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.     

Molecular evolution of serpins: homologous structure of the human alpha 1-antichymotrypsin and alpha 1-antitrypsin genes

alpha 1-Antichymotrypsin belongs to a supergene family that includes alpha 1-antitrypsin, antithrombin III, ovalbumin, and angiotensinogen. The human chromosomal alpha 1-antichymotrypsin gene has been cloned and its molecular structure established. The gene is approximately 12 kb in length and contains five exons and four introns. The locations of the introns within the alpha 1-antichymotrypsin gene are identical with those of the human alpha 1-antitrypsin and angiotensinogen genes. Other members of this supergene family contain introns located at nonhomologous positions of the genes. The homologous organization of the alpha 1-antichymotrypsin and alpha 1-antitrypsin genes corresponds with the high degree of homology between their protein sequences and suggests that these loci arose by recent gene duplication. A model is presented for the evolution of both the genomic structure and the protein sequences of the serine protease inhibitor superfamily.     

Intron structure of the human antithrombin III gene differs from that of other members of the serine protease inhibitor superfamily

Antithrombin III (ATIII) plays an integral role in the coagulation system by inhibiting thrombin and several other activated clotting factors. Inherited deficiency of ATIII is quite common and can result in life-threatening thrombotic complications. In order to understand the basis of ATIII deficiency, we have isolated and characterized the normal human ATIII gene from a recombinant Charon 4A bacteriophage genomic library. The ATIII gene contains six exons and five introns distributed over approximately 19 kilobases of DNA. The positions of introns in the ATIII gene were compared with other members of the serine protease inhibitor family which share 17-31% amino acid homology. When aligned to achieve maximal protein homology, only one of the ATIII introns corresponded to the four introns of rat angiotensinogen or human alpha 1-antitrypsin. Similarly, only one ATIII intron was homologous to the seven introns of chicken ovalbumin. We present two testable models to explain the discrepancy in intron positions among members of the serine protease inhibitor superfamily of genes.     

TNF-alpha-induced self expression in human lung endothelial cells is inhibited by native and oxidized alpha1-antitrypsin

Endothelial cells are among the main physiological targets of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). In endothelial cells TNF-alpha elicits a broad spectrum of biological effects including differentiation, proliferation and apoptosis. alpha1-antitrypsin (AAT), an endogenous inhibitor of serine proteases plays a vital role in protecting host tissue from proteolytic injury at sites of inflammation. Recently, it has been shown that AAT can be internalized by pulmonary endothelial cells, raising speculation that it may modulate endothelial cell function in addition to suppressing protease activity. Using Affymetrix microarray technology, real time PCR and ELISA methods we have investigated the effects of AAT on un-stimulated and TNF-alpha stimulated human primary lung microvascular endothelial cell gene expression and protein secretion. We find that AAT and TNF-alpha generally induced expression of distinct gene families with AAT exhibiting little activity in terms of inflammatory gene expression. Approximately 25% of genes up regulated by TNF-alpha were inhibited by co-administration of AAT including TNF-alpha-induced self expression. Surprisingly, the effects of AAT on TNF-alpha-induced self expression was inhibited equally well by oxidized AAT, a modified form of AAT, which lacks serine protease inhibitor activity. Overall, the pattern of gene expression regulated by native and oxidized AAT was similar with neither inducing pro-inflammatory gene expression. These findings suggest that inhibitory effects of native and oxidized forms of AAT on TNF-alpha stimulated gene expression may play an important role in limiting the uncontrolled endothelial cell activation and vascular injury in inflammatory disease.     

Alpha1-antitrypsin, old dog, new tricks. Alpha1-antitrypsin exerts in vitro anti-inflammatory activity in human monocytes by elevating cAMP

Regulation of serine protease activity is considered to be the sole mechanism for the function of alpha1-antitrypsin (AAT). However, recent reports of the anti-inflammatory effects of AAT are hard to reconcile with this classical mechanism. We discovered that two key activities of AAT in vitro, namely inhibition of endotoxin-stimulated tumor necrosis factor-alpha and enhancement of interleukin-10 in human monocytes, are mediated by an elevation of cAMP and activation of cAMP-dependent protein kinase A. As expected with this type of mechanism, the AAT-mediated rise in cAMP and the impact on endotoxin-stimulated tumor necrosis factor-alpha and interleukin-10 was enhanced when the catabolism of cAMP was blocked by the phosphodiesterase inhibitor rolipram. These effects were still observed with modified forms of AAT lacking protease inhibitor activity.     

A review and comparison of the murine alpha1-antitrypsin and alpha1-antichymotrypsin multigene clusters with the human clade A serpins

The major human plasma protease inhibitors, alpha(1)-antitrypsin and alpha(1)-antichymotrypsin, are each encoded by a single gene, whereas in the mouse they are represented by clusters of 5 and 14 genes, respectively. Although there is a high degree of overall sequence similarity within these groupings, the reactive-center loop (RCL) domain, which determines target protease specificity, is markedly divergent. The literature dealing with members of these mouse serine protease inhibitor (serpin) clusters has been complicated by inconsistent nomenclature. Furthermore, some investigators, unaware of the complexity of the family, have failed to distinguish between closely related genes when measuring expression levels or functional activity. We have reviewed the literature dealing with the mouse equivalents of human alpha(1)-antitrypsin and alpha(1)-antichymotrypsin and made use of the recently completed mouse genome sequence to propose a systematic nomenclature. We have also examined the extended mouse clade "a" serpin cluster at chromosome 12F1 and compared it with the syntenic region at human chromosome 14q32. In summarizing the literature and suggesting a standardized nomenclature, we aim to provide a logical structure on which future research may be based.     

Primary structure of human alpha 2-antiplasmin, a serine protease inhibitor (serpin)

The plasma protein alpha 2-antiplasmin is the main physiological inhibitor of the serine protease plasmin, which is responsible for the dissolution of fibrin clots. We have determined the primary structure of mature human alpha 2-antiplasmin by DNA sequencing of overlapping cDNA fragments prepared from human liver mRNA. cDNA clones were identified by hybridization with a 48-base pair deoxyoligonucleotide probe deduced from the sequence of a 16-amino acid peptide of alpha 2-antiplasmin. Mature human alpha 2-antiplasmin contains 452 amino acids. It is homologous (23-28%) with five other proteins belonging to the serine protease inhibitor (serpin) superfamily. Its reactive site, i.e. the peptide bond cleaved by reaction with its primary target enzyme, plasmin, consists of Arg364-Met365. This dipeptide corresponds to the reactive site Met358-Ser359 of the archetypal serpin, alpha 1-antitrypsin.     

Human C1 inhibitor: primary structure, cDNA cloning, and chromosomal localization

The primary structure of human C1 inhibitor was determined by peptide and DNA sequencing. The single-chain polypeptide moiety of the intact inhibitor is 478 residues (52,869 Da), accounting for only 51% of the apparent molecular mass of the circulating protein (104,000 Da). The positions of six glucosamine-based and five galactosamine-based oligosaccharides were determined. Another nine threonine residues are probably also glycosylated. Most of the carbohydrate prosthetic groups (probably 17) are located at the amino-terminal end (residues 1-120) of the protein and are particularly concentrated in a region where the tetrapeptide sequence Glx-Pro-Thr-Thr, and variants thereof, is repeated 7 times. No phosphate was detected in C1 inhibitor. Two disulfide bridges connect cysteine-101 to cysteine-406 and cysteine-108 to cysteine-183. Comparison of the amino acid and cDNA sequences indicates that secretion is mediated by a 22-residue signal peptide and that further proteolytic processing does not occur. C1 inhibitor is a member of the large serine protease inhibitor (serpin) gene family. The homology concerns residues 120 through the C-terminus. The sequence was compared with those of nine other serpins, and conserved and nonconserved regions correlated with elements in the tertiary structure of alpha 1-antitrypsin. The C1 inhibitor gene maps to chromosome 11, p11.2-q13. C1 inhibitor genes of patients from four hereditary angioneurotic edema kindreds do not have obvious deletions or rearrangements in the C1 inhibitor locus. A HgiAI DNA polymorphism, identified following the observation of sequence variants, will be useful as a linkage marker in studies of mutant C1 inhibitor genes.     

Characterization of a cDNA for human protein C inhibitor. A new member of the plasma serine protease inhibitor superfamily

A cDNA library in lambda-phage lambda gt11 containing DNA inserts prepared from human liver mRNA was screened with monoclonal antibodies to human protein C inhibitor. Six positive clones were isolated from 6 X 10(6) phages and plaque purified. The cDNA in the phage containing the largest insert, which hybridized to a DNA probe prepared on the basis of the amino-terminal amino acid sequence of the mature inhibitor, was sequenced. This cDNA insert contained 2106 base pairs coding for a 5'-noncoding region, a 19-amino acid signal peptide, a 387-amino acid mature protein, a stop codon, and a long 3'-noncoding region of 839 base pairs. Based on the amino acid sequence of the carboxyl-terminal peptide released by cleavage of protein C inhibitor by activated protein C as well as by thrombin, the reactive site peptide bond of protein C inhibitor is Arg354-Ser355. Five potential carbohydrate-binding sites were found in the mature protein. The high homology of the amino acid sequence of protein C inhibitor to the other known inhibitors clearly demonstrates that protein C inhibitor is a member of the superfamily of serine protease inhibitors including alpha 1-antichymotrypsin, alpha 1-antitrypsin, antithrombin III, ovalbumin, and angiotensinogen. Based on the difference matrices for these proteins, we present possible phylogenetic trees for these proteins.     

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.     

An alpha-1 antitrypsin genetic variant from India

The highly polymorphic human alpha-1 antitrypsin (AAT) gene codes for the most abundant circulating plasma serine protease inhibitor. Previously, genetic variants of the AAT gene were reported from different regions of the world. In the present study, the AAT gene was characterized in an Indian sample. The AAT gene was isolated and cloned from a liver biopsy sample through RT-PCR and the full-length gene was sequenced. Nucleotide sequence comparison with the human genome and the AAT sequences available in the GenBank (NCBI) demonstrated four unique variations--(i) an A to G variation at position 286 (Thr96Ala), (ii) an A to G variation at position 839 (Asp280Gly), (iii) a T to C variation at position 1182 that did not result in any change in the protein sequence (TTT to TTC both code for Phe) and (iv) an A to C variation at position 1200 (Glu400Asp) that resulted in replacement by an amino acid of similar nature. Other variations found were T to C at position 710 (Val273Ala) and T to C position 863 (Val288Glu), which were also reported earlier. In conclusion, this study reports the entire 1257 bp nucleotide sequence of protein coding region of the human AAT gene from an Indian sample. This preliminary finding is significant, as it reports for the first time the AAT gene sequence in the Indian sample.     

Retarded protein folding of deficient human alpha 1-antitrypsin D256V and L41P variants

alpha(1)-Antitrypsin is the most abundant protease inhibitor in plasma and is the archetype of the serine protease inhibitor superfamily. Genetic variants of human alpha(1)-antitrypsin are associated with early-onset emphysema and liver cirrhosis. However, the detailed molecular mechanism for the pathogenicity of most variant alpha(1)-antitrypsin molecules is not known. Here we examined the structural basis of a dozen deficient alpha(1)-antitrypsin variants. Unlike most alpha(1)-antitrypsin variants, which were unstable, D256V and L41P variants exhibited extremely retarded protein folding as compared with the wild-type molecule. Once folded, however, the stability and inhibitory activity of these variant proteins were comparable to those of the wild-type molecule. Retarded protein folding may promote protein aggregation by allowing the accumulation of aggregation-prone folding intermediates. Repeated observations of retarded protein folding indicate that it is an important mechanism causing alpha(1)-antitrypsin deficiency by variant molecules, which have to fold into the metastable native form to be functional.     

Screening of alpha-1-antitrypsin gene by denaturing gradient gel electrophoresis (DGGE)

Alpha-1-antitrypsin (AAT) is a serine protease inhibitor whose deficiency could cause emphysema and liver disease and, as recently described, could be a risk factor for lung cancer development. Alpha-1-antitrypsin inhibits a variety of proteases but its primary target is neutrophil elastase, an extracellular endopeptidase capable of degrading most protein components of the extracellular matrix. Inhibition of neutrophil elastase by AAT has an important role in maintaining the integrity of connective tissue. The gene encoding for AAT spans over 12.2 kb, consists of seven exons and is highly polymorphic. Therefore several methods for mutation screening of alpha-1-antitrypsin gene have been developed. Method described here is based on denaturing gradient gel electrophoresis (DGGE). This method is highly efficient and reliable and allows rapid analysis of entire coding region of alpha-1-antitrypsin gene, including splice junction sites. Previously described DGGE based analysis of AAT gene included overnight electrophoresis of individually amplified fragments. The optimization of the method described in this paper is directed towards the shortening of the duration of electrophoresis and amplification of fragments in multiplex reaction in order to make the analysis less time-consuming and therefore more efficient.     

Tissue-specific expression of the human alpha 1-antitrypsin gene is controlled by multiple cis-regulatory elements.

Human alpha 1-antitrypsin (AAT) is expressed in the liver, and a 318 bp fragment immediately flanking the CAP site of the gene was found to be sufficient to drive the expression of a reporter gene (CAT) specifically in hepatoma cells. The enhancing activity however, was orientation-dependent. The DNA fragment was separated into a distal region and a proximal region. A "core enhancer" sequence GTGGTTTC is present within the distal region and is capable of activity enhancement in both orientations when complemented by the proximal region in the sense orientation. The results strongly suggest that there are multiple cis-acting elements in the human AAT gene that confer cell specificity for its expression. Nuclear proteins prepared from the hepatoma cells bound specifically to the proximal region in a band-shifting assay that was resistant to competition by the globin promoter DNA. Foot-printing analysis showed a protected domain within the proximal region that contains a nearly perfect palindromic sequence TGGTTAATATTCACCA, which may be important in the regulation of AAT expression in the liver.     

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.     

The relationship between rat major acute phase protein and the kininogens

The rat major acute phase protein (alpha 1-MAP) is a cysteine protease inhibitor. The stoichiometry of the interaction between the inhibitor and enzyme was shown to be 1:2. A cDNA clone specific for rat alpha 1-MAP was isolated from a cDNA library prepared from an inflamed rat liver RNA template. The 1458-base pair insert was sequenced and positively identified by alignment with a partial amino acid sequence obtained by radiosequence analysis of the primary translation product for alpha 1-MAP. Complete sequence analysis determined the alpha 1-MAP cDNA coded for the entire protein with the exception of the first four amino acids of the signal peptide, all of which were identified by radiosequencing. The coding sequence spans 1282 nucleotides, followed by 115 base pairs of a 3' untranslated region. Two putative active sites, suggested by the enzyme-inhibitor ratio, have been identified by analysis of internal duplications of the alpha 1-MAP sequence and the alignment of these regions with the sequences of several low molecular weight cysteine protease inhibitors. A computer homology analysis of the protein sequence revealed a 59.3% overall identity between rat alpha 1-MAP and bovine low molecular weight (LMW) kininogen. The homology included the signal peptide regions. LMW kininogen is a precursor of bradykinin. alpha 1-MAP does contain a bradykinin sequence; the flanking amino acids are different, however. Evidence for the expression of the LMW and a high molecular weight kininogen from the same gene, and the high degree of homology between these proteins and the rat acute phase protein suggest that all three proteins belong to a precisely regulated gene family.