhibitor of neutrophil elastase as alpha 1AT purified from plasma. Importantly, TG1(E.coli) inhibited human neutrophil elastase with an association rate constant of 1.3 +/- 0.4X10(7) M-1 sec-1, similar to that of normal plasma alpha 1AT (1.1 +/- 0.1, p greater than 0.2). Furthermore, when TG1(E.coli) was added to alpha 1AT-deficient plasma obtained from homozygous alpha 1AT type Z individuals, the TG1(E.coli) remained functional and augmented the anti-neutrophil elastase activity of the serum proportional to the amount of TG1(E.coli) added. These observations suggest that if sufficient amounts of recombinant DNA methodology-produced alpha 1AT molecules could be safely delivered to the alveolar structures of alpha 1AT-deficient individuals, they would function to protect the alveolar walls from elastolytic attack.     

Purification and biochemical characterization of recombinant alpha 1-antitrypsin variants expressed in Escherichia coli

Site-directed variants of alpha 1-antitrypsin (alpha 1AT) expressed in a recombinant strain of Escherichia coli have been isolated with an overall process yield of 50% following tangential flow ultrafiltration, anion-exchange, immobilized metal affinity, and hydrophobic interaction chromatography. The primary structure of the purified variants including the integrity of the N- and C-termini has been verified by electrospray mass spectrometry of the intact molecules (44 kDa) for two of the variants (alpha 1AT Leu-358 and alpha 1AT Ala-357, Arg-358). Complementary classical peptide mapping and automated amino acid sequencing have verified 75% of the primary sequence of alpha 1AT Ala-357, Arg-358. Isoelectric focusing in an immobilized pH gradient revealed some microheterogeneity which proved to be reproducible from one purification batch to another. The isolated variants of alpha 1AT did not show any signs of proteolytic degradation during the purification process and proved to be fully active against their target proteases. The described process also allowed the complete removal of endotoxins from the preparations, opening the possibility to evaluate these novel protease inhibitors for their in vivo efficacy in different animal models of human disease.     

Role of ANG II in coronary capillary angiogenesis at the insulin-resistant stage of a NIDDM rat model

With the use of Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of human non-insulin-dependent diabetes mellitus (NIDDM), we assessed whether ANG II is involved in coronary capillary angiogenesis at the insulin-resistant stage of NIDDM (20 wk of age). In OLETF rats, ANG II labeling and angiotensin type 1 (AT(1)) receptor expression in coronary vessels were increased more than in nondiabetic controls. A marked increase in vascular expression of vascular endothelial growth factor (VEGF) at both mRNA and protein levels was found in OLETF rats. The increased expression level of VEGF was associated with accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) activated by increased advanced glycation end products (AGEs). Morphometric analysis showed a significantly increased total coronary capillary density, which was a result of arterialization of the venular capillary portion in OLETF rats. Treatment of OLETF rats with candesartan, an AT(1) receptor blocker, inhibited vascular expressions of VEGF, HIF-1alpha, and AGEs, and ameliorated the morphometric changes. These results suggest a key role of ANG II in the pathogenesis of the coronary capillary remodeling in this NIDDM model.