Human tissue factor: cDNA sequence and chromosome localization of the gene

A human placenta cDNA library in lambda gt11 was screened for the expression of tissue factor antigens with rabbit polyclonal anti-human tissue factor immunoglobulin G. Among 4 million recombinant clones screened, one positive, lambda HTF8, expressed a protein that shared epitopes with authentic human brain tissue factor. The 1.1-kilobase cDNA insert of lambda HTF8 encoded a peptide that contained the amino-terminal protein sequence of human brain tissue factor. Northern blotting identified a major mRNA species of 2.2 kilobases and a minor species of approximately 3.2 kilobases in poly(A)+ RNA of placenta. Only 2.2-kilobase mRNA was detected in human brain and in the human monocytic U937 cell line. In U937 cells, the quantity of tissue factor mRNA was increased severalfold by exposure of the cells to phorbol 12-myristate 13-acetate. Additional cDNA clones were selected by hybridization with the cDNA insert of lambda HTF8. These overlapping isolates span 2177 base pairs of the tissue factor cDNA sequence that includes a 5'-noncoding region of 75 base pairs, an open reading frame of 885 base pairs, a stop codon, a 3'-noncoding region of 1141 base pairs, and a poly(A) tail. The open reading frame encodes a 33-kilodalton protein of 295 amino acids. The predicted sequence includes a signal peptide of 32 or 34 amino acids, a probable extracellular factor VII binding domain of 217 or 219 amino acids, a transmembrane segment of 23 amino acids, and a cytoplasmic tail of 21 amino acids. There are three potential glycosylation sites with the sequence Asn-X-Thr/Ser.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human thrombomodulin: complete cDNA sequence and chromosome localization of the gene

A human umbilical vein endothelial cell cDNA library in lambda gt11 was screened for expression of thrombomodulin antigens with affinity-purified rabbit polyclonal anti-thrombomodulin immunoglobulin G (IgG) and mouse monoclonal anti-human thrombomodulin IgG. Among 7 million recombinant clones screened, 12 were recognized by both antibodies. Two of these, lambda HTm10 and lambda HTm12, were shown to encode thrombomodulin by comparison of the amino acid sequence deduced from the nucleotide sequence to the amino acid sequence determined directly from tryptic peptides of thrombomodulin. Thrombomodulin mRNA was estimated to be 3.7 kilobases in length by Northern blot analysis of endothelial cell and placental poly(A)+ RNA. Thrombomodulin mRNA was not detected in human brain, HepG2 hepatoma cells, or the monocytic U937 cell line. Additional cDNA clones were selected by hybridization with the 1.2-kilobase insert of lambda HTm10. One isolate, lambda HTm15, contained a 3693 base pair cDNA insert with an apparent 5'-noncoding region of 146 base pairs, an open reading frame of 1725 base pairs, a stop codon, a 3'-noncoding region of 1779 base pairs, and a poly(A) tail of 40 base pairs. The cDNA sequence encodes a 60.3-kDa protein of 575 amino acids. The predicted protein sequence includes a signal peptide of approximately 21 amino acids, an amino-terminal ligand-binding domain of approximately 223 amino acids, an epidermal growth factor (EGF) homology region of 236 amino acids, a serine/threonine-rich segment of 34 amino acids, a membrane-spanning domain of 23 amino acids, and a cytoplasmic tail of 38 amino acids. The EGF-homology region consists of six tandemly repeated EGF-like domains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence of the cDNA and gene for angiogenin, a human angiogenesis factor

Human cDNAs coding for angiogenin, a human tumor derived angiogenesis factor, were isolated from a cDNA library prepared from human liver poly(A) mRNA employing a synthetic oligonucleotide as a hybridization probe. The largest cDNA insert (697 base pairs) contained a short 5'-noncoding sequence followed by a sequence coding for a signal peptide of 24 (or 22) amino acids, 369 nucleotides coding for the mature protein of 123 amino acids, a stop codon, a 3'-noncoding sequence of 175 nucleotides, and a poly(A) tail. The gene coding for human angiogenin was then isolated from a genomic lambda Charon 4A bacteriophage library employing the cDNA as a probe. The nucleotide sequence of the gene and the adjacent 5'- and 3'-flanking regions (4688 base pairs) was then determined. The coding and 3'-noncoding regions of the gene for human angiogenin were found to be free of introns, and the DNA sequence for the gene agreed well with that of the cDNA. The gene contained a potential TATA box in the 5' end in addition to two Alu repetitive sequences immediately flanking the 5' and 3' ends of the gene. The third Alu sequence was also found about 500 nucleotides downstream from the Alu sequence at the 3' end of the gene. The amino acid sequence of human angiogenin as predicted from the gene sequence was in complete agreement with that determined by amino acid sequence analysis. It is about 35% homologous with human pancreatic ribonuclease, and the amino acid residues that are essential for the activity of ribonuclease are also conserved in angiogenin. This provocative finding is thought to have important physiological implications.     

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.     

Molecular cloning, sequencing, and expression of human myocardial fatty acid ethyl ester synthase-III cDNA

Fatty acid ethyl ester synthase-III (FAEES-III), previously purified to homogeneity from human heart, metabolizes ethanol nonoxidatively. Using a derived partial amino acid sequence and corresponding oligonucleotide probes, the cDNA for this enzyme has been cloned from a human heart lambda gtll library. Of the five positive clones obtained, one contained a complete coding region (630 base pairs) and the entire 3'-noncoding region (41 base pairs). From this nucleotide sequence the complete 210 amino acid sequence of FAEES-III (Mr 23,307) is reported. Comparison of its amino acid sequence with that of glutathione S-transferase pi-1 suggests that they belong to the same gene family since they differ in only six nucleotides and four amino acids. The sequence of FAEES-III was also compared with those of placental glutathione S-transferase and the basic glutathione S-transferase. FAEES-III was 84% homologous with placental glutathione S-transferase but only less than 10% homologous with the basic glutathione S-transferase. Northern blots demonstrate expression of FAEES-III mRNA in normal human liver, placenta, and heart. In all cases, the mRNA for the enzyme is 0.7 kilobase in size. MCF-7 cells transfected with FAEES-III cDNA have a 14-fold increase in synthase activity and a 12-fold increase in glutathione S-transferase (GST) activity compared with control cells. MCF-7 cells transfected with GST pi-1 cDNA have a 13-fold increase in GST activity compared with control cells but no increase in synthase activity. When the supernatant of COS-7 cells transfected with FAEES-III cDNA were immunoblotted with rabbit FAEES-III antibody, a band at 24 kilodaltons was demonstrated. Thus, we have obtained the first cDNA and amino acid sequence for a human FAEES-III which also has significant GST activity, and we have identified 4 residues potentially responsible for conferring ethanol recognition to GSTs.     

Structure of the gene for human plasminogen activator inhibitor-2. The nearest mammalian homologue of chicken ovalbumin

Plasminogen activator inhibitor-2 (PAI-2) can regulate the formation of plasmin by inhibiting urokinase and tissue plasminogen activator. PAI-2 is induced in monocytes and endothelium by inflammatory mediators, and it is made in the placenta during pregnancy. PAI-2 is a member of the serine protease inhibitor gene family, and it is particularly similar to chicken ovalbumin. Like ovalbumin, PAI-2 is secreted without cleavage of a signal peptide. To determine the structure of the PAI-2 gene, two bacteriophage lambda human genomic DNA libraries were screened with PAI-2 cDNA probes. Characterization of three positive clones shows that the human PAI-2 gene spans 16.5 kilobases and has eight exons. The 5'-untranslated sequence of the PAI-2 mRNA is 77 base pairs in length as suggested by primer extension and S1 nuclease mapping. The eukaryotic consensus sequence TATAAAA is found 22 base pairs 5' of the proposed cap site. The PAI-2 gene is on chromosome 18q21-23 as determined by hybridization to flow-sorted chromosomes and by in situ hybridization. There appear to be two common PAI-2 alleles that differ by six nucleotides in exons 1, 4, and 8. The structure of the PAI-2 gene is quite different from that of PAI-1 although these two inhibitors have common target protease specificity. In contrast, the structure of the PAI-2 gene is very similar to that of the chicken ovalbumin gene. When protein sequences are aligned to obtain maximal identity, six of the seven intron positions in the PAI-2 gene are identical to those in the chicken ovalbumin gene. We conclude that PAI-2 is the closest mammalian homologue of avian ovalbumin.     

Cloning of a complete cDNA encoding human aromatase: immunochemical identification and sequence analysis

A complete cDNA clone encoding a human aromatase was isolated from a human placental cDNA library in lambda gt11. An antibody to the polypeptide specified by the isolated clone was prepared, and Western blot analysis and antibody inhibition experiments of human placental aromatase activity confirmed the identification of the clone as aromatase cDNA. The isolated aromatase cDNA clone of 3030 bp with two unique EcoRI sites contained a 3'-noncoding region of 1397 bp, an open reading frame of 1509 bp encoding 503 amino acid residues, and a 5'-noncoding region of 124 bp. Analysis of the amino acid sequence of aromatase and comparison of aromatase with other forms of cytochrome P-450 indicated that this enzyme is a unique form of the cytochrome P-450 superfamily.     

Cloning and nucleotide sequence of placental hGH-V cDNA

We have previously demonstrated the presence in human placenta and maternal serum of a GH variant, called human placental growth hormone (hPGH). We have also shown that the hGH-V gene is expressed at the placental level thus possibly coding for hPGH. The hGH-V cDNA has now been isolated from a lambda gt 11 human placenta cDNA library. Its sequence has been determined which firmly establishes the GH-V gene mode of splicing as well as the GH-V protein structure. Our data give final evidence of placental hGH-V gene expression and reinforce the hypothesis of identity between the hGH-V protein and hPGH.     

Heparin cofactor II: cDNA sequence, chromosome localization, restriction fragment length polymorphism, and expression in Escherichia coli

Heparin cofactor II (HCII) is an inhibitor of thrombin in plasma that is activated by dermatan sulfate or heparin. An apparently full-length cDNA for HCII was isolated from a human liver lambda gt11 cDNA library. The cDNA consisted of 2215 base pairs (bp), including an open-reading frame of 1525 bp, a stop codon, a 3'-noncoding region of 654 bp, and a poly(A) tail. The deduced amino acid sequence contained a signal peptide of 19 amino acid residues and a mature protein of 480 amino acids. The sequence of HCII demonstrated homology with antithrombin III and other members of the alpha 1-antitrypsin superfamily. Blot hybridization of an HCII probe to DNA isolated from sorted human chromosomes indicated that the HCII gene is located on chromosome 22. Twenty human leukocyte DNA samples were digested with EcoRI, PstI, HindIII, KpnI, or BamHI, and Southern blots of the digests were probed with HCII cDNA fragments. A restriction fragment length polymorphism was identified with BamHI. A slightly truncated form of the cDNA, coding for Met-Ala instead of the N-terminal 18 amino acids of mature HCII, was cloned into the vector pKK233-2 and expressed in Escherichia coli. The resultant protein of apparent molecular weight 54,000 was identified on an immunoblot with 125I-labeled anti-HCII antibodies. The recombinant HCII formed a complex with 125I-thrombin in a reaction that required the presence of heparin or dermatan sulfate.     

cDNA for Mo3, a monocyte activation antigen, encodes the human receptor for urokinase plasminogen activator.

We have cloned the cDNA for Mo3, an activation Ag expressed by human monocytes and myelomonocytic cell lines after stimulation by PMA, LPS, muramyl dipeptide, certain cytokines, and cAMP agonists. We have previously shown that Mo3 expression in vivo is associated predominantly with macrophages in inflammatory sites. Mo3 is a highly glycosylated protein of about 50 kDa in monocytes and U-937 cells and is anchored to the plasma membrane by glycosyl-phosphatidylinositol linkage. We purified Mo3 protein by cleavage from the U-937 cell surface with phosphatidylinositol-specific phospholipase C, followed by affinity chromatography using a mAb. An internal peptide sequence was determined and used to design oligonucleotide probes for screening an expression cDNA library. Nucleotide sequencing indicated that the complete coding sequence encodes 335 amino acids, including a predicted signal peptide of 22 residues and a hydrophobic C-terminal portion that is probably cleaved during formation of the GPI linkage. The resulting mature protein of about 290 amino acids is consistent with the 29-kDa molecular mass of deglycosylated Mo3. A Northern blot of RNA from U-937 cells revealed a 1.5-kb band that was induced by PMA treatment. Mo3 cDNA was transfected into Cos cells and surface expression of Mo3 was detected by ELISA using various anti-Mo3 mAb. We performed a computer search of the National Biomedical Research Foundation database and found that Mo3 is identical to the human receptor for the urokinase plasminogen activator (uPA-R). Purified soluble Mo3, as well as anti-Mo3 antibodies, were able to block uPA binding to its receptor on U-937 cells, indicating that Mo3 is indeed uPA-R. The use of these anti-Mo3 antibodies may be helpful in assessing the role of uPA-R in processes such as inflammation and tumor invasion.     

Recombinant bovine alpha-lactalbumin obtained by limited proteolysis of a fusion protein expressed at high levels in Escherichia coli

A cDNA clone containing the entire coding region for bovine pre-alpha-lactalbumin (LA) together with 27 base pairs of 5'-noncoding and 268 base pairs of 3'-noncoding sequences was isolated from a bovine mammary cDNA plasmid library in the Okayama-Berg vector system using a synthetic oligonucleotide probe and sequenced. The coding segment for mature LA was subcloned into the T7 expression system of Studier and co-workers (Studier, F.W., and Moffatt, B.A. (1986) J. Mol. Biol. 189, 113-130; Rosenberg, A.H., Lade, B.N., Chui, D.S., Lin, S.W., Dunn, J.J., and Studier, F.W. (1987) Gene (Amst.) 56, 125-135) and expressed as a 21-kDa fusion protein that consisted of the mature bovine LA sequence connected to the NH2-terminal 50 residues of human cathepsin D by a linker sequence containing protease cleavage sites. This fusion protein was expressed in an insoluble form and accumulated to about 50% of the total bacterial protein within 3 h after induction of T7 RNA polymerase synthesis. The protein was solubilized, purified by gel filtration, and converted to an active form by treatment with mixtures of reduced and oxidized glutathione in the presence of Ca2+. The maximum specific activity of the fusion protein was about 25% of that of native LA, suggesting that the attachment of an NH2-terminal extension sterically hinders but does not prevent the interaction with galactosyltransferase. The extension also does not block the binding of the regulatory Ca2+ ion that is required for folding from the reduced denatured state. Trypsin cleaved the folded fusion protein specifically at a Lys-Glu bond at the junction with the mature LA sequence to give a product indistinguishable in structure and activity from native LA.     

Human microsomal xenobiotic epoxide hydrolase. Complementary DNA sequence, complementary DNA-directed expression in COS-1 cells, and chromosomal localization

A lambda gt11 expression library constructed from human liver mRNA was screened with an antibody against human microsomal xenobiotic epoxide hydrolase. The clone pheh32 contains an insert of 1742 base pairs with an open reading frame coding for a protein of 455 amino acids with a calculated Mr of 52,956. The nucleotide sequence is 77% similar to the previously reported rat xenobiotic epoxide hydrolase cDNA sequence. The deduced amino acid sequence of the human epoxide hydrolase is 80% similar to the previously reported rabbit and 84% similar to the deduced rat protein sequence. The NH2-terminal amino acids deduced from the human xenobiotic epoxide hydrolase cDNA are identical to the published 19 NH2-terminal amino acids of the purified human xenobiotic epoxide hydrolase protein. Northern blot analysis revealed a single mRNA band of 1.8 kilobases. Southern blot analysis indicated that there is only one gene copy/haploid genome. The human xenobiotic epoxide hydrolase gene was assigned to the long arm of human chromosome 1. Several restriction fragment length polymorphisms were observed with the human epoxide hydrolase cDNA. pheh32 was expressed as enzymatically active protein in cultured monkey kidney cells (COS-1).     

Primary structure and gene localization of human prolidase

Complementary DNA clones of prolidase (imidodipeptidase, EC 3.4.13.9) were isolated from human liver and placental cDNA libraries. Two clones named lambda PL21 and lambda PP6 from the liver and placental cDNA libraries, respectively, were analyzed in detail. The first clone, lambda PL21, carried a cDNA insert of 1.7 kilobase pairs and covered all the coding region of human prolidase mRNA. The second clone, lambda PP6, contained a 1.8-kilobase insert with a full-length 3'-untranslated region. Comparison of the amino acid sequence predicted from the nucleotide sequence of the cDNA insert of the two clones with the partial amino acid sequence determined by Edman degradation of peptides derived from human erythrocyte prolidase established that both clones code for human prolidase. The amino terminus of the human mature enzyme is blocked and seems to begin with the sequence X-Ala-Ala-Ala. Presumably no processing occurs at the carboxyl terminus. The mature enzyme is composed of 492 residues, corresponding to Mr 54,305. The sequence of prolidase is unique and not similar to any known protein, except for a significant similarity to regions of F1-ATPase alpha and beta subunits from various sources. The gene has been mapped to the short arm of chromosome 19 (19p13.2). Elucidation of the complete amino acid sequence and the gene location of prolidase should provide the basis for understanding structure-function relationships and also inherited disorders caused by deficiency of this metabolically important enzyme.     

Cloning and characterization of a cDNA coding for the lipoprotein-associated coagulation inhibitor shows that it consists of three tandem Kunitz-type inhibitory domains

Human plasma contains a lipoprotein-associated coagulation inhibitor (LACI) which inactivates factor Xa directly, and in a Xa-dependent fashion also inhibits the VIIa-tissue factor complex of the extrinsic coagulation pathway. Rabbit polyclonal anti-LACI antiserum was used to screen human placental and fetal liver lambda gt11 cDNA libraries for the expression of LACI antigens. Immunologically positive clones were further tested for their ability to bind 125I-factor Xa. Seven clones were obtained which are immunologically and functionally active. The longest cDNA insert (lambda P9) of these isolates is 1.4 kilobases (kb) while other clones are 1.0 kb in length. Nucleotide sequence analysis shows that lambda P9 consists of 1431 bases that include a 5'-noncoding sequence of 132 nucleotides, an open reading frame of 912 nucleotides, and a 3'-noncoding region of 387 nucleotides. The open reading frame encodes a signal peptide of 28 residues followed by a 32-kilodalton protein of 276 residues. The predicted sequence of mature LACI contains 18 cysteines and three potential N-linked glycosylation sites. The amino acid sequence analysis of purified LACI's NH2 terminus and two of its proteolytic fragments match exactly those deduced from the cDNA sequence, indicating that the cDNA codes for LACI. The translated amino acid sequence of LACI shows several discernible domains, including a highly negatively charged NH2 terminus, three tandem Kunitz-type inhibitory domains, and a highly positively charged carboxyl terminus. Northern blot analysis shows that the following liver-derived cell lines, Chang liver, HepG2 hepatoma, and SK hepatoma all, contain two major species of mRNA (1.4 and 4.4 kb) which hybridize with LACI cDNA.     

Molecular cloning, characterization, and expression of a human 14-kDa lectin

Full length cDNAs coding for a 14-kDa beta-galactoside binding lectin have been isolated from HL-60 cells and human placenta. Oligonucleotide probes based on a pentapeptide present in several partial sequences of homologous human lectins were used to screen a lambda GT10 HL-60 cDNA library. The HL-60 cDNA clones that were isolated were used to design a synthetic primer representing the 3'-untranslated region of the HL-60 lectin. This primer was then used to synthesize a lambda GT10 human placenta cDNA library, and restriction fragments of the HL-60 cDNA clones were used to screen the library. The cDNA clones for both HL-60 and placenta lectin had identical sequences with short 5'- and 3'-untranslated regions and coded for a 135-amino acid protein which lacks a hydrophobic signal peptide sequence. Biochemical data show that, despite the presence of a possible N-linked glycosylation site, the protein is not glycosylated. Northern and Southern blot analyses indicate that the 14-kDa lectin is encoded for by a single gene. The lectin cDNA was expressed in Escherichia coli and biologically active protein was purified from cell lysates by affinity chromatography.     

Characterization of cytochrome b(5) in the ascidian Polyandrocarpa misakiensis and budding-specific expression

A cDNA for cytochrome b(5) was cloned from a cDNA library of buds of the ascidian, Polyandrocarpa misakiensis, by a hybridization method involving a digoxigenin-labeled cDNA probe of human soluble cytochrome b(5). The nucleotide sequence of the cDNA for the ascidian cytochrome b(5) (Pmb5) consisted of about 1,800 base pairs including 5'- and 3'-noncoding regions, and a coding sequence of 405 base pairs. The amino acid sequence of 135 residues deduced from the coding nucleotide sequence exhibited 54% identity and 76% similarity to chicken cytochrome b(5). A highly conserved amino acid sequence was observed in the amino-terminal domain of 96 residues containing two heme-binding histidine residues. The putative soluble form of the recombinant Pmb5 expressed in Escherichia coli was purified to homogeneity by column chromatographies on an anion-exchanger and gel filtration. The purified Pmb5 showed the typical absorption spectrum of cytochrome b(5) with an asymmetric peak at 556 nm and a shoulder at 560 nm upon reduction with NADH and NADH-cytochrome b(5) reductase. The low temperature spectrum of the dithionite-reduced form of the protein contained the split peaks at 551 and 555 nm, this spectrum being very similar to that of mammalian liver cytochrome b(5). Expression of Pmb5 in the ascidian was examined immunohistochemically with a monoclonal antibody against the Pmb5. Apparently high level expression of Pmb5 was found in the developing buds, but the levels of cytochrome b(5) in the parents and juvenile adults were very low. This is the first report on the characterization of Pmb5, and the increased expression of Pmb5 in the ascidian.