Infestin, a thrombin inhibitor presents in Triatoma infestans midgut, a Chagas' disease vector: gene cloning, expression and characterization of the inhibitor.

This work describes the purification, gene cloning and expression of infestin, a thrombin inhibitor from midguts of Triatoma infestans. Infestin is located in the midgut and its purification was performed by anion-exchange and affinity chromatographies. The N-terminal sequence and the sequence of tryptic peptides were determined. Using RT-PCR, total RNA and infestin cDNA information, a DNA fragment was cloned which encodes a multi non-classical Kazal-type serine protease inhibitor. Isolated native infestin has two non-classical Kazal-type domains and shows an apparent molecular mass of 13 kDa, while its gene codes for a protein with four non-classical Kazal-type domains corresponding to an apparent molecular mass of 22 kDa. Two recombinant infestins, r-infestin 1-2 and r-infestin 1-4, were constructed using the vector pVT102U/alpha and expressed in S. cerevisiae. Native and r-infestin 1-2 showed very similar inhibitory activities towards thrombin and trypsin with dissociation constants of 43.5 and 25 pM for thrombin and 2.0 and 3.1 nM for trypsin, respectively. No other serine protease of the blood coagulation cascade was inhibited by the r-infestin 1-2. Surprisingly, r-infestin 1-4 inhibited not only thrombin and trypsin (K(i) of 0.8 and 5.2 nM, respectively), but also factor XIIa, factor Xa and plasmin (K(i) of 78 pM, 59.2 and 1.1 nM, respectively).

Bauhinia bauhinioides plasma kallikrein inhibitor: interaction with synthetic peptides and fluorogenic peptide substrates related to the reactive site sequence.

A serine proteinase inhibitor was purified from Bauhinia bauhinioides seeds after extraction with 0.15M NaCl by ion-exchange column chromatography on DEAE-Sephadex, gel filtration on Superose 12 column, Mono Q chromatography or alternatively by affinity chromatography on trypsin- Sepharose. The inhibitor is a single polypeptide chain with molecular mass 20 kDa by gel filtration on Superose 12, but was resolved into two peaks by ion - exchange chromatography on Mono Q (FPLC system). The main eluted peak inhibits trypsin (Ki = 0.6 nM), plasma kallikrein (Ki = 0.35 nM), plasmin (Ki = 33.1 nM), and weakly chymotrypsin (Ki = 2,700 nM), being the most effective plasma kallikrein inhibitor isolated from Bauhinia seeds. Therefore, it was denominated Bauhinia bauhinioides kallikrein inhibitor (BbKI). Activity is thermolabile and on trypsin inhibition optimum pH is 8.0. BbKI displays high homology to other plant Kunitz inhibitors, except for the absence of disulfide bridges, and the only cysteine residue is at the C-terminal position (residue 154) characterizes a distinct member of the Kunitz family. The affinity of the inhibitor to trypsin was confirmed by adsorption to trypsin-Sepharose resin and by isolation of the trypsin-inhibitor complex by gel filtration. Peptides with variations around the reactive site of BbKI (GLPVRFESPLRINIIKESY) were synthesized containing a quenched fluorogenic group. Trypsin but not plasma kallikrein substrates, these peptides strongly inhibited plasma kallikrein.

Leucaena leucocephala serine proteinase inhibitor: primary structure and action on blood coagulation, kinin release and rat paw edema.

A serine proteinase inhibitor isolated from Leucaena leucocephala seeds (LlTI) was purified to homogeneity by acetone fractionation, ion exchange chromatography, gel filtration and reverse phase chromatography (HPLC). SDS-PAGE indicated a protein with M(r) 20000 and two polypeptide chains (alpha-chain, M(r) 15000, and beta-chain, M(r) 5000), the sequence being determined by automatic Edman degradation and by mass spectroscopy. LlTI is a 174 amino acid residue protein which shows high homology to plant Kunitz inhibitors, especially those double chain proteins purified from the Mimosoideae subfamily. LlTI inhibits plasmin (K(i) 3.2 x 10(-10) M), human plasma kallikrein (K(i) 6.3 x 10(-9) M), trypsin (K(i) 2.5 x 10(-8) M) and chymotrypsin (K(i) 1.4 x 10(-8) M). Factor XIIa activity is inhibited but K(i) was not determined, and factor Xa, tissue kallikrein and thrombin are not inhibited by LlTI. The action of LlTI on enzymes that participate in the blood clotting extrinsic pathway is confirmed by the prolongation of activated partial thromboplastin time, used as clotting time assay. The inhibition of the fibrinolytic activity of plasmin was confirmed on the hydrolysis of fibrin plates. LlTI inhibits kinin release from high molecular weight kininogen by human plasma kallikrein in vitro and, administered intravenously, causes a decrease in paw edema induced by carrageenin or heat in male Wistar rats. In addition, lower concentrations of bradykinin were found in limb perfusion fluids of LlTI-treated rats.

Human plasma kallikrein and tissue kallikrein binding to a substrate based on the reactive site of a factor Xa inhibitor isolated from Bauhinia ungulata seeds.

Kunitz type Bauhinia ungulata factor Xa inhibitor (BuXI) was purified from B. ungulata seeds. BuXI inactivates factor Xa and human plasma kallikrein (HuPK) with Ki values of 18.4 and 6.9 nM, respectively. However, Bauhinia variegata trypsin inhibitor (BvTI) which is 70% homologous to BuXI does not inhibit factor Xa and is less efficient on HuPK (Ki = 80 nM). The comparison between BuXI and BvTI reactive site structure indicates differences at Met59, Thr66 and Met67 residues. The hydrolysis rate of quenched fluorescence peptide substrates based on BuXI reactive site sequence, Abz-VMIAALPRTMFIQ-EDDnp (leading peptide), by HuPK and porcine pancreatic kallikrein (PoPK) is low, but hydrolysis is enhanced with Abz-VMIAALPRTMQ-EDDnp, derived from the leading peptide shortened by removing the dipeptide Phe-Ileu from the C-terminal portion, for HuPK (Km = 0.68 microM, k(cat)/Km = 1.3 x 10(6) M(-1) s(-1)), and the shorter substrate Abz-LPRTMQ-EDDnp is better for PoPK (Km = 0.66 microM, k(cat)/Km = 2.2 x 10(3) M(-1) s(-1)). The contribution of substrate methionine residues to HuPK and PoPK hydrolysis differs from that observed with factor Xa. The determined Km and k(cat) values suggest that the substrates interact with kallikreins the same as an enzyme and inhibitor interacts to form complexes.

Functional phage display of leech-derived tryptase inhibitor (LDTI): construction of a library and selection of thrombin inhibitors.

The recombinant phage antibody system pCANTAB 5E has been used to display functionally active leech-derived tryptase inhibitor (LDTI) on the tip of the filamentous M13 phage. A limited combinatorial library of 5.2 x 10(4) mutants was created with a synthetic LDTI gene, using a degenerated oligonucleotide and the pCANTAB 5E phagemid. The mutations were restricted to the P1-P4' positions of the reactive site. Fusion phages and appropriate host strains containing the phagemids were selected after binding to thrombin and DNA sequencing. The variants LDTI-2T (K8R, I9V, S10, K11W, P12A), LDTI-5T (K8R, I9V, S10, K11S, P12L) and LDTI-10T (K8R, I9L, S10, K11D, P12I) were produced with a Saccharomyces cerevisiae expression system. The new inhibitors, LDTI-2T and -5T, prolong the blood clotting time, inhibit thrombin (Ki 302 nM and 28 nM) and trypsin (Ki 6.4 nM and 2.1 nM) but not factor Xa, plasma kallikrein or neutrophil elastase. The variant LDTI-10T binds to thrombin but does not inhibit it. The relevant reactive site sequences of the thrombin inhibiting variants showed a strong preference for arginine in position P1 (K8R) and for valine in P1' (I9V). The data indicate further that LDTI-5T might be a model candidate for generation of active-site directed thrombin inhibitors and that LDTI in general may be useful to generate specific inhibitors suitable for a better understanding of enzyme-inhibitor interactions.

Primary structure of Dioclea glabra trypsin inhibitor, DgTI, a Bowman-Birk inhibitor.

A novel serine proteinase inhibitor, DgTI, was purified from Dioclea glabra seeds by acetone precipitation, and ion-exchange and reverse phase chromatography. The inhibitor belongs to the Bowman-Birk family, and its primary sequence, determined by Edman degradation and mass spectrometry, of 67 amino acids is: SSGPCCDRCRCTKSEPPQCQCQDVRLNSCHSACEACVCSHSMPGLCSCLDITHFCHEPCKSSGDDED++ +. Although two reactive sites were determined by susceptibility to trypsin (Lys(13) and His(40)), the inhibitory function was assigned only to the first site. The inhibitor forms a 1:1 complex with trypsin, and Ki is 0.5 x 10(-9) M. Elastase, chymotrypsin, kallikreins, factor Xa, thrombin, and plasmin were not inhibited. By its properties, DgTI is a Bowman-Birk inhibitor with structural and inhibitory properties between the class of Bowman-Birk type I (with a fully active second reactive site), and Bowman-Birk type II (devoid of second reactive site).

A novel phospholipase A2, BJ-PLA2, from the venom of the snake Bothrops jararaca: purification, primary structure analysis, and its characterization as a platelet-aggregation-inhibiting factor.

This paper describes the isolation and primary structure analysis of a new phospholipase A2 with platelet-aggregation-inhibiting activity from the venom of Bothrops jararaca. The protein, named BJ-PLA2, was isolated by means of ammonium sulfate precipitation and anion-exchange and reversed-phase chromatographies and behaved as a homogeneous single-chain protein on SDS-PAGE. Its amino acid sequence was determined by N-terminal sequencing and analysis of overlapped chemical and proteolytic fragments by automated Edman degradation and mass spectometry determination. BJ-PLA2 consists of 124 amino acid residues and has the structural features of snake venom class II phospholipases A2. Chemical modification with p-bromophenacylbromide caused complete loss of enzymatic activity and partially affected the platelet-aggregation-inhibiting activity of BJ-PLA2.

Sequence of a new Bowman-Birk inhibitor from Torresea acreana seeds and comparison with Torresea cearensis trypsin inhibitor (TcTI2).

TaTI (Torresea acreana trypsin inhibitor), a new member of the Bowman-Birk trypsin inhibitor family, was purified from seeds of Torresea acreana, one of the two known species of Torresea, a Brazilian native Leguminosae of the Papilionoideae subfamily. Purification was performed by acetone fractionation, anion-exchange chromatography, and gel filtration. The TaTI appears as M(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.

Amino acid sequence of a lectin-like protein from Lachesis muta stenophyrs venom.

The primary structure of the lectin-like protein from Lachesis muta stenophyrs venom was deduced from analysis of the N-terminus and the sequence of peptides obtained after digestion with trypsin, Arg-C enzyme, Staphylococcus aureus V8 protease and endoproteinase Asp-N. Peptides generated by cleavage of the lectin with cyanogen bromide and o-iodosobenzoic acid were also sequenced. Comparison of the complete 135 amino acid residues sequence with those of the lectin from the venom of Crotalus atrox, with platelet coagglutinin from Bothrops jararaca beta-fragment and with the anticoagulant B protein chain from Trimeresurus flavoviridis venom, revealed 92, 46 and 29% identity, respectively. Significant homology was also found with C-type carbohydrate-recognition domain-like structures from invertebrate and vertebrate lectins. To our knowledge, this is the second known primary structure of a lectin-like protein from snake venom.

Plant serine proteinase inhibitors. Structure and biochemical applications on plasma kallikrein and related enzymes.

The action of two Bowman-Birk and several plant Kunitz-type inhibitors were studied on trypsin, chymotrypsin, plasma kallikrein and factor XII. The primary structure of some of them was completely defined. The results showed that the Bowman-Birk type inhibitors, although potent inhibitors for trypsin (Ki in the range of 1-2 nM), are not able to inhibit plasma kallikrein. Factor XII (Ki = 1.4 microM) and chymotrypsin (Ki = 5.0 nM) are inhibited by Torresea cearensis trypsin inhibitor (TcTI) but not by Dioclea glabra trypsin inhibitor (DgTI). Both inhibitors reactive site regions are highly homologous, and the amino acid residues in P1 position are the same, Lys and His; major differences are in the charge of the C-terminal portion of the molecules. The studied Kunitz-type inhibitors were all able to inhibit plasma kallikrein (Ki between 4 and 80 nM), with the exception of Schizolobium parahyba chymotrypsin inhibitor (SpCI), that is specific for chymotrypsin. All Kunitz-type inhibitors inactivate chymotrypsin, but with a dissociation constant in the range of 0.1 to 0.6 microM. Factor XIIf is inhibited with Ki in the range of 0.1 microM. Bauhinia bauhinioides trypsin inhibitor (BbTI) did not promote factor XIIf inhibition. The Kunitz-type inhibitors are a highly homologous, sharing 60% identity in the N-terminal portion of the loop containing the reactive site, and 28.6% identity in the C-terminal portion of the same loop.