Identification of two sites of sulfation of human heparin cofactor II

Tyrosine sulfate was identified as a constituent of human heparin cofactor II by analysis of sulfate-labeled protein secreted by a human hepatoma-derived cell line and of purified protein from human plasma. Alkaline hydrolysis of heparin cofactor II released tyrosine sulfate as demonstrated by anion-exchange high performance liquid chromatography of hydrolysates. Two sites of sulfation were identified, and the amino acid sequences of the sites were established by sequential Edman degradation of sulfate-containing tryptic peptides that were isolated by reverse-phase high performance liquid chromatography. Each peptide contains only a single tyrosine residue so that the sites of sulfation can be assigned unambiguously. The two sites of sulfation are separated by 13 residues and represent an internal sequence repeat in the heparin cofactor II molecule. The two sites have the following sequences. Glu56-Asp-Asp-Asp-Tyr(SO4)-Leu-Asp62 Glu69-Asp-Asp-Asp-Tyr(SO4)-Ile-Asp75 Sulfate-labeled heparin cofactor II formed a covalent complex with thrombin in a heparin-dependent manner. Thus, the sulfate-containing form of the protein was shown to be biologically active. The characteristic sulfate-containing segment of heparin cofactor II, which contains 17 acidic amino acid residues over a span of 30 residues, may contribute to the unique properties of this thrombin inhibitor.     

Trichuris suis: A secretory serine protease inhibitor

A trypsin inhibitor was identified in extracts of adult Trichuris suis and culture fluids from 24-h in vitro cultivation of adult parasites. The inhibitor was isolated by acid precipitation, affinity chromatography (trypsin-agarose), and reverse phase HPLC as a single polypeptide with a molecular weight estimated at 6.6 kDa by laser desorption mass spectrometry. The purified inhibitor associated strongly with trypsin (equilibrium dissociation inhibitory constant (K(j)) of 3.07 nM) and chymotrypsin (K(j) = 24.5 nM) and was termed TsTCI. Elastase, thrombin, and factor Xa were not inhibited. The cDNA-derived amino acid sequence of the mature TsTCI consisted of 61 residues including 8 cysteine residues with a molecular mass of 6.687 kDa. The N-terminal region of TsTCI (46 residues) showed limited homology (36%) to a protease inhibitor from the hemolymph of the honeybee Apis mellifera, which is considered to be a member of the Ascaris inhibitor family. However, TsTCI did not display sequence homology with other members of this family or the distinctive cysteine residue pattern which distinguishes this family. However, similarity of a region of TsTCI (11 residues) with the reactive site regions of inhibitors from the nematodes Ascaris suum, Anisakis simplex, and Ancylostoma caninum was apparent.     

Purification and partial amino acid sequence of a bovine cartilage-derived collagenase inhibitor

An inhibitor of mammalian collagenase from bovine scapular cartilage has been purified to homogeneity. The inhibitor, extracted from cartilage using 2 M NaCl, was applied to an A-1.5m gel filtration column. Inhibitor eluted at an apparent Mr of 28,000. Further purification was achieved by ion exchange chromatography, gel filtration, and reverse-phase high performance liquid chromatography. A purification of greater than 1,000-fold was achieved. The inhibitor was judged homogeneous by the appearance of a single band on a silver-stained 15% sodium dodecyl sulfate-polyacrylamide gel. Reduced inhibitor had an Mr of 27,400, unreduced inhibitor had an Mr of 23,900. NH2-terminal sequence data were obtained for the first 45 residues. The bovine cartilage-derived inhibitor exhibits greater than 65% homology over the first 23 residues with a collagenase inhibitor purified from human skin fibroblasts maintained in cell culture. This is the first demonstration that collagenase inhibitors extracted directly from tissue may be similar to those obtained from culture medium.     

Variegin, a novel fast and tight binding thrombin inhibitor from the tropical bont tick

Tick saliva contains potent antihemostatic molecules that help ticks obtain their enormous blood meal during prolonged feeding. We isolated thrombin inhibitors present in the salivary gland extract from partially fed female Amblyomma variegatum, the tropical bont tick, and characterized the most potent, variegin, one of the smallest (32 residues) thrombin inhibitors found in nature. Full-length variegin and two truncated variants were chemically synthesized. Despite its small size and flexible structure, variegin binds thrombin with strong affinity (K(i) approximately 10.4 pM) and high specificity. Results using the truncated variants indicated that the seven residues at the N terminus affected the binding kinetics; when removed, the binding characteristics changed from fast to slow. Further, the thrombin active site binding moiety of variegin is in the region of residues 8-14, and the exosite-I binding moiety is within residues 15-32. Our results show that variegin is structurally and functionally similar to the rationally designed thrombin inhibitor, hirulog. However, compared with hirulog, variegin is a more potent inhibitor, and its inhibitory activity is largely retained after cleavage by thrombin.     

Trichuris suis: a secretory chymotrypsin/elastase inhibitor with potential as an immunomodulator

A serine protease inhibitor, termed TsCEI, was purified from adult-stage Trichuris suis by acid precipitation, affinity chromatography (elastase-agarose), and reverse-phase HPLC. The molecular weight of TsCEI was estimated at 6.437 kDa by laser desorption mass spectrometry. TsCEI potently inhibited both chymotrypsin (K(i) = 33.4 pM) and pancreatic elastase (K(i) = 8.32 nM). Neutrophil elastase, chymase (mouse mast cell protease-1, mMCP-1), and cathepsin G were also inhibited by TsCEI, whereas trypsin, thrombin, and factor Xa were not. The cDNA-derived amino acid sequence of the mature TsCEI consisted of 58 residues including 9 cysteine residues with a molecular mass of 6.196 kDa. TsCEI displayed 48% sequence identity to a previously characterized trypsin/chymotrypsin inhibitor of T. suis, TsTCI. TsCEI showed 36% sequence identity to a protease inhibitor from the hemolymph of the honeybee Apis mellifera. Sequence similarity was also detected with the trypsin/thrombin inhibitor of the European frog Bombina bombina, the elastase isoinhibitors of the nematode Anisakis simplex, and the chymotrypsin/elastase and trypsin inhibitors of the nematode Ascaris suum. The inhibitors of T. suis, an intestinal parasite of swine, may function as components of a parasite defense mechanism by modulating intestinal mucosal mast cell-associated, protease-mediated, host immune responses.     

Purification, cloning, and synthesis of a novel salivary anti-thrombin from the mosquito Anopheles albimanus.

An anti-thrombin peptide (anophelin) was isolated from the salivary glands of the mosquito Anopheles albimanus through molecular sieving and reverse-phase high-performance liquid chromatography. The purified peptide inhibited thrombin-induced platelet aggregation, thrombin esterolytic activity on a synthetic substrate, and thrombin cleavage of fibrinogen. The purified anti-thrombin had a molecular mass of 6342.4 Da. Its amino terminus was blocked, but internal sequence yielded three peptide sequences, which were used to design oligonucleotide probes for polymerase chain reaction amplification of salivary gland cDNA and isolation of the full-length clone. Analysis of the sequence of anophelin shows no similarities to any other anti-thrombin peptides. Anophelin was successfully synthesized and characterized to be a tight-binding, specific, and novel inhibitor of thrombin.     

嵌合水蛭肽的构建与活性分析

血管成形术或动脉粥样斑块破裂等因素所致血管壁损伤而引起的血栓形成过程中 ,血小板的激活和凝血酶的形成起着关键作用 .因此 ,抗血小板和抗凝是治疗血栓的两个重要方面 .血小板膜糖蛋白GPⅡb Ⅲa受体拮抗剂 ,如含Arg Gly Asp(RGD)序列的多肽 ,在临床上已显示了良好的抗血小板     

Purification and characterization of a trypsin inhibitor from Putranjiva roxburghii seeds

A highly stable and potent trypsin inhibitor was purified to homogeneity from the seeds of Putranjiva roxburghii belonging to Euphorbiaceae family by acid precipitation, cation-exchange and anion-exchange chromatography. SDS-PAGE analysis, under reducing condition, showed that protein consists of a single polypeptide chain with molecular mass of approximately 34 kDa. The purified inhibitor inhibited bovine trypsin in 1:1 molar ratio. Kinetic studies showed that the protein is a competitive inhibitor with an equilibrium dissociation constant of 1.4x10(-11) M. The inhibitor retained the inhibitory activity over a broad range of pH (pH 2-12), temperature (20-80 degrees C) and in DTT (up to100 mM). The complete loss of inhibitory activity was observed above 90 degrees C. CD studies, at increasing temperatures, demonstrated the structural stability of inhibitor at high temperatures. The polypeptide backbone folding was retained up to 80 degrees C. The CD spectra of inhibitor at room temperature exhibited an alpha, beta pattern. N-terminal amino acid sequence of 10 residues did not show any similarities to known serine proteinase inhibitors, however, two peptides obtained by internal partial sequencing showed significant resemblance to Kunitz-type inhibitors.     

Production and Characterization of Hirudin Variant-1 by SUMO Fusion Technology in E. coli

Hirudin is the most potent non-covalent inhibitor of thrombin. Several expression systems have been used to produce recombinant hirudin for pharmaceutical purposes. However, high expression of active hirudin in Escherichia coli cytoplasm has not been successful owing to the fact that heterogenetic small peptide is easily degraded in the cell. To solve this problem, we constructed a recombinant form of the hirudin variant-1 (HV1) as a fusion protein with the small ubiquitin-related modifier gene (SUMO) by use of over-lap PCR. The fusion gene His₆-SUMO-HV1 was highly expressed in E. coli BL21 (DE3) in which the SUMO-HV1 accounts for over 30% of the soluble fraction. The fusion protein was purified by Ni?CNTA affinity chromatography and cleaved by a SUMO-specific protease Ulp1 to release the HV1 with natural N-terminal. The recombinant HV1 (rHV1) was further purified by Ni?CNTA affinity chromatography and then by Q anion-exchange chromatography. N-terminal sequencing result demonstrated the purified rHV1 had the same N-terminal sequence as the native hirudin. MALDI-TOF/MS analysis indicated that the molecular weight of the purified rHV1 protein was 6939.161 Da, which was similar to the theoretical molecular weight of rHV1 6,944 Da. The Chromozym TH assay result showed that the anti-thrombin activity of purified rHV1 was 8,800 ATU/mg and comparable to the specific activity of native hirudin.     

Cloning and Expression of a New Gene Encoding an Antifungal Protein in Phytolacca americana

protein (Pa-AFP) with molecular weight about 4 kD was purified from the seeds of Phytolacca americana L. , which obviously inhibits the growth of Rhizoctonia solani Kiihn in vitro. The authors isolated mRNA from the seeds of pokeberry and designed a degenerate PCR primer according to the N-terminal sequence of the purified protein. The full-length cDNA encoding Pa-AFP was cloned by RT-PCR and 5'-RACE and sequenced. The deduced amino acid sequence indicates that a preprotein with 65 amino acid residues is firstly translated and then processed to a mature protein with 38 amino acids. The DNA encoding the mature protein was subcloned into expression vector pGEX-4T1, and expressed efficiently in E. coli BL21 as a GST- Pa-AFP fusion protein. The fusion protein was purified by glutathione-Sepharose 4B affinity colmnn chromatography. The purified fusion protein was specifically digested by thrombin and the Pa-AFP was further purified by filtration column chromatography.     

Dipetalogastin, a potent thrombin inhibitor from the blood-sucking insect. Dipetalogaster maximus cDNA cloning, expression and characterization.

A cDNA coding for the thrombin inhibitor dipetalogastin has been isolated from a stomach library of Dipetalogaster maximus, a blood-sucking insect. The open reading frame of the cloned inhibitor cDNA codes for a protein of 344 amino-acid residues. Sequence analysis reveals the existence of three repeated homologous main regions, indicating that the inhibitor consists of three domains. Each domain shows a double-headed structure with an internal sequence homology like rhodniin, the thrombin inhibitor from the blood-sucking insect Rhodnius prolixus. Peptide sequence comparisons of the deduced amino-acid sequence exhibit a high homology of the domains I and II to the natural inhibitor dipetalogastin from the stomach content of D. maximus and to rhodniin, respectively. Significant sequence similarities to Kazal-type inhibitors, like the conserved sequence CGXDXXTYXNXC and several cysteine residues, indicate that the thrombin inhibitor from D. maximus is a further blood-sucking insect which belongs to the Kazal-type family (besides rhodniin). A biologically active recombinant protein corresponding to domain II of the dipetalogastin cDNA was expressed in Escherichia coli. The isolated recombinant dipetalogastin with a molecular mass of 12.91 kDa has proved to be a specific thrombin inhibitor similar to its natural counterpart as well as rhodniin and hirudin. The Ki value of the recombinant dipetalogastin was determined to be 49.3 +/- 22.28 fM.     

Primary structure and reactive site of a novel wheat proteinase inhibitor of subtilisin and chymotrypsin

The proteinase inhibitor WSCI, active in inhibiting bacterial subtilisin and a number of animal chymotrypsins, was purified from endosperm of exaploid wheat (Triticum aestivum, c.v. San Pastore) by ion exchange chromatography and its complete amino acid sequence was established by automated Edman degradation. WSCI consists of a single polypeptide chain of 72 amino acid residues, has a molecular mass of 8126.3 Da and a pl of 5.8. The inhibition constants (Ki) for Bacillus licheniformis subtilisin and bovine pancreatic alpha-chymotrypsin are 3.92 x 10(-9) M and 7.24 x 10(-9) M, respectively. The inhibitor contains one methionine and of tryptophan residue and has a high content of essential amino acids (41 over a total of 72 residues), but no cysteines. The primary structure of WSCI shows high similarity with barley subtilisin-chymotrypsin isoinhibitors of the Cl-2 type and with maize subtilisinchymotrypsin inhibitor MPI. Significant degrees of similarity were also found between sequences of WSCI and of other members of the potato inhibitor I family of the serine proteinase inhibitors. The wheat inhibitor WSCI has a single reactive site (the peptide bond between methionyl-48 and glutamyl-49 residues) as identified by affinity chromatography and sequence analysis.     

Sequence of a new Bowman-Birk inhibitor fromTorresea acreana seeds and comparison withTorresea cearensis trypsin inhibitor (TcTI2)

TaTI (Torresea acreana trypsin inhibitor), a new member of the Bowman-Birk trypsin inhibitor family, was purified from seeds ofTorresea acreana, one of the two known species ofTorresea, a Brazilian native Leguminosae of the Papilionoideae subfamily. Purification was performed by acetone fractionation, anion-exchange chromatography, and gel filtration. The TaTI appears asM _r 7000 in SDS-PAGE under reducing conditions. There are 63 amino acid residues present in the TaTI sequence, which was confirmed by mass spectrometry (8388 daltons). The putative reactive sites residues were Lys-15 and Arg-42 at the first and second site, respectively. The antibodies raised against TcTI2,Torresea cearensis trypsin inhibitor 2, showed a cross-reaction with TaTI, but not with other Bowman-Birk inhibitors purified from Leguminosae. The inhibition constants of TaTI and TcTI2 were comparable when measured against trypsin, chymotrypsin, and factor XIIa, but not on plasmin. The latter was tenfold more effectively inhibited by TcTI2 then by TaTI. Neither TaTI nor TcTI2 affects thrombin, plasma kallikrein, or factor Xa.     

Sequence of a new Bowman-Birk inhibitor fromTorresea acreana seeds and comparison withTorresea cearensis trypsin inhibitor (TcTI2)

TaTI (Torresea acreana trypsin inhibitor), a new member of the Bowman-Birk trypsin inhibitor family, was purified from seeds ofTorresea acreana, one of the two known species ofTorresea, a Brazilian native Leguminosae of the Papilionoideae subfamily. Purification was performed by acetone fractionation, anion-exchange chromatography, and gel filtration. The TaTI appears asM _r 7000 in SDS-PAGE under reducing conditions. There are 63 amino acid residues present in the TaTI sequence, which was confirmed by mass spectrometry (8388 daltons). The putative reactive sites residues were Lys-15 and Arg-42 at the first and second site, respectively. The antibodies raised against TcTI2,Torresea cearensis trypsin inhibitor 2, showed a cross-reaction with TaTI, but not with other Bowman-Birk inhibitors purified from Leguminosae. The inhibition constants of TaTI and TcTI2 were comparable when measured against trypsin, chymotrypsin, and factor XIIa, but not on plasmin. The latter was tenfold more effectively inhibited by TcTI2 then by TaTI. Neither TaTI nor TcTI2 affects thrombin, plasma kallikrein, or factor Xa.     

Bovine plasma protein C inhibitor with structural and functional homologous properties to human plasma protein C inhibitor

Bovine plasma protein C inhibitor was purified; it was then characterized in comparison with human protein C inhibitor. The specific inhibitory activity of the purified inhibitor for bovine activated protein C was 8,500 times that of the inhibitor in plasma. The purified inhibitor showed a single band with Mr 56,000 by SDS-PAGE at pH 7.0, and two bands at pH 8.8, a major one with Mr 56,000 and a minor one with Mr 105,000, under both unreduced and reduced conditions. The pI range of the inhibitor was between 4.4 and 6.1. The Mr of the inhibitor was reduced by treatment with neuraminidase, O-glycanase, and also with glycopeptidase-A, suggesting that the inhibitor has both Asn-linked and Ser/Thr-linked carbohydrate chains. Twenty-seven of the NH2-terminal 49 amino acid residues of the bovine inhibitor, which lacks the first 4 residues from the NH2-terminal amino acid sequence of human inhibitor, were identical to those of the human inhibitor. The bovine inhibitor inhibited bovine and human activated protein C, human thrombin, Factor Xa, Factor XIa, and plasma kallikrein with Ki = 1.0, 5.2, 2.6, 3.0, 1.3 X 10(-8) M, and 4.5 X 10(-9) M, respectively. The inhibitory rates for activated protein C and thrombin were accelerated significantly in the presence of heparin or negatively charged dextran sulfate. However, the acceleration by heparin or dextran sulfate for the inhibition of Factor Xa, Factor XIa, and plasma kallikrein was not significant. The bovine inhibitor did not inhibit human Factor XIIa or plasmin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the interactions of a bifunctional inhibitor with alpha-thrombin by molecular modelling and peptide synthesis.

A potent thrombin inhibitor, [D-Phe45, Arg47] hirudin 45-65, that contains an active site-directed sequence D-Phe-Pro-Arg-Pro, an exosite specific fragment hirudin 55-65 (H55-65) and a linker portion hirudin 49-54, was designed based on the hirudin sequence [DiMaio et al. (1990) J. Biol. Chem., 265, 21698-21798]. A three-dimensional model of the complex between the B-chain of human thrombin and the inhibitor [D-Phe45, Arg47] hirudin 45-65 was constructed using molecular modelling starting from the X-ray C alpha coordinates of the thrombin-hirudin complex and the NMR-derived structure of the thrombin-bound hirudin 55-65. The contribution of the H49-54 fragment to the thrombin-inhibitor interaction was deduced by examining a series of analogs containing single glycine substitution and analogs with reduced number of residues within the linker. The results were consistent with the molecular modelling observations i.e. the H49-54 fragment serves the role of a spacer in the binding interaction and could be replaced by four glycine residues. The studies on the interaction of the exosite-directed portion of the inhibitor with thrombin using a series of synthetic H55-65 analogs demonstrated that residues AspH55 to ProH60 play a major role in binding to human thrombin where the side chains of PheH56, IleH59 and GluH57 showed critical contributions. Molecular modelling suggested that these side chains may contribute to inter- and intramolecular hydrophobic and electrostatic interactions, respectively.     

A serine protease inhibitor from the anemone Radianthus macrodactylus: isolation and physicochemical characteristics

A serine protease inhibitor with a molecular mass of 6106 +/- 2Da (designated as InhVJ) was isolated from the tropical anemone Radianthus macrodactylus by a combination of liquid chromatography methods. The molecule of InhVJ consists of 57 amino acid residues, has three disulfide bonds, and contains no Met or Trp residues. The N-terminal amino acid sequence of the inhibitor (19 aa residues) was established. It was shown that this fragment has a high degree of homology with the N-terminal amino acid sequences of serine protease inhibitors from other anemone species, reptiles, and mammals. The spatial organization of the inhibitor at the levels of tertiary and secondary structures was studied by the methods of UV and CD spectroscopy. The specific and molar absorption coefficients of InhVJ were determined. The percentage of canonical secondary structure elements in the polypeptide was calculated. The inhibitor has a highly ordered tertiary structure and belongs to mixed alpha/beta or alpha + beta polypeptides. It was established that InhVJ is highly specific toward trypsin (Ki 2.49 x 10(-9) M) and alpha-chymotrypsin (Ki 2.17 x 10(-8) M) and does not inhibit other proteases, such as thrombin, kallikrein, and papain. The inhibitor InhVJ was assigned to the family of the Kunitz inhibitor according to its physicochemical properties.     

Transgenic rabbit producing human growth hormone in milk

The gene construct WAP(6xHisThr):hGH containing the entire human growth hormone gene (hGH) under the rat whey acidic protein (WAP) promoter regulating the expression in mammary glands of mammals was prepared. The 5' end of the gene was modified by the addition of a sequence encoding six histidine residues and a sequence recognized by thrombin. The gene construct was introduced by microinjection into the male pronucleus of a fertilized oocyte. The founder male rabbit was obtained with the transgene mapping to chromosome 7. The presence of the growth hormone was confirmed in samples of milk collected during the lactation of F1 generation females. The growth hormone can be easily purified by affinity chromatography and cleavage by thrombin to an active form.     

Purification of granulin-like polypeptide from the blood-sucking leech, Hirudo nipponia.

A cysteine-rich (approximately 20%), low molecular weight (MW 6 kDa) polypeptide has been isolated from the Korean blood-sucking leech, Hirudo nipponia. From its amino acid composition and N-terminal amino acid sequence analysis, the new protein is similar to granulin (or epithelin), and so it has been named leech granulin. The leech granulin behaved as a thrombin inhibitor in the purification steps of size-exclusion, ion-exchange, chromatofocusing, and reverse-phase high-performance liquid chromatography. The leech granulin is an acidic peptide (pI 3.75) containing high cysteine residues with a unique sequence similar to granulins or epithelins isolated from other organisms. For the first time, a granulin-like polypeptide having thrombin inhibitory activity has been isolated from a leech species.     

Electromagnetic radiation and behavioural response of ticks: an experimental test

Blood-sucking arthropods have different types of anticoagulants to allow the ingestion of a blood meal from their hosts. In this study, five anticoagulants prolonging the activated partial thromboplastin time were resolved from the salivary gland crude extract of the camel tick Hyalomma dromedarii by chromatography on diethylaminoethyl (DEAE)-cellulose column. They were designated P1, P2, P3, P4 and P5 according to their elution order. P5 was found to be a potent thrombin inhibitor and purified by ultrafiltration through two centrifugal concentrators of 50 and 30 kDa molecular weight cut-off (MWCO), respectively. The camel tick salivary gland thrombin inhibitor was purified 60.6 folds with a specific activity of 564 units/mg protein. It turned out to be homogenous on native-PAGE with molecular weight of 36 kDa as detected on 12% SDS-PAGE. It inhibits bovine thrombin competitively with K _i value of 0.55 μM. A task for the future will be the elucidation of this thrombin inhibitor structure to allow its application in thrombosis treatment.