Expression of active recombinant human alpha 1-antitrypsin in transgenic rabbits.

A DNA construct containing the human alpha 1-antitrypsin gene including 1.5 and 4 kb of 5' and 3' flanking sequences, was microinjected into the pronucleus of rabbit embryos. The recombinant human protein was (a) expressed in the blood circulation of F0 and F1 transgenic rabbits at an average concentration of 1 mg ml-1, (b) shown to be fully active and (c) shown to be separable from its rabbit counterpart. Transgenic rabbits might represent a novel source of human proteins of therapeutic interest.     

Relationship between protein structure and methionine oxidation in recombinant human alpha 1-antitrypsin

alpha 1-Antitrypsin is a metastable and conformationally flexible protein that belongs to the serpin family of protease inhibitors. Although it is known that methionine oxidation in the protein's active site results in a loss of biological activity, there is little specific knowledge regarding the reactivity of each of the protein's methionine residues. In this study, we have used peptide mapping to study the oxidation kinetics of each of alpha 1-antitrypsin's methionines in alpha 1-AT((C232S)) as well as M351L and M358V mutants. These kinetic studies establish that Met1, Met226, Met242, Met351, and Met358 are reactive with hydrogen peroxide at neutral pH and that each reactive methionine is oxidized in a bimolecular, rather than coupled, mechanism. Analysis of Met226, Met351, and Met358 oxidation provides insights regarding the structure of alpha 1-antitrypsin's active site that allow us to relate conformation to experimentally observed reactivity. The relationship between solution pH and methionine oxidation was also examined to evaluate methionine reactivity under conditions that perturb the native structure. Methionine oxidation data show that at pH 5, global conformational changes occur that alter the oxidation susceptibility of each of alpha 1-antitrypsin's 10 methionine residues. Between pH 6 and 9, however, more localized conformational changes occur that affect primarily the reactivity of Met242. In sum, this work provides a detailed analysis of methionine oxidation in alpha 1-antitrypsin and offers new insights into the protein's solution structure.     

N-glycosylation and biological activity of recombinant human alpha1-antitrypsin expressed in a novel human neuronal cell line

Human alpha‐1‐antitrypsin (A1AT) is a protease inhibitor that is involved in the protection of lungs from neutrophil elastase enzyme that drastically modifies tissue functioning. The glycoprotein consists of 394 amino acids and is N‐glycosylated at Asn‐46, Asn‐83, and Asn‐247. A1AT deficiency is currently treated with A1AT that is purified from human serum. In view of therapeutic applications, rA1AT was produced using a novel human neuronal cell line (AGE1.HN®) and we investigated the N‐glycosylation pattern as well as the in vitro anti‐inflammatory activity of the recombinant glycoprotein. rA1AT (300 mg/L) was biologically active as analyzed using elastase assay. The N‐glycan pool, released by PNGase F digestion, was characterized using 2D‐HPLC, MALDI‐TOF mass spectrometry, and by exoglycosidase digestions. A total of 28 N‐glycan structures were identified, ranging from diantennary to tetraantennary complex‐type N‐glycans. Most of the N‐glycans were found to be (α1–6) core‐fucosylated and part of them contain the Lewis X epitope. The two major compounds are a monosialylated diantennary difucosylated glycan and a disialylated diantennary core‐fucosylated glycan, representing 25% and 18% of the total N‐glycan pool, respectively. Analysis of the site‐specificity revealed that Asn‐247 was mainly occupied by diantennary N‐glycans whereas Asn‐46 was occupied by di‐, and triantennary N‐glycans. Asn‐83 was exclusively occupied by sialylated tri‐ and tetraantennary N‐glycans. Next, we evaluated the anti‐inflammatory activity of rA1AT using A1AT purified from human serum as a reference. rA1AT was found to inhibit the production of TNF‐α in neutrophils and monocytes as commercial A1AT does. Biotechnol. Bioeng. 2011;108:2118–2128. © 2011 Wiley Periodicals, Inc.     

The reactivity and oxidation pathway of cysteine 232 in recombinant human alpha 1-antitrypsin

Oxidative damage to the sulfur-containing amino acids, methionine and cysteine, is a major concern in biotechnology and medicine. alpha1-Antitrypsin, which is a metastable and conformationally flexible protein that belongs to the serpin family of protease inhibitors, contains nine methionines and a single cysteine in its primary sequence. Although it is known that methionine oxidation in the protein active site results in a loss of biological activity, there is little specific knowledge regarding the reactivity of its unpaired thiol, Cys-232. In this study, the thiol-modifying reagent NBD-Cl (7-chloro-4-nitrobenz-2-oxa-1,3-diazole) was used to label peroxide-modified alpha1-antitrypsin and demonstrate that the Cys-232 in vitro oxidation pathway begins with a stable sulfenic acid intermediate and is followed by the formation of sulfinic and cysteic acid in successive steps. pH-dependent reactivity with hydrogen peroxide showed that Cys-232 has a pK(a) of 6.86 +/- 0.05, a value that is more than 1.5 pH units lower than that of a typical protein thiol. pH-induced conformational changes in the region surrounding Cys-232 were also examined and indicate that mildly acidic conditions induce a conformation that enhances Cys-232 reactivity. In summary, this work provides new insights into alpha1-antitrypsin reactivity in oxidizing environments and shows that a unique structural environment renders its unpaired thiol, Cys-232, its most reactive amino acid.     

Application of vortex flow adsorption technology to intein-mediated recovery of recombinant human alpha1-antitrypsin

Vortex flow is a secondary flow pattern that appears above a critical rotation rate in the annular gap between an inner rotating solid cylinder and an outer stationary cylindrical shell. By suspending adsorbent resin in the vortices, a novel unit operation, vortex flow adsorption (VFA), is created. In VFA, the rotation of the inner cylinder facilitates the fluidization of the adsorbent resin. Similar to expanded bed processes, VFA has high fluid voidage so that it can be used to recover biochemical products directly from fermentation broths or cell homogenates without removing cells or cell debris first. In this study, recombinant human alpha1-antitrypsin (alpha1-AT) was expressed in Escherichia coli as a fusion with a modified intein containing a chitin-binding domain. Therefore, the fusion protein can be recovered by chitin resin affinity adsorption. The intein can be induced to undergo in vitro peptide bond cleavage to specifically release alpha1-AT from the bound fusion protein. The capture efficiency of the fusion protein, 26.2%, was obtained in the VFA process. In addition, the specific activity of alpha1-AT was dramatically improved from 0.3 to 205.2 EIC/(mg total protein) after adsorption and cleavage. Therefore, vortex flow adsorption is an integrative technology to combine the primary clarification, concentration, and purification steps in conventional downstream processing into a single unit operation to efficiently recover and purify biochemical products.     

Targeting a recombinant adenovirus vector to HCC cells using a bifunctional Fab-antibody conjugate

We developed a specific adenoviral gene delivery system with monoclonal antibody (mAb) AF-20 that binds to a 180 kDa antigen highly expressed on human hepatocellular carcinoma (HCC) cells. A bifunctional Fab-antibody conjugate (2Hx-2-AF-20) was generated through AF-20 mAb crosslinkage to an anti-hexon antibody Fab fragment. Uptake of adenoviral particles and gene expression was examined in FOCUS HCC and NIH 3T3 cells by immunofluorescence; beta-galactosidase expression levels were determined following competitive inhibition of adenoviral CAR receptor by excess fibre knob protein. The chimeric complex was rapidly internalized at 37 degrees C, and enhanced levels of reporter gene expression was observed in AF-20 antigen positive HCC cells, but not in AF-20 antigen negative NIH 3T3 control cells. Targeting of recombinant adenoviral vectors to a tumor associated antigen by a bifunctional Fab-antibody conjugate is a promising approach to enhance specificity and efficiency of gene delivery to HCC.     

Secretory expression of human alpha1 -antitrypsin in Saccharomyces cerevisiae using galactose as a gratuitous inducer

Human alpha1 -antitrypsin (alpha1-AT) was expressed and secreted in Saccharomyces cerevisiae carrying the gal1 and reg1-501 mutations under the control of GAL10 promoter. Increasing galactose from 0 to 0.5 g/l increased the extracellular concentration of alpha1-AT, though the secretion efficiency was decreased. The final titre of alpha1-AT secreted and the secretion efficiency were 17.9 mu g/ml and 24.8% respectively, with 0.5 g galactose/l. © Rapid Science Ltd. 1998     

Purification and properties of normal human alpha 1-antitrypsin

A relatively gentle purification method has been devised for the major serine-protease inhibitor in human plasma. The product, a single chain glycoprotein of 50,000 molecular weight, is obtained in 25% yield. It contains 11.5% carbohydrate by weight, a single residue of cysteine and two of tryptophan. N-terminal glutamate or glutamine was found by dansylation. Isoelectric focusing at high resolution in acrylamide gels revealed three major and several minor protein species. Several possible mechanisms to account for this isoelectric microheterogeneity are discussed.     

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.     

The isolation of a clone for human alpha 1-antitrypsin and the detection of alpha 1-antitrypsin in mRNA from liver and leukocytes

A recombinant clone containing an insert complementary to alpha 1-antitrypsin (alpha 1-AT) mRNA has been isolated from a human adult liver cDNA library. The clone was selected by direct screening of recombinants with a synthetic oligodeoxynucleotide 17 bases in length corresponding to the known partial DNA sequence of the gene. The insert size of the clone is 250 base pairs. The DNA sequence of the clone has been determined and agrees with the published partial DNA sequence. There is one nucleotide difference from the published sequence, causing a single amino acid change at position 376 where aspartate replaces glutamate. The clone has been used to detect alpha 1-AT mRNA sequences in human liver and in a mixed leukocyte population containing monocytes and lymphocytes. A single mRNA approximately 1,400 nucleotides in length is observed in both leukocytes and liver. Leukocytes contain only 0.15% as much alpha 1-AT mRNA as liver.     

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.     

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.     

Metastability in the inhibitory mechanism of human alpha1-antitrypsin

Metastability of the native form of proteins has been recognized as a mechanism of biological regulation. The energy-loaded structure of the fusion protein of influenza virus and the strained native structure of serpins (serine protease inhibitors) are typical examples. To understand the structural basis and functional role of the native metastability of inhibitory serpins, we characterized stabilizing mutations of alpha1-antitrypsin in a region presumably involved in complex formation with a target protease. We found various unfavorable interactions such as overpacking of side chains, polar-nonpolar interactions, and cavities as the structural basis of the native metastability. For several stabilizing mutations, there was a concomitant decrease in the inhibitory activity. Remarkably, some substitutions at Lys-335 increased the stability over 6 kcal mol-1 with simultaneous loss of activity over 30% toward porcine pancreatic elastase. Considering the location and energetic cost of Lys-335, we propose that this lysine plays a pivotal role in conformational switch during complex formation. Our current results are quite contradictory to those of previously reported hydrophobic core mutations, which increased the stability up to 9 kcal mol-1 without any significant loss of activity. It appears that the local strain of inhibitory serpins is critical for the inhibitory activity.     

Effects of glycosylation on the folding and stability of human, recombinant and cleaved alpha 1-antitrypsin.

The equilibrium unfolding transitions for the human M form of alpha 1-antitrypsin have been determined using a number of techniques reflecting changes in tryptophan fluorescence lifetime and quenching, exposure of tryptophan to solvent, secondary structure and the Stokes' radius of the protein. The denaturation curves are more complex than is usual for globular proteins and indicate the presence of multiple equilibrium intermediates in the presence of denaturant. This is in marked contrast to the more co-operative transition of the cleaved inhibitor. In addition, a recombinant non-glycosylated alpha 1-antitrypsin has been shown to have a closely similar conformation to the human M protein and to exhibit very similar reversible unfolding transitions, and hence similar stability and co-operativity. Differences in tryptophan environment are reflected in the dequenching of tryptophan fluorescence and reduced asymmetry in the near ultraviolet circular dichroism of the non-glycosylated protein, suggesting direct interaction of glycosyl residues with a tryptophan. Both the M type and the recombinant protein exhibit similar patterns of folding, with rapid collapse to a compact intermediate reminiscent of the widely observed molten globule state that folds more slowly to the native protein. The papain-cleaved M form also folds through a similar compact state in the absence of the C-terminal peptide that results from cleavage. It is concluded that part of the C-terminal 36 residue peptide interacts strongly with the main body of the protein in the folded inhibitor. This interaction will also be important during early stages of folding of the intact protein to direct the folding pathway. The lack of glycosylation leads to an increase in aggregation of the recombinant protein upon refolding, especially after extended denaturation times. The more rapid turnover of the recombinant protein in vivo is shown not to be due to a lower thermodynamic stability, but may be associated with a lower kinetic stability arising from the increased tendency to aggregation.