High-level expression of active human alpha1-antitrypsin in transgenic tobacco chloroplasts

We have produced human alpha1-antitrypsin (A1AT), a major therapeutic protein, in genetically engineered tobacco plastids. Four different expression vectors have been evaluated which encode A1AT under the control of various 5′ and 3′ plastid expression elements. The use of heterologous promoter and terminator sequences derived from the corn and soybean plastid genomes leads to simpler and predictable recombinant genome patterns, avoiding unwanted recombination products between introduced and resident tobacco sequences. High level expression of unglycosylated A1AT, representing up to 2% of total soluble proteins, has been measured in leaves of transgenic tobacco lines. Some heterogeneity in the recombinant A1AT is detected after 2D protein separation, but the chloroplast-made protease inhibitors are fully active and bind to porcine pancreatic elastase.     

The human alpha-1-antitrypsin gene is efficiently expressed from two tissue-specific promotors in transgenic mice.

Alpha 1-antitrypsin (alpha 1AT) present in large amounts in human serum and synthesized predominantly in hepatocytes, is the most abundant protease inhibitor and alpha 1AT mutant proteins are associated with hereditary disorders. To investigate the regulation of the normal human alpha 1AT gene, we have microinjected fertilized mouse eggs with a 17.5 kb DNA fragment containing the entire gene with 7 kb 5' and 0.3 kb 3' flanking sequences. We show that this DNA fragment contains all the information for efficient, accurate and tissue-specific expression. High serum concentration of the human protein was found in three independent transgenic mouse lines. The human alpha 1AT RNA is transcribed efficiently in liver, kidney and macrophages and we demonstrate that two different promoters are used for the expression in liver and macrophages of the transgenic mice.     

Regional localization of human gene loci on chromosome 9: studies of somatic cell hybrids containing human translocations.

Somatic cell hybrids were derived from the fusion of (1) Chinese hamster cells deficient in hypoxanthine guanine phosphoribosyltransferase (HPRT) and human cells carrying an X/9 translocation and (2) Chinese hamster cells deficient in thymidine kinase (TK) and human cells carrying a 17/9 translocation. Several independent primary hybrid clones from these two series of cell hybrids were analyzed cytogenitically for human chromosome content and electrophoretically for the expression of human markers known to be on human chromosome 9. The results allow the assignment of the loci for the enzymes galactose-1-phosphate uridyltransferase (GALT), soluble aconitase (ACONs), and adenylate kinase-3 (AK3) to the short arm of chromosome 9 (p11 to pter) and the locus for the enzyme adenylate kinase-1 (AK1) to the distal end of the long arm of human chromosome 9 (hand q34). Earlier family studies have shown that the locus for AK1 is closely linked to the ABO blood group locus and to the locus of the nail-patella (Np) syndrome. Thus the regional localization of AK1 locus permits the localization of the AK1-Np-ABO linkage group.     

Expression of human hepatic genes in somatic cell hybrids

Four diploid human cell types (lymphocytes, fibroblasts, amniotic fluid cells, and hepatocytes) were fused to mouse hepatoma cells, HH. HH synthesized and secreted several liver-specific gene products including albumin, transferrin, and alpha-fetoprotein. The resulting interspecific hybrids were compared to determine whether or not the pattern of human hepatic gene expression was similar when these various cells were fused with the mouse hepatoma line. The expression of six human hepatic genes was examined, including albumin, alpha-fetoprotein, ceruloplasmin, transferrin, alpha-1-antitrypsin, and haptoglobin. Albumin was most frequently expressed while alpha-fetoprotein was not detected in any of the hybrids studied. The patterns of expression of human serum proteins differed between the hybrid series. Hybrids derived from human fibroblasts produced primarily albumin, while those derived from lymphoblastoid cells and amniocytes had a higher frequency of clones secreting alpha-1-antitrypsin. The findings reported here suggest that the frequency of hybrid clones expressing human hepatic gene products and the array of proteins produced are influenced by the histogenetic state of the human parental cell type.     

Inhibition of human pancreatic proteinases by human plasma alpha2-antiplasmin and antithrombin

Human plasma alpha1-antitrypsin inhibits human pancreatic trypsin, chymotrypsin and elastase, which are massively released into the blood stream during acute pancreatitis. To examine whether the plasma proteins of individuals with genetic deficiency of alpha1-antitrypsin are protected against the deleterious action of these enzymes by other inhibitors, we have tested their inhibition by alpha2-antiplasmin and antithrombin. We have determined the inhibition rate constants kass and calculated d(t), the in vivo inhibition time. Surprisingly, trypsin is inhibited faster by alpha2-antiplasmin [kass=2.5 x 10(6) M(-1)S(-1), d(t)=2.3 s] and antithrombin [kass=1.7 x 10(5) M(-1)s(-1), d(t)=5.8 s] than by alpha1-antitrypsin [d(t)=17 s or 116 s in alpha1-antitrypsin-sufficient or alpha1-antitrypsin-deficient individuals, respectively]. Low molecular weight heparin accelerates the inhibition of trypsin by antithrombin by a factor of 16 [d(t)=0.36 s]. Antithrombin and alpha2-antiplasmin are not physiological inhibitors of chymotrypsin and elastase. These enzymes are, however, physiologically inhibited by alpha1-antitrypsin and alpha1-antichymotrypsin even in alpha1-antitrypsin-deficient individuals. We conclude that (i) low molecular weight heparin may be helpful in the management of acute pancreatitis, and (ii) genetically determined alpha1-antitrypsin deficiency probably does not lead to a significantly increased risk of plasma protein degradation during this disease.     

Isolation of a cDNA clone for human antithrombin III

Antithrombin III (ATIII) is an important plasma protease inhibitor with a central role in the coagulation system. On the basis of its protein sequence, ATIII is one member of a "super family" of protease inhibitors that includes alpha 1-antitrypsin and chicken ovalbumin. An increased risk of thromboembolism is associated with inherited ATIII deficiency. To study the structure and expression of the human ATIII gene, we have isolated complementary (cDNA) clones for ATIII from human liver mRNA. ATIII cDNA clones were identified by hybridization to a mixture of synthetic oligodeoxynucleotides encoding amino acids 251-256 of the ATIII protein sequence. The largest cDNA clone (1.4 kilobases) included the coding region of ATIII mRNA from codon 10 through a 3'-untranslated region. Comparison of ATIII cDNA clones from two different sources revealed a sequence polymorphism at an internal PstI restriction site. Analysis of both total genomic DNAs and an ATIII gene cloned in a bacteriophage Charon 4A showed that the ATIII gene is present once per haploid genome and is distributed over 10-16 kilobases of DNA. Computer-assisted comparison of the cDNA sequence with those for baboon alpha 1-antitrypsin and chicken ovalbumin revealed homologies consistent with their inclusion in the protease inhibitor superfamily.     

Structure of human alpha 2-plasmin inhibitor deduced from the cDNA sequence

We have isolated three cDNA clones for human alpha 2-plasmin inhibitor (alpha 2-PI). Two clones are from human hepatoma cell line, Hep G2, and cover the entire protein coding region plus the 3'-flanking region up to the poly(A) sequence, and the other clone is from human liver and contains the carboxyl-terminal half. The total length of the cDNAs is 2.29 kb, corresponding to more than 95% of the full-length mRNA. alpha 2-PI seems to consist of 452 amino acid residues plus 39 amino acid residues for the signal peptide. The amino acid sequence shows 23 to 28% homology to those of five other protease inhibitors, plasminogen activator inhibitor (PAI), protein C inhibitor (PCI), alpha 1-antitrypsin (alpha 1-AT), antithrombin III (AT III), and alpha 1-antichymotrypsin (alpha 1-AC). alpha 2-PI seems to be the most distantly related among these inhibitors. Comparison of the phylogenetic trees of proteases and their inhibitors indicates that four proteases, namely elastase (or trypsin), chymotrypsin, plasminogen activator, and thrombin, may have evolved concurrently with the corresponding inhibitors. However, alpha 2-PI and PCI seem to have evolved asynchronously from their substrates. The data suggest that alpha 2-PI may originally have inhibited some protease other than plasmin, and protein C may have had an inhibitor different from the present one early in its evolutionary history.     

Structure of the human alpha 1-acid glycoprotein gene: sequence homology with other human acute phase protein genes.

We have determined the sequence coding for human alpha 1-acid glycoprotein from two independently isolated cDNA clones and a genomic clone. The aminoacid sequences deduced from the three clones, deriving from three different individuals, are identical. Southern blot analysis on human DNA indicates that there are at least two genes coding for alpha 1-AGP. We propose that alpha 1-AGP found in plasma is a mixture of the products of these two different genes. This is the simpler explanation for the heterogeneity in the aminoacid composition in purified alpha 1-AGP observed by Schmid et al. (1). DNA sequence comparison with cDNA clones coding for human alpha 1-antitrypsin and haptoglobin shows a conserved sequence within the 5' untranslated region which may play a role in the acute phase response.     

Sequence homology between human alpha 1-antichymotrypsin, alpha 1-antitrypsin, and antithrombin III

alpha 1-Antichymotrypsin mRNA was isolated by specific polysome immunoprecipitation from turpentine-treated baboon liver. The highly enriched mRNA was used for synthesis and cloning of the corresponding cDNA. Baboon alpha 1-antichymotrypsin cDNA clones were identified by hybrid-selected translation, and the insert DNA fragment from one of the putative clones was used as a probe to screen a human liver cDNA library comprised of 40 000 independent transformants. One of the human cDNA clones was unambiguously identified to contain alpha 1-antichymotrypsin DNA sequences by comparison of its 5'-terminal nucleotide sequence with the N-terminal amino acid sequence of the protein. This cDNA clone, designated phACT235, contains 1524 base pairs of human DNA, which was sequenced in its entirety. The inserted DNA codes for a 25 amino acid signal peptide sequence and the entire mature alpha 1-antichymotrypsin of 408 amino acid residues. Comparison of the amino acid sequence of alpha 1-antichymotrypsin with that of the human alpha 1-antitrypsin has revealed a homology level similar to that between chymotrypsin and trypsin.     

cDNA cloning of human inter-alpha-trypsin inhibitor discloses three different proteins

Inter-alpha-trypsin inhibitor (ITI) is a serum protein of unknown function. Part of the molecule (formerly called HI30) is closely related to a tumor-derived protein acting as a growth factor for endothelial cells. We screened a human liver cDNA expression library with antibodies raised against human ITI and isolated several clones which could be divided into three groups according to their DNA sequences. The cDNA of the first group codes for a protein composed of alpha 1-microglobulin (alpha 1M) and urinary trypsin inhibitor (UTI) and is identical to that encoded by a clone originally found by screening a human liver cDNA library with oligonucleotides derived from amino-acid sequences of the two Kunitz-type domains of UTI. The proteins derived from the cDNA of the second and the third group of clones are distantly related to each other, but unrelated to the protein derived from group 1 clones. Partial amino-acid sequencing of ITI isolated from serum allowed the verification of large parts of the cDNA-derived amino-acid sequences. The results favour the view that ITI is not a single chain protein, but rather a very tight complex of several components or a mixture of such complexes.     

Enhanced specificity in immunoscreening of expression cDNA clones using radiolabeled antigen overlay

A highly sensitive and specific method has been developed for immunoscreening clones from an expression cDNA library. The procedures utilize a radiolabeled antigen detection method described originally for the immunoblotting of plasma proteins (5). Screening of rat alpha 1-antitrypsin clones was used. Comparison between Western blots of alpha 1-antitrypsin using both labeled antigen and protein A detection methods showed that the former yielded lower background and greater sensitivity than the latter. Further, this technique was shown to have a lower detection limit of less than 20 ng through Western blot analysis of varying concentrations of alpha 1-antitrypsin. The procedures are based on the expression of the protein by cDNA clones containing the DNA inserts in the correct reading frame. Following the transfer of phage proteins to nitrocellulose membranes, the bivalent antibodies bind monovalently to both nitrocellulose-bound-antigen in the phage lysates and radiolabeled antigen. The radiolabeled antigen overlay method is superior to the protein A detection method in sensitivity, specificity and reproducibility. This improved method can be applied in general for screening expression cDNA libraries, provided that the specific antiserum and radiolabeled antigen are available.     

Presence of alpha1-antitrypsin and transferrin in human follicular fluid--correlation with fertilization

We purified two proteins with molecular masses of approximately 50 kDa and 80 kDa with N-terminal sequences similar to those of alpha1-antitrypsin (a1AT) and transferrin indicating that they are identical to or highly homologous to these proteins. Proteins from human follicular fluid were purified after ammonium sulfate fractionation followed by water dialysis and High Performance Liquid Chromatography. The fraction of peak 3 showed a single band on electrophoresis and its N-terminal amino acid sequence was similar to that of human serum transferrin. The fraction of peak 10 proved to be a glycoprotein and its N-terminal amino acid sequence was similar to that of human serum a1AT. There are indications that transferrin may be involved in the fertilization process. Sperm motion was assessed employing computer-assisted semen analysis. The addition of purified protein to prepared sperm samples from normospermic men significantly increases the straight-line velocity (VSL), the amplitude of lateral head displacement (ALH) and the number of progressively motile sperm. a1AT does not seem to have a stimulatory effect on sperm motility.     

RNA structural elements for expression in Escherichia coli. Alpha 1-antitrypsin synthesis using translation control elements based on the cII ribosome-binding site of phage lambda

Analysis of a series of lambda cII::alpha 1-antitrypsin (alpha 1AT) gene fusions of different sizes showed that increased alpha 1AT expression correlated with the stabilisation of a particular computer-predicted RNA secondary structure. Moreover, significant synthesis of unfused alpha 1AT was achieved by reconstruction of this conformation to permit interaction between the upstream region of the ribosome-binding site and the first part of the alpha 1AT coding sequence. This high-level expression was dependent upon certain silent point mutations in the coding sequence, indicating that RNA primary and secondary structure determinants can operate in concert to dictate the efficiency of protein synthesis.     

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.     

Alterations in the mouse and human proteome caused by Huntington's disease

Huntington's disease is an autosomal dominantly inherited disease that usually starts in midlife and inevitably leads to death. In our effort to identify proteins involved in processes upstream or downstream of the disease-causing huntingtin, we studied the proteome of a well established mouse model by large gel two-dimensional electrophoresis. We could demonstrate for the first time at the protein level that alpha1-antitrypsin and alphaB-crystalline both decrease in expression over the course of disease. Importantly, the alpha1-antitrypsin decrease in the brain precedes that in liver and testes in mice. Reduced expression of the serine protease inhibitors alpha1-antitrypsin and contraspin was found in liver, heart, and testes close to terminal disease. Decreased expression of the chaperone alphaB-crystallin was found exclusively in the brain. In three brain regions obtained post-mortem from Huntington's disease patients, alpha1-antitrypsin expression was also altered. Reduced expression of the major urinary proteins not found in the brain was seen in the liver of affected mice, demonstrating that the disease exerts its influence outside the brain of transgenic mice at the protein level. Maintaining alpha1-antitrypsin and alphaB-crystallin availability during the course of Huntington's disease might prevent neuronal cell death and therefore could be useful in delaying the disease progression.     

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.     

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.