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.     

Assignment of the human antithrombin III structural gene to chromosome 1q23-25

The human antithrombin III (ATIII) structural gene was mapped by in situ hybridization and quantitative analysis of ATIII gene dosage in DNA isolated from carriers of chromosome 1 deletions. These studies indicate that the ATIII structural gene maps to human chromosome q23-q25 and so is likely identical to AT3.     

Human and mouse S-protein mRNA detected in Northern blot experiments and evidence for the gene encoding S-protein in mammals by Southern blot analysis

Summary Human S-protein is a serum glycoprotein that binds and inhibits the activated complement complex, mediates coagulation through interaction with antithrombin III and plasminogen activator inhibitor I, and also functions as a cell adhesion protein through interactions with extracellular matrix and cell plasma membranes. A full length cDNA clone for human S-protein was isolated from a lambda gt11 cDNA library of mRNA from the HepG2 hepatocellular carcinoma cell line using mixed oligonucleotide sequences predicted from the amino-terminal amino acid sequence of human S-protein. The cDNA clone in lambda was subcloned into pUC18 for Southern and Northern blot experiments. Hybridization with radiolabeled human S-protein cDNA revealed a single copy gene encoding S-protein in human and mouse genomic DNA. In addition, the S-protein gene was detected in monkey, rat, dog, cow and rabbit genomic DNA. A 1.7 Kb mRNA for S-protein was detected in RNA from human liver and from the PLC/PRF5 human hepatoma cell line. No S-protein mRNA was detected in mRNA from human lung, placenta, or leukocytes or in total RNA from cultured human embryonal rhabdomyosarcoma (RD cell line) or cultured human fibroblasts from embryonic lung (IMR90 cell line) and neonatal foreskin. A 1.6 Kb mRNA for S-protein was detected in mRNA from mouse liver and brain. No S-protein mRNA was detected in mRNA from mouse skeletal muscle, kidney, heart or testis.     

A new member of the plasma protease inhibitor gene family.

A 2.1-kb cDNA clone representing a new member of the protease inhibitor family was isolated from a human liver cDNA library. The inhibitor, named human Leuserpin 2 (hLS2), comprises 480 amino acids and contains a leucine residue at its putative reactive center. HLS2 is about 25-28% homologous to three human members of the plasma protease inhibitor family: antithrombin III, alpha 1-antitrypsin and alpha 1-antichymotrypsin. A comparison with published partial amino acid sequences shows that hLS2 is closely related to the thrombin inhibitor heparin cofactor II.     

Molecular cloning and nucleotide sequence of cDNA encoding human muscle glycogen debranching enzyme.

cDNA comprising the entire length of the human muscle glycogen debranching enzyme was cloned and its nucleotide sequence determined. The debrancher mRNA includes a 4545-base pair coding region and a 2371-base pair 3'-nontranslated region. The calculated molecular mass of the debrancher protein derived from cDNA sequence is 172,614 daltons, consistent with the estimated size of purified protein (Mr 165,000 +/- 500). A partial amino acid sequence (13 internal tryptic peptides with a total of 213 residues) determined on peptides derived from purified porcine muscle debrancher protein confirmed the identity of the cDNA clone. Comparison of the amino acid sequence predicted from the human glycogen debrancher cDNA with the partial protein sequence of the porcine debrancher revealed a high degree (88%) of interspecies sequence identity. RNA blot analysis showed that debrancher mRNA in human muscle, lymphoblastoid cells, and in porcine muscle are all similar in size (approximately 7 kilobases). Two patients with inherited debrancher deficiency had a reduced level of debrancher mRNA, whereas two other patients had no detectable abnormality in RNA blots. The isolation of the debrancher cDNA and determination of its primary structure is an important step toward defining the structure-function relationship of this multifunctional enzyme and in understanding the molecular basis of the type III glycogen storage disease.     

Enzymatic modification of heparan sulfate on a biochip promotes its interaction with antithrombin III

A heparan sulfate glycosaminoglycan chain, biotinylated at its reducing-end, was bound to a streptavidin-coated biochip. Surface plasmon resonance spectroscopy showed a low affinity interaction with antithrombin III (ATIII) when it was flowed over a surface containing heparan sulfate. ATIII bound tightly with high affinity when the same surface was enzymatically modified to using 3-O-sulfotransferase isoform 1 (3-OST-1) in the presence of 3'-phosphoadenosine 5'-phosphosulfate (PAPS). The 3-OST-1 enzyme is involved in heparan sulfate biosynthesis and introduces a critical 3-O-sulfo group into this glycosaminoglycan affording the appropriate pentasaccharide sequence capable of high affinity binding to ATIII. This experiment demonstrates the specific structural modification of a glycosaminoglycan bound to a biochip using a biosynthetic enzyme, suggesting a new approach to rapid screening glycosaminoglycan-protein interactions.     

cDNA sequence of human class III alcohol dehydrogenase

A human placental cDNA library was screened using oligonucleotide probes based on the peptide sequence of the human class III alcohol dehydrogenase. An incomplete cDNA clone covering most of the coding sequence of class III alcohol dehydrogenase was isolated from a human placental cDNA library. This was subsequently used as a probe to obtain a full-length clone from a human testicular library. The cDNA sequence codes for a protein that is identical to the enzyme purified from human liver. Southern analysis of human genomic DNA suggests that it may contain more than a single copy per haploid genome.     

Chicken chromosomal protein HMG-14 and HMG-17 cDNA clones: isolation, characterization and sequence comparison

A cDNA clone coding for the chicken high-mobility group 14 (HMG-14) mRNA has been isolated from a chicken-liver cDNA library by screening with two synthetic oligodeoxynucleotide pools whose sequences were derived from the partial amino acid sequence of the HMG-14 protein. A chicken HMG-17 cDNA clone was also isolated in a similar fashion. Comparison of the two chicken HMG cDNA clones to the corresponding human cDNA sequences shows that chicken and human HMG-14 mRNAs and polypeptides are considerably less similar than are the corresponding HMG-17 sequences. In fact, the chicken HMG-14 is almost as similar to the chicken HMG-17 in amino acid sequence as it is to mammalian HMG-14 polypeptides. HMG-14 and HMG-17 mRNAs seem to contain a conserved sequence element in their 3'-untranslated regions whose function is at present unknown. The chicken HMG-14 and HMG-17 genes, in contrast to their mammalian counterparts, appear to exist as single-copy sequences in the chicken genome, although there appear to exist one or more additional sequences which partially hybridize to HMG-14 cDNA. Chicken HMG-14 mRNA, about 950 nucleotides in length, was detected in chicken liver RNA but was below our detection limits in reticulocyte RNA.     

3-Ketoacyl-acyl carrier protein synthase III from spinach (Spinacia oleracea) is not similar to other condensing enzymes of fatty acid synthase.

A cDNA clone encoding spinach (Spinacia oleracea) 3-ketoacyl-acyl carrier protein synthase III (KAS III), which catalyzes the initial condensing reaction in fatty acid biosynthesis, was isolated. Based on the amino acid sequence of tryptic digests of purified spinach KAS III, degenerate polymerase chain reaction (PCR) primers were designed and used to amplify a 612-bp fragment from first-strand cDNA of spinach leaf RNA. A root cDNA library was probed with the PCR fragment, and a 1920-bp clone was isolated. Its deduced amino acid sequence matched the sequences of the tryptic digests obtained from the purified KAS III. Northern analysis confirmed that it was expressed in both leaf and root. The clone contained a 1218-bp open reading frame coding for 405 amino acids. The identity of the clone was confirmed by expression in Escherichia coli BL 21 as a glutathione S-transferase fusion protein. The deduced amino acid sequence was 48 and 45% identical with the putative KAS III of Porphyra umbilicalis and KAS III of E. coli, respectively. It also had a strong local homology to the plant chalcone synthases but had little homology with other KAS isoforms from plants, bacteria, or animals.     

Identification and isolation of transcribed human X chromosome DNA sequences

A human X chromosome specific phage library has been used as a source of X-specific genomic DNA clones which hybridize with cellular RNA. Random cDNA clones were mapped for X chromosome sequence localization and 8 were identified as hybridizing to X chromosome Hind III fragments. All eight also hybridized with autosomal Hind III fragments. The X chromosome genomic sequences corresponding to two of these cDNA clones were isolated from a phage library constructed with the Hind III endonuclease digest products of X enriched DNA. One genomic DNA segment, localized to the short area of the X, shared sequence homology with at least one region of the human Y chromosome. The methodology developed represents a rapid means to obtain a specific genomic DNA clone from a single chromosome when multiple different genomic loci homologous to an expressed DNA sequence exist.     

Cloning and sequence analysis of cDNA clones for bovine aortic-endothelial-cell transglutaminase.

A cDNA clone encoding transglutaminase was isolated from a bovine-endothelial-cell cDNA library using oligonucleotide probes designed based on partial amino acid sequences of the purified protein. Sequencing of the cDNA insert revealed an open reading frame of 2061 bp coding for a protein of 687 amino acids. The sequence of bovine endothelial-cell transglutaminase was 88, 82, 80, 37, 37 and 37% identical with that of human endothelial, rat macrophage, guinea-pig liver, human and rat keratinocyte transglutaminases, and the human blood-coagulation factor XIIIa subunit, respectively. The cDNA clone was hybridized to a single mRNA species of 3.9 kb in the liver, lung, spleen and heart but not hybridized to RNA from the brain. Northern-blot analysis of mRNA from retinoid-treated cultured vascular endothelial cells revealed that retinoids were able to induce a large increase in the transglutaminase mRNA levels.     

Cloning of the Full-Length cDNA of Porcine Antithrombin III and Comparison with its Human Homolog

The characterization of porcine antithrombin III (ATIII)—a highly powerful anticoagulant—is essential for using porcine liver in xenotransplantation applications. The objective of this study was to clarify the functions of porcine ATIII through comparison with human ATIII. We cloned porcine ATIII and compared its important functional sites with those of human ATIII. The full-length cDNA of porcine ATIII was cloned by screening a porcine liver cDNA library, and the ATIII activities of 23 pigs were determined. The full-length cDNA of porcine ATIII spanned 1498 bp and encoded 463 amino acids. Porcine ATIII shared 87.67% nucleotide identity and 89.06% amino acid identity with human ATIII. Complete identity was found at active center Arg393–Ser394, and remarkably high similarities were found at 2 critical heparin-binding sites (residues 41 through 49 and 114 through 156) and in some key residues involved in heparin binding. An ATIII assay found no significant difference between porcine and human plasma. The high level of similarity between porcine ATIII and human ATIII suggests that porcine ATIII will function in a manner similar to human ATIII in xenotransplantation.Abbreviation: ATIII, antithrombin IIIAntithrombin III (ATIII) is a single-chain glycoprotein found in mammalian plasma that inhibits thrombin and other serine proteinases involved in the blood coagulation cascade, such as factor IX, factor X,²⁰ and plasmin. ATIII is considered the most powerful serine proteinase inhibitor (serpin) and the most important contributor to the anticoagulation system.¹¹ Previous studies have provided detailed knowledge of human ATIII¹ and have identified 2 essential activities as prerequisites for its effective function: (1) recognizing and attacking target proteases and (2) interacting with its cofactor, heparin.²The pig has played an important role in biomedical research,^3,6,15,33 especially as a large animal model for surgical experiments and a promising candidate for xenotransplantation.^9,12Although knowledge of the physiologic features of the porcine coagulation system is important for its successful application, differences (if any) between human and porcine coagulation factors have not been studied in detail.¹⁶ Considering the unknown properties of porcine ATIII when it is secreted into human blood and interacts with human thrombin and heparin after liver or hepatocyte xenotransplantation, there is a clear need for elucidating the properties of porcine ATIII.Treatment with high doses of recombinant human ATIII prevents coagulopathy and protects renal xenografts from early injury in the pig-to-baboon model.⁸ Heparin-dependent inhibition of human factor Xa by porcine arterial endothelial cells is blocked completely by neutralizing ATIII but is unaffected by the antitissue factor pathway inhibitor antibody.¹⁸ These results suggest that human ATIII is an effective anticoagulant in the xenotransplantation model, but porcine tissue factor pathway inhibitor and human Xa are incompatible.¹⁸ However, porcine ATIII has not been evaluated with regard to xenotransplantation models. Consequently, in the present study, we cloned and characterized the full-length cDNA of porcine ATIII. We then compared sequence and function of porcine ATIII cDNA with those of human ATIII to gain insight into the molecular compatibility of porcine and human coagulation-related molecules.     

Molecular cloning of human plasma glutathione peroxidase gene and its expression in the kidney.

A genomic clone containing the human plasma glutathione peroxidase (GSH-Px) gene has been isolated using a rat plasma GSH-Px cDNA as a probe. The partial nucleotide sequence of the clone completely matched the sequence of the human plasma GSH-Px cDNA. The results of Southern blot hybridization indicate that the human plasma GSH-Px gene consists of at least 4 exons and 3 introns, and spans about 12 kb. RNA blot analysis demonstrated that the human plasma GSH-Px gene is expressed in the kidney.     

Human pepsinogen C (progastricsin). Isolation of cDNA clones, localization to chromosome 6, and sequence homology with pepsinogen A

The entire pepsinogen C (PGC) coding sequence was determined by analysis of a series of five overlapping cDNA clones identified in a library constructed from human gastric mucosa poly(A+) RNA. A partial cDNA clone was initially identified using a 256-fold degenerate oligonucleotide probe for amino acid residues 4-12 of pepsin C, and subsequently 4 additional clones were identified upon rescreening with a probe complementary to the 5' region of the original cDNA clone. Northern analysis of gastric mucosa poly(A+) RNA with a PGC cDNA probe revealed an mRNA 1.5-kilobase species that was indistinguishable from that detected with a human pepsinogen A (PGA) cDNA probe. In contrast, the PGC and PGA cDNA probes detected distinct genomic restriction fragments indicating there was no detectable cross-hybridization under high stringency conditions. The PGC gene was localized to human chromosome 6 by analysis of a panel of human x mouse somatic cell hybrids. The regions containing the active site aspartyl groups of PGC are conserved in relationship to several other aspartic proteinases. We propose that the absence of detectable immunologic cross-reactivity between the two groups of human pepsinogens, A and C, results from divergent evolution of sequences located on the surface of the zymogens in contrast to the strongly conserved active site regions located within the binding cleft of the enzymes that are inaccessible for antigenic recognition.     

A peptide model for the heparin binding site of antithrombin III.

A peptide model for the heparin binding site of antithrombin III (ATIII) was synthesized to elucidate the structural consequences of heparin binding. This peptide [ATIII(123-139)] and a sequence-permuted analogue (ATIII random) showed similar conformational behavior (as analyzed by circular dichroism spectroscopy) in aqueous and organic media. In the presence of heparin, however, the peptide ATIII(123-139) assumed a stable conformation, whereas peptide ATIII random did not. Complex formation was saturable and sensitive to salt. The ATIII(123-139)-heparin complex contained beta-structure, rather than helical structure. This finding is incompatible with current models of heparin binding and suggests that heparin binding may induce nonnative structures at the binding site which could, in turn, lead to activation of ATIII. The peptide ATIII(123-139) was able to inhibit the binding of ATIII by heparin, consistent with the notion that this peptide may be a model for the heparin binding site.