中华硬蜱凝血酶抑制剂的分离纯化与活性

通过凝胶过滤和反相高压液相层析。从200只半饱吸血的中华硬蜱(Ixodide sinesis)唾液腺中分离纯化得到一凝血酶抑制剂。用飞行质谱测定其分子量为6．356kDa,与其他蜱类来源的凝血酶抑制剂分子量不同,提示为一新蛋白分子。该抑制剂对凝血酶有强烈的抑制活性,对激活的第X因子和胰蛋白酶有微弱的抑制活性。其发现将为发展疫苗、生物控制中华硬蜱提供资料和目标抗原。     

A tick B-cell inhibitory protein from salivary glands of the hard tick, Hyalomma asiaticum asiaticum

Some studies done to date suggest that B-cell inhibitory factor occurred in tick saliva. In this study, a novel protein having B-cell inhibitory activity was purified and characterized from the salivary glands of the hard tick, Hyalomma asiaticum asiaticum. This protein was named B-cell inhibitory factor (BIF). The cDNA encoding BIF was cloned by cDNA library screening. The predicted protein from the cDNA sequence is composed of 138 amino acids including the mature BIF. No similarity was found by Blast search. The lipopolysaccharide-induced B-cell proliferation was inhibited by BIF. This is the first report of the identification and characterization of B-cell inhibitory protein from tick. The current study facilitates the study of identifying the interaction among tick, Borrelia burgdorferi, the causative agent of Lyme disease, and host.     

cDNA sequence and predicted primary structure of the gamma subunit from the ATP synthase from Chlamydomonas reinhardtii

The 1701-base nucleotide sequence (not including the poly(A) tail) of a cDNA for the gamma subunit of the ATP synthase from Chlamydomonas reinhardtii was determined. A start translation sequence, 23 bases in from the 5' end, initiates an 1074-base-long open reading frame. The sequence of the first 21 amino acids at the amino-terminal end of the mature gamma subunit from C. reinhardtii was determined and compared to the deduced amino acid sequence of the open reading frame. From this it was determined that the mature protein contains 323 amino acids, with the first 35 amino acids probably being part of the transit peptide. The length of the mature protein is the same as that for the mature gamma subunit from spinach, for which only a few of the amino acids of the transit peptide are known. The similarity of the two mature proteins at the nucleotide level is 56% while at the amino acid level it is 77%. In addition, the 3 cysteines, which in spinach are involved in the energy-linked catalytic functions of the ATP synthase, are conserved in the predicted amino acid sequence for the gamma subunit from C. reinhardtii. In contrast, the mature C. reinhardtii gamma subunit contains 3 additional cysteine residues not found in the spinach gamma subunit.     

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.     

Identification of a novel transforming growth factor-beta (TGF-beta 5) mRNA in Xenopus laevis

A novel transforming growth factor-beta (TGF-beta) mRNA of about 3.0 kilobases, which encodes a putative protein of 382 amino acids, has been identified in amphibians by cDNA cloning. This mRNA, which we designate as TGF-beta 5, is developmentally regulated and highly expressed beginning at early neurula (stage 14) and in many adult tissues in Xenopus laevis. Following the first methionine, the putative precursor protein has a hydrophobic region, approximately 22 amino acids long, which probably represents a signal sequence, similar to that found in TGF-beta s 1-3. The precursor also has potential sites for glycosylation, integrin binding (RGD), and a tetrabasic amino acid (RKKR) site for potential cleavage of the precursor peptide to a biologically active protein. The putative mature protein consists of 112 amino acids with 9 cysteines and has 76, 66, 69, and 72% identity to TGF-beta s 1-4, respectively.     

Cloning of cDNAs coding for the heavy chain region and connecting region of human factor V, a blood coagulation factor with four types of internal repeats

Human factor V is a high molecular weight plasma glycoprotein that participates as a cofactor in the conversion of prothrombin to thrombin by factor Xa. Prior to its participation in the coagulation cascade, factor V is converted to factor Va by thrombin generating a heavy chain and a light chain, and these two chains are held together by calcium ions. A connecting region originally located between the heavy and light chains is liberated during the activation reaction. In a previous study, a cDNA of 2970 nucleotides that codes for the carboxyl-terminal 938 amino acids of factor V was isolated and characterized from a Hep G2 cDNA library [Kane, W. H., & Davie, E. W. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6800-6804]. This cDNA has been used to obtain additional clones from Hep G2 and human liver cDNA libraries. Furthermore, a Hep G2 cDNA library prepared with an oligonucleotide from the 5' end of these cDNAs was screened to obtain overlapping cDNA clones that code for the amino-terminal region of the molecule. The composite sequence of these clones spans 6911 nucleotides and is consistent with the size of the factor V message present in Hep G2 cells (approximately 7 kilobases). The cDNA codes for a leader sequence of 28 amino acids and a mature protein of 2196 amino acids. The amino acid sequence predicted from the cDNA was in complete agreement with 139 amino acid residues that were identified by Edman degradation of cyanogen bromide peptides isolated from the heavy chain region and connecting region of plasma factor V.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of two forms of cyclophilin from the hard tick Haemaphysalis longicornis

We have shown here the identification and characterization of two cyclophilin, cyclophilin A (CyPA) and B (CyPB), from the ixodid tick, Haemaphysalis longicornis. Both CyPA and CyPB contain the conserved peptidyl-prolyl isomerase (PPIase) domain, with CyPA consisting of 188 amino acids and CyPB of 216 amino acids. CyPA and CyPB share 50–67% amino acid sequence identity with the cyclophilins of other organisms, and are found in multiple organs throughout the developmental stages of ixodid ticks. In addition, recombinant CyPA and CyPB exhibited PPIase activity that could be inhibited by the cyclic peptides cyclosporin A (CsA). Silencing of CyPA through RNA interference has led to a significant reduction in the body weight of engorged ticks and their failure to lay eggs, in contrast to CyPB whose silencing did not result in any detectable phenotypic changes. Our results indicate that CyPA represents the major cyclophilin protein in H. longicornis involved in blood ingestion, viability, and oocyte development. This is the first report of cyclophilin proteins from ixodid and argasid ticks.     

The primary structure of the iron-sulfur subunit of ubiquinol-cytochrome c reductase from Neurospora, determined by cDNA and gene sequencing

The primary structure of the iron-sulfur subunit of ubiquinol-cytochrome c reductase from Neurospora mitochondria was determined by cDNA and genomic DNA sequencing. A first cDNA was identified from a cDNA bank cloned in Escherichia coli by hybridization selection of mRNA, cell-free protein synthesis and immunoadsorption. Further cDNA and geonomic DNA were identified by colony filter hybridization. The N-terminal sequence of the mature protein was determined by automated Edman degradation. From the sequence a molecular mass of 24749 Da results for the precursor protein and of 21556 Da for the mature protein. The presequence consists of 32 amino acids with four arginines as the only charged residues. The mature protein consists of 199 amino acids. It is characterized by a small N-terminal hydrophilic part of 29 residues, a hydrophobic stretch of 25 residues and a large C-terminal hydrophilic domain of 145 residues. The only four cysteines of the protein, which are assumed to bind the 2 Fe-2S cluster, are located in a moderate hydrophobic region of this large domain. Cysteines 3 and 4 are unusually arranged in that they are separated by only one proline. From sequence data the arrangement of the subunit in the membrane is deduced.     

Molecular cloning,expression, and characterization of bovine tissue factor pathway inhibitor-2

Human tissue factor pathway inhibitor-2 (TFPI-2) is a matrix-associated Kunitz-type serine proteinase inhibitor that is secreted by all cells of the vasculature, and presumably plays a role in the regulation of plasmin-mediated matrix remodeling. In this report, we describe the cloning and expression of a full-length cDNA for bovine TFPI-2 that exhibits 72% sequence identity with that of human TFPI-2. Following a 22 residue signal peptide, the mature protein contains 212 amino acids with 18 cysteines, three putative N-glycosylation sites, and one putative O-glycosylation site. The deduced sequence of mature bovine TFPI-2 revealed a short acidic amino-terminal region, three tandem Kunitz-type domains, and a carboxy-terminal tail highly enriched in basic amino acids. Recombinant bovine TFPI-2 was expressed in HEK 293 cells and resolved into two isoforms, designated as alpha-TFPI-2 (M(r) 33 kDa) and beta-TFPI-2 (M(r) 31 kDa), which presumably represent differentially glycosylated forms of the inhibitor. Similar to human TFPI-2, both bovine TFPI-2 isoforms exhibited strong inhibitory activity towards trypsin and plasmin, and weak inhibitory activity towards the factor VIIa-tissue factor complex.     

A novel antimicrobial peptide from salivary glands of the hard tick, Ixodes sinensis

A novel antimicrobial peptide named as ixosin was isolated from the salivary glands of the hard tick, Ixodes sinensis, by gel filtration, ion exchange chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). Its amino acid sequence was determined as GLHKVMREVLGYERNSYKKFFLR by Edman degradation and its molecular weight was 2870.5 analyzed by fast atom bombardment (FAB) mass spectrometry. This is the first antimicrobial peptide from ticks that lacks cysteine in its primary structure. The cDNA encoding ixosin was cloned by cDNA library screening. The predicted protein from the cDNA sequence composed of 79 amino acids including mature ixosin. Purified ixosin exerted its antimicrobial activities against bacteria and fungi. No similarity was found by BLAST search to any database entries and, thus, our findings describe a novel antimicrobial peptide.     

Amino acid sequence and structure modeling of savignin,a thrombin inhibitor from the tick,Ornithodoros savignyi

The full-length gene of savignin, a potent thrombin (E.C. 3.4.21.5) inhibitor from the tick Ornithodoros savignyi has been cloned and sequenced. Both 5' and 3' UTR's, a signal peptide from the translated amino acid sequence and an unusual poly-adenylation signal (AATACA) has been identified. The translated protein sequence shows high identity (63%) with ornithodorin, the thrombin inhibitor from the tick, Ornithodoros moubata. Molecular modeling using the structure of ornithodorin as reference gave a structure with an RMSD of 0.25 A for the full-length protein, 0.11 A for the N-terminal BPTI-like domain and 0.11 A for the C-terminal BPTI-like domain, indicating that maximum deviation occurs in the mobile bridge (0.18 A) between the two domains. Docking of savignin to thrombin shows that the interaction is similar to the ornithodorin-thrombin complex. The N-terminal amino acid residues of savignin bind inside the active site cleft, while the C-terminal domain of savignin has a net negative electrostatic potential and interacts with the basic fibrinogen recognition exosite of thrombin through hydrogen bonds and hydrophobic interactions. These results correlate with kinetic data obtained, which showed that savignin is a competitive, slow, tight-binding inhibitor that requires thrombin's fibrinogen-binding exo-site for optimal inhibition.     

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.     

Purification and cloning of a novel antimicrobial peptide from salivary glands of the hard tick, Ixodes sinensis

A novel antimicrobial peptide named as ixosin-B was isolated from the salivary glands of the hard tick, Ixodes sinensis, by gel filtration, ion exchange chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). Its amino acid sequence was determined as QLKVDLWGTRSGIQPEQHSSGKSDVRRWRSRY by Edman degradation. The cDNA encoding ixosin-B was cloned by cDNA library screening. The predicted protein from the cDNA sequence composed of 89 amino acids including mature ixosin-B. Purified ixosin-B exerted its antimicrobial activities against bacteria and fungi. No similarity was found by BLAST search to any database entries and, thus, our findings describe a novel antimicrobial peptide. It is also the fourth family of antimicrobial peptide from hard ticks.     

Cloning, primary structure and properties of a novel human integrin beta subunit.

The originally described integrin beta subunits that define the three subfamilies of integrin heterodimers are beta 1, beta 2 and beta 3. In this paper, we describe the isolation of a cDNA coding for a novel human integrin beta subunit, designated as beta 5. The beta 5 cDNA was isolated from a human thymic epithelial cell library, using oligonucleotide probes that were designed from a region highly conserved among the known beta 1, beta 2 and beta 3 sequences. The beta 5 cDNA codes for 799 (or 796) amino acids, including a 23 amino acid leader sequence. There are 776 (or 773) amino acids in the mature protein, which includes a long extracellular domain of 696 amino acids, a transmembrane domain and an intracellular C-terminal domain of 57 amino acids. The beta 5 sequence resembled the known beta 3, beta 1 and beta 2 sequences by 55, 43 and 38%, respectively, including conservation of 56/56 cysteines. Rabbit antiserum was prepared against a 20 amino acid synthetic peptide predicted from the beta 5 C-terminal sequence. This serum immunoprecipitated a beta 5 protein that was 100,000 Mr (reduced) and 95,000 Mr (nonreduced). Only a single alpha subunit was detected in association with beta 5, and that alpha subunit was immunochemically indistinguishable from the alpha v subunit previously found as part of the vitronectin receptor complex. By immunoprecipitation, beta 5 was most prevalent on carcinoma cell lines, was also present on hepatoma and fibroblast cell lines, and was absent from lymphoblastoid cells and platelets.     

The acid-stable proteinase inhibitor of human mucous secretions (HUSI-I, antileukoprotease). Complete amino acid sequence as revealed by protein and cDNA sequencing and structural homology to whey proteins and Red Sea turtle proteinase inhibitor

The complete amino acid sequence of human antileukoprotease has been determined by direct sequencing of the inhibitory active protein isolated from seminal plasma (HUSI-I) and by sequence analysis of cDNA reverse-transcribed from mRNA prepared from cervical tissue. The inhibitor (Mr 11726) consists of 107 amino acid residues including 16 cysteines presumably forming disulfide bonds. The molecule comprises two consecutive domains which are homologous to each other, to the second domain of the basic protease inhibitor from Red Sea turtle (chelonianin) and to both domains of the whey proteins of rat and mouse. Both domains contain a pattern of cysteines known as the 'four-disulfide-core' that has also been found in wheat germ agglutinin and neurophysin.