Fluorescence-detected polymerization kinetics of human α1-antitrypsin

The time dependence of the humanα ₁-antitrypsin polymerization process was studied by means of the intrinsic fluorescence stopped-flow technique as well as the fluorescence-quenching-resolved spectra (FQRS) method and native PAGE. The polymerization was induced by mild denaturing conditions (1 M GuHCl) and temperature. The data show that the dimer formation reaction under mild conditions was followed by an increase of fluorescence intensity. This phenomenon is highly temperature sensitive. The structure ofα ₁-antitrypsin dimer resembles the conformation of antithrombin III dimer. In the presence of the denaturant the polymerization process is mainly limited to the dimer state. Theα ₁-antitrypsin activity measurements confirm monomer-to-dimer transition under these conditions. These results are in contrast to the polymerization process induced by temperature, where the dimer state is an intermediate step leading to long-chain polymers. On the basis of stopped-flow and electrophoretic data it is suggested that both C-sheet as well as A-sheet mechanisms contribute to the polymerization process under mild conditions.     

Origin of the multiple components of human α1-antitrypsin

Multiple components of human ₁-antitrypsin were separated by preparative starch gel electrophoresis, and the sialic acid contents of these components were determined. The acidic components contained more sialic acid per molecule than the basic components. The molecular sizes of these components were identical, excluding the possibility of polymerization of the inhibitor in the formation of the multiple components. Consequently, the multiple components of the inhibitor are primarily due to differences in the sialic acid content of each component. Three major components contain eight, seven, and six sialic acid residues per molecule, respectively.This work was supported by Grant HL-17535 from the National Institutes of Health.     

Intracellular precursor forms of transferrin, α1-acid glycoprotein,and α1-antitrypsin in human liver

Ion-exchange chromatography of dialyzed human plasma and of buffer extracts of acetone-dried powder from human liver was used to analyze 13 different plasma proteins which are synthesized in the liver. Specific intracellular forms which differ from the plasma forms were found for transferrin, α₁-acid glycoprotein, α₁-antitrypsin, and albumin. The intracellular forms were labeled earlier than the plasma forms, when liver slices were incubated with radioactive leucine, suggesting that they are precursor forms of the proteins in the bloodstream. The liver form of transferrin was found to have the same molecular weight and N-terminus as the plasma form, but it differed from the plasma form by the absence of sialic acid. For α₁-acid glycoprotein two different liver forms were observed, both of which had lower molecular weights than the plasma form. One of these liver forms was analyzed further. Its polypeptide chain was found to have a blocked N-terminus, as does the plasma form. However, in contrast to the plasma form, it did not contain sialic acid. Its content of N-acetyl glucosamine was about one-third and the content of neutral hexoses about two-thirds of that found in the plasma form. Circular dichroism spectra were similar for liver and plasma forms and indicated a predominant β structure with very little α-helix content for both.     

The reaction of tetranitromethane with human plasma α1-antitrypsin

• 1.1. Three of the 7 tyrosyl residues of plasma α₁-antitrypsin were nitrated with C(NO₂₄.2
• 2.2. Nitration of α₁-antitrypsin with C(NO₂)₄ resulted in some degree of polymerization as shown by gel filtration on Sephadex G-100 column.
• 3.3. The monomeric form was the major reaction product and the overall yield of the monomeric nitro-α₁-antitrypsin was approximately 65 % of the nitrated material.
• 4.4. The monomeric nitro-α₁-antitrypsin retained the ability to form complexes with either trypsin or chymotrypsin as judged by cellulose acetate membrane electrophoresis, and about 85 % of the inhibitory capacity of the native α₁-antitrypsin against both enzymes.

     

Polymorphism of α1-antitrypsin in a Portuguese population

Summary Serum Pi phenotypes were studied in 219 samples. The MM phenotype was the most common as in all other populations. The frequencies of Pi^S and Pi^Z were high considering other populations.Pi^F was not detected.This investigation was supported by a grant from Instituto de Alta Cultura (Project LMC.-10).     

Carbohydrate composition of purified human liver α-L-Fucosidase

Summary Human liver -L-fucosidase was purified to apparent homogeneity and analyzed for carbohydrate content primarily by gas-liquid chromatography (glc). The enzyme is about 7% carbohydrate by weight and contains the following sugars (residues per 50,000 molecular weight subunit): mannose (8.3), glucosamine (4.3) (presumably N-acetylated), sialic acid (1.6) and glucose (1.6). Galactose (0.8) and L-fucose (1.8) were also found but their presence may be due to artifacts of the purification procedure.     

The reaction of human plasma α1-antitrypsin with cyanogen bromide

• 1.1. Human plasma α₁-antitrypsin was subjected to cyanogen bromide cleavage and the peptide fragments were separated into 7 fractions.
• 2.2. The F-4 fraction was further separated into 2 peptides, F-4a and F-4b containing 19 and 24 amino acid residues, respectively.
• 3.3. The F-5 fraction contained only 1 major peptide consisting of 11 amino acid residues.
• 4.4. The F-7 fraction was composed of 2 fragments, F-7a (homoserine) and F-7b, (a tripeptide).
• 5.5. The amino acid sequences of peptide F-4b and F-5 were determined.
• 6.6. The two 1/2 cystine residues in the native α₁-antitrypsin occur in a disulfide linkage.

     

Production of functional human α1-antitrypsin by plant cell culture

Recombinant human α₁-antitrypsin (rAAT) was expressed and secreted from transgenic rice cell suspension cultures in its biologically active form. This was accomplished by transforming rice callus tissues with an expression vector, p3D-AAT, containing the cDNA for mature human AAT protein. Regulated expression and secretion of rAAT from this vector was achieved using the promoter, signal peptide, and terminator from a rice α-amylase gene Amy3D. The Amy3D gene of rice is tightly controlled by simple sugars such as sucrose. It was possible, therefore, to induce the expression of the rAAT by removing sucrose from the cultured media or by allowing the rice suspension cells to deplete sucrose catabolically. Although transgenic rice cell produced a heterogeneous population of the rAAT molecules, they had the same N-terminal amino acids as those found in serum-derived (native) AAT from humans. This result indicates that the rice signal peptidase recognizes and cleaves the novel sequence between the Amy3D signal peptide and the first amino acid of the mature human AAT. The highest molecular weight band seen on Western blots (AAT top band) was found to have the correct C-terminal amino acid sequence and normal elastase binding activity. Staining with biotin-concanavalin A and avidin horseradish peroxidase confirmed the glycosylation of the rAAT, albeit to a lesser extent than that observed with native AAT. The rAAT, purified by immunoaffinity chromatography, had the same association rate constant for porcine pancreatic elastase as the native AAT. Thermostability studies revealed that the rAAT and native AAT decayed at the same rate, suggesting that the rAAT is correctly folded. The productivity of rice suspension cells expressing rAAT was 4.6–5.7 mg/g dry cell. Taken together, these results support the use of rice cell culture as a promising new expression system for production of biologically active recombinant proteins. Received: 18 January 1999 / Received revision: 26 April 1999 / Accepted: 1 May 1999     

Chemical modifications of lysyl and arginyl residues of human plasma α1-antitrypsin

Reactions of human plasma α₁-antitrypsin (α₁-AT) with reagents known to modify the lysyl residues [citraconic anhydride, acetic anhydride, 2,4,6-trinitrobenzenesulfonic acid (TNBS)] and arginyl residues [1,2-cyclohexanedione (CHD) and phenylglyoxal (PGO)] in proteins have been studied. Native and modified human plasma α₁-AT preparations were tested for their inhibitory activities against trypsin and α-chymotrypsin. TNBS was utilized to modify and quantitate free amino groups (?-NH₂ groups of lysine residues) in human plasma α₁-AT. The number of lysine residues determined by the TNBS spectrophotometric procedure agreed well with that found by amino acid analyses. Both the trypsin-inhibitory and chymotrypsin-inhibitory activities of α₁-AT were destroyed by modification with TNBS. CHD was employed to modify the arginyl residues of α₁-AT. Neither the trypsin-inhibitory nor the chymotrypsin-inhibitory activity of α₁-AT was affected by modification of its arginyl residues. Amino acid analyses of the CHD-treated α₁AT revealed that only the arginine residues were modified. PGO was also utilized for the modification of the arginyl residues in α₁-AT. Both the trypsininhibitory and chymotrypsin-inhibitory activities of α₁-AT were destroyed after modification. However, amino acid analyses showed that not only the arginyl, but also the lysyl residues of the PGO-treated inhibitor were modified. The side reaction of PGO with the lysyl residues could explain the loss of inhibitory activities. Reaction of a α₁-AT with citraconic anhydride resulted in an extensive modification of the amino groups accompanied by a 100% loss in inhibitory activity against both trypsin and α-chymotrypsin. Comparable results were observed when acetic anhydride was utilized as the acylating reagent. With the exception of the citraconylated α₁AT, all of the other chemically modified α₁-AT derivatives studied presently retained their immunological reactivities against antisera to native α₁-AT. Regeneration of about 60% of the PGO-blocked arginyl residues in α₁-AT did not lead to any recovery of the proteinase inhibitory activities. Full recovery of trypsin-inhibitory and immunological activities were achieved when about 50% of the citraconylated amino groups were deblocked. The CHD-treated α₁-AT still retained the capacity to form complexes with both trypsin and chymotrypsin. On the other hand, the other chemically modified α₁-AT derivatives have completely lost the ability to form complexes with the enzymes. Recovery of the ability to form complexes with the enzymes was, however, recovered when about 50% of the citraconylyl groups was removed from the α₁-AT molecule. Based on these modification studies, it is concluded that α₁-AT is a lysyl inhibitor type (i.e., the reactive site is Lys-X bond) and that the interaction of α₁-AT with trypsin or chymotrypsin very likely involves or requires the same site as in the case of the soybean trypsin inhibitor (Kunitz).     

N-glycan analysis of human α1-antitrypsin produced in Chinese hamster ovary cells

Human alpha-1-antitrypsin (α1AT) is a glycoprotein with protease inhibitor activity protecting tissues from degradation. Patients with inherited α1AT deficiency are treated with native α1AT (nAT) purified from human plasma. In the present study, recombinant α1AT (rAT) was produced in Chinese hamster ovary (CHO) cells and their glycosylation patterns, inhibitory activity and in vivo half-life were compared with those of nAT. A peptide mapping analysis employing a deglycosylation reaction confirmed full occupancy of all three glycosylation sites and the equivalency of rAT and nAT in terms of the protein level. N-glycan profiles revealed that rAT contained 10 glycan structures ranging from bi-antennary to tetra-antennary complex-type glycans while nAT displayed six peaks comprising majorly bi-antennary glycans and a small portion of tri-antennary glycans. In addition, most of the rAT glycans were shown to have only core α(1?-?6)-fucose without terminal fucosylation, whereas only minor portions of the nAT glycans contained core or Lewis X-type fucose. As expected, all sialylated glycans of rAT were found to have α(2?-?3)-linked sialic acids, which was in sharp contrast to those of nAT, which had mostly α(2?-?6)-linked sialic acids. However, the degree of sialylation of rAT was comparable to that of nAT, which was also supported by an isoelectric focusing gel analysis. Despite the differences in the glycosylation patterns, both α1ATs showed nearly equivalent inhibitory activity in enzyme assays and serum half-lives in a pharmacokinetic experiment. These results suggest that rAT produced in CHO cells would be a good alternative to nAT derived from human plasma.     

Development, preparation, and testing of VAQTA®, a highly purified hepatitis A vaccine

Manufacture of VAQTA^®, an inactivated hepatitis A vaccine, uses state-of-the-art technologies in cell culture and bioprocessing science, which have made it possible to routinely produce the vaccine at manufacturing scale. VAQTA^® consists of an attenuated strain of hepatitis A virus that is highly purified and formaldehyde-inactivated, then formulated with an aluminum hydroxide adjuvant. Process development and scale-up have resulted in a well-characterized vaccine manufacturing process with appropriate in-process controls to assure consistent performance, and a reproducible, well-defined product. Results are presented from a series of manufacturing demonstration lots to show consistency, as well as comparability to clinical lots prepared at an earlier stage in development.     

Multiple molecular forms of serum α1-antitrypsin

Total soluble proteins, peroxidase, and peroxidase isozymes were examined in polyploid series of fern gametophytes and sporophytes. A distinctive pattern of protein bands was associated with gametophytes and sporophytes and the pattern did not vary within each phenotype with increases in the genome. Peroxidase activity per cell increased in direct proportion to increases in the genome and was determined to be gene dosage related. Slight differences in the patterns of peroxidase isozyme bands were associated with increases in the chromosome complement in both series of plants, but major variations were found between gametophyte and sporophyte. Quantitative analysis of peroxidase activity in each band revealed both increases and decreases in individual isozymes as ploidy increased. These findings suggest the involvement of regulatory mechanisms controlling isozyme activity.     

Purification of phenotypically unaltered α-1-antitrypsin

A method is presented for the purification of α-1-antitrypsin from normal human plasma. The method results in recovery of an average of 12.1% of the trypsin-inhibiting capacity of the serum. The electrophoretic heterogeneity that characterizes the phenotype of α-1-antitrypsin in serum is unchanged. Thus, the phenotypic heterogeneity is not dependent upon serum factors. In addition, the material has 85–100% biologic activity and migrates as a single band in polyacrylamide gel electrophoresis with sodium dodecylsulfate and migrates as a band with a cathodic shoulder at pH 7.5 in polyacrylamide gel electrophoresis.     

Molecular basis of α1-antitrypsin deficiency revealed by the structure of a domain-swapped trimer

α(1)-Antitrypsin (α1AT) deficiency is a disease with multiple manifestations, including cirrhosis and emphysema, caused by the accumulation of stable polymers of mutant protein in the endoplasmic reticulum of hepatocytes. However, the molecular basis of misfolding and polymerization remain unknown. We produced and crystallized a trimeric form of α1AT that is recognized by an antibody specific for the pathological polymer. Unexpectedly, this structure reveals a polymeric linkage mediated by domain swapping the carboxy-terminal 34 residues. Disulphide-trapping and antibody-binding studies further demonstrate that runaway C-terminal domain swapping, rather than the s4A/s5A domain swap previously proposed, underlies polymerization of the common Z-mutant of α1AT in vivo.     

Recombinant production of native human α-1-antitrypsin protein in the liver HepG2 cells

Objectives

Alpha-1 antitrypsin (A1AT) deficiency is associated with emphysema and liver disease. Only plasma-derived A1AT protein is available for augmentation therapy. Recombinant A1AT (recA1AT) protein expressed in various types of available hosts are either non-glycosylated or aberrantly glycosylated resulting into reduced stability and biological activity. To overcome these limitations, we have used the human liver HepG2 cell line to produce recA1AT protein.

Results

HepG2 cells were transfected by A1AT cDNA and cell populations were generated that stably overexpressed A1AT protein. Real-time RT-PCR and rocket immunoelectrophoresis of cell culture supernatants indicated that the transfection resulted more than two-fold increase in A1AT production compared to that of control parental cells. Immunoblot analysis showed that both plasma and HepG2-produced A1AT proteins have identical molecular weight in either glycosylated or deglycosylated form. Partial digestion with PNGase F indicated that the three N-glycosylation sites of recA1AT, like the native A1AT protein in plasma, are occupied. Recombinant A1AT also like the native A1AT was thermostable and could efficiently inhibit trypsin proteolytic activity against BSA and BAPNA chromogenic substrate. The recombinant HepG2 cells cultured in media containing B27 serum free supplement released recA1AT at the same level as in the serum containing media.

Conclusions

RecA1AT production in HepG2 cells grown under serum free condition at a large scale could provide a reliable source of the native protein suitable for therapeutic use in human.

     

Expression of modified gene encoding functional human α-1-antitrypsin protein in transgenic tomato plants

Transgenic plants offer promising alternative for large scale, sustainable production of safe, functional, recombinant proteins of therapeutic and industrial importance. Here, we report the expression of biologically active human alpha-1-antitrypsin in transgenic tomato plants. The 1,182 bp cDNA sequence of human AAT was strategically designed, modified and synthesized to adopt codon usage pattern of dicot plants, elimination of mRNA destabilizing sequences and modifications around 5' and 3' flanking regions of the gene to achieve high-level regulated expression in dicot plants. The native signal peptide sequence was substituted with modified signal peptide sequence of tobacco (Nicotiana tabacum) pathogenesis related protein PR1a, sweet potato (Ipomoea batatas) sporamineA and with dicot-preferred native signal peptide sequence of AAT gene. A dicot preferred translation initiation context sequence, 38 bp alfalfa mosaic virus untranslated region were incorporated at 5' while an endoplasmic reticulum retention signal (KDEL) was incorporated at 3' end of the gene. The modified gene was synthesized by PCR based method using overlapping oligonucleotides. Tomato plants were genetically engineered by nuclear transformation with Agrobacterium tumefaciens harbouring three different constructs pPAK, pSAK and pNAK having modified AAT gene with different signal peptide sequences under the control of CaMV35S duplicated enhancer promoter. Promising transgenic plants expressing recombinant AAT protein upto 1.55% of total soluble leaf protein has been developed and characterized. Plant-expressed recombinant AAT protein with molecular mass of around approximately 50 kDa was biologically active, showing high specific activity and efficient inhibition of elastase activity. The enzymatic deglycosylation established proper glycosylation of the plant-expressed recombinant AAT protein in contrast to unglycosylated rAAT expressed in E. coli ( approximately 45 kDa). Our results demonstrate feasibility for high-level expression of biologically active, glycosylated human alpha-1-antitrypsin in transgenic tomato plants.     

Mysteries of α1-antitrypsin deficiency: emerging therapeutic strategies for a challenging disease

The classical form of α1-antitrypsin deficiency (ATD) is an autosomal co-dominant disorder that affects ~1 in 3000 live births and is an important genetic cause of lung and liver disease. The protein affected, α1-antitrypsin (AT), is predominantly derived from the liver and has the function of inhibiting neutrophil elastase and several other destructive neutrophil proteinases. The genetic defect is a point mutation that leads to misfolding of the mutant protein, which is referred to as α1-antitrypsin Z (ATZ). Because of its misfolding, ATZ is unable to efficiently traverse the secretory pathway. Accumulation of ATZ in the endoplasmic reticulum of liver cells has a gain-of-function proteotoxic effect on the liver, resulting in fibrosis, cirrhosis and/or hepatocellular carcinoma in some individuals. Moreover, because of reduced secretion, there is a lack of anti-proteinase activity in the lung, which allows neutrophil proteases to destroy the connective tissue matrix and cause chronic obstructive pulmonary disease (COPD) by loss of function. Wide variation in the incidence and severity of liver and lung disease among individuals with ATD has made this disease one of the most challenging of the rare genetic disorders to diagnose and treat. Other than cigarette smoking, which worsens COPD in ATD, genetic and environmental modifiers that determine this phenotypic variability are unknown. A limited number of therapeutic strategies are currently available, and liver transplantation is the only treatment for severe liver disease. Although replacement therapy with purified AT corrects the loss of anti-proteinase function, COPD progresses in a substantial number of individuals with ATD and some undergo lung transplantation. Nevertheless, advances in understanding the variability in clinical phenotype and in developing novel therapeutic concepts is beginning to address the major clinical challenges of this mysterious disorder.KEY WORDS: α1-antitrypsin deficiency, Autophagy, Liver disease     

Induction of α1-antitrypsin mRNA and cloning of its cDNA

Local inflammation was inflicted in a baboon by turpentine administration in order to induce the plasma level of α1-antitrypsin, an acute phase protein synthesized in the liver. Comparison of the α1-antitrypsin mRNA activity in the induced and non-induced baboon liver indicated that the “acute phase” response to chemical-inflicted inflammation is mediated through an increase in the steady-state level of cellular mRNA. Alpha-1-antitrypsin was then enriched from the induced baboon liver to a purity of greater than 90% by specific immunoprecipitation of polysomes. Double-stranded DNA was synthesized from the enriched mRNA and inserted into the $\text{Pst}$ I site of pBR322. Recombinant clones containing α1-antitrypsin cDNA sequences were identified by hybridselected translation and confirmed by DNA sequence analysis.