Maria Luiza Vilela Oliva

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.

Primary structure of a Kunitz-type trypsin inhibitor from Enterolobium contortisiliquum seeds.

A trypsin inhibitor was isolated from Enterolobium contortisiliquum seeds. Starting with a saline extract, ECTI (E. contortisiliquum trypsin inhibitor) was purified as a homogeneous protein by acetone precipitation, ion-exchange chromatography (DEAE-Sephadex A-50), gel filtration (Sephadex G-75 and Superose 12) and reversed phase HPLC (mu-Bondapak C-18). The amino acid sequence was determined by automatic degradation and by DABITC/PITC microsequence analysis of the reduced and carboxymethylated protein and also of purified peptides derived from the protein by cleavage with iodosobenzoic acid and by enzymic digestion with trypsin, chymotrypsin and Staphylococcus aureus V8 protease. ECTI contains 174 amino acid residues in two polypeptide chains, an alpha-chain consisting of 134 residues and a beta-chain made up of 40 residues. The inhibitor displays a high degree of sequence identity with other Kunitz-type proteinase inhibitors isolated from the Mimosoideae subfamily. The reactive site was identified (by homology) as the arginine-isoleucine peptide bond at position 64-65. ECTI inhibits trypsin and chymotrypsin in the stoichiometric ratio of 1:1 and also Factor XIIa, plasma kallikrein and plasmin, but not thrombin and Factor Xa.

A novel subclassification for Kunitz proteinase inhibitors from leguminous seeds.

Kunitz-type trypsin inhibitors from legume seeds have been characterized structurally. The presence of Cys-Cys in single or double chains shows a new pattern of proteins structurally not so closely related to STI. Therefore, briefly, with regard to cysteine content, plant Kunitz proteinase inhibitors may be classified into four groups: no Cys-Cys at all, one, two and more than two Cys residues. Functional properties and diversity of these proteins are also briefly discussed.

Coagulant and antibacterial activities of the water-soluble seed lectin from Moringa oleifera

Aims:  The aim of this work was to analyse the coagulant and antibacterial activities of lectin isolated from Moringa oleifera seeds that are used for water treatment.

Kunitz-type Bauhinia bauhinioides inhibitors devoid of disulfide bridges: isolation of the cDNAs, heterologous expression and structural studies.

Abstract Bauhinia bauhinoides cruzipain inhibitor (BbCI) and Bauhinia bauhinioides kallikrein inhibitor (BbKI) are cysteine and serine proteinase inhibitors structurally homologous to plant Kunitz-type inhibitors, but are devoid of disulfide bridges. Based on cDNA sequences, we found that BbKI and BbCI are initially synthesized as a prepropeptide comprising an N-terminal signal peptide (19 residues), the mature protein (164 residues) and a C-terminal targeting peptide (10 residues). Partial cDNAs encoding the mature enzymes plus N-terminal His-tags and thrombin cleavage sites were expressed in E. coli and the soluble proteins were purified by one-step nickel affinity chromatography. After thrombin cleavage, both proteins exhibited potent inhibitory activities toward their cognate proteinases like the wild-type proteins. BbCI inhibits human neutrophil elastase (K i(app) 5.3 nM), porcine pancreatic elastase (K i(app) 40 nM), cathepsin G (K i(app) 160 nM) and the cysteine proteinases cruzipain (K i(app) 1.2 nM), cruzain (K i(app) 0.3 nM) and cathepsin L (K i(app) 2.2 nM), while BbKI strongly inhibits plasma kallikrein (K i(app) 2.4 nM) and plasmin (K i(app) 33 nM). Circular dichroism spectra of BbCI and BbKI were in agreement with the β-trefoil fold described for Kunitz inhibitors. The inhibitory potency of both BbCI- and BbKI-type inhibitors suggests that other, non-covalent interactions may compensate for the lack of disulfide bridges.

Structure of cruzipain/cruzain inhibitors isolated from Bauhinia bauhinioides seeds.

Abstract The saline extract of Bauhinia bauhinioides dry seeds was shown to inhibit cruzipain, a cysteine proteinase from Trypanosoma cruzi. The inhibitory activity was assigned to a protein with 164 amino acid residues and molecular mass of 18 034 Da that was purified by chromatography on DEAESephadex, trypsinSepharose (removal of trypsin inhibitors), Mono Q and a reversed phase C[4] column. The primary structure is homologous to other plant Kunitztype inhibitors, but it lacks cysteine residues and therefore the disulfide bridges. No methionine residue was identified by amino acid sequencing. The inhibition of cruzipain fits into a slowtight binding mechanism with a low dissociation constant (K 1.2 nM). The studied Bauhinia protein also inhibits cruzain (K 0.3 nM), a Cterminally truncated recombinant species of cruzipain. Cathepsin L, a cysteine proteinase with high homology to cruzipain, is also inhibited (K 0.22 nM), but not cathepsin B, papain, bromelain or ficin.

Purification of a lectin with antibacterial activity from Bothrops leucurus snake venom.

A novel lectin was isolated from Bothrops leucurus snake venom using a combination of affinity and gel filtration chromatographies. The lectin (BlL) agglutinated glutaraldehyde-treated rabbit and human erythrocytes with preference for rabbit erythrocytes. Galactose, raffinose, lactose, fetal bovine serum and casein inhibited lectin-induced rabbit erythrocyte agglutination. BlL, with a molecular mass of 30 kDa and composed of two subunits of 15 kDa, showed dependence on calcium. BlL is an acidic protein with highest activity over the pH range of 4.0-7.0 and stable under heating to 70°C. Fluorescence emission spectra showed tryptophan residues partially buried within the lectin structure. The percentages of secondary structure revealed by circular dichroism were 1% α-helix, 44% β-sheet, 24% β-turn and 31% unordered. BlL showed effective antibacterial activity against Gram-positive bacteria Staphylococcus aureus, Enterococcus faecalis and Bacillus subtilis with minimal inhibitory concentrations of 31.25, 62.25 and 125 μg/mL, respectively. In conclusion, B. leucurus snake venom contains a galactoside-binding lectin with antibacterial activity.

In vivo characterization of Lopap, a prothrombin activator serine protease from the Lonomia obliqua caterpillar venom.

Increasing occurrence of hemorrhagic syndrome in man, caused by contact with Lonomia obliqua caterpillars, has been reported in Southern Brazil in the past 10 years. The L. obliqua venom causes a severe consumptive coagulopathy, which can lead to hemorrhagic syndrome. L. obliqua prothrombin activator protease (Lopap) is a 69-kDa prothrombin activator serine protease isolated from L. obliqua caterpillar bristle extract, which is able to evoke thrombus formation, unclottable blood, and fibrinogen depletion when injected into the blood stream of rats. The purified protein generated thrombin from prothrombin, able to clot purified human fibrinogen and plasma. A decrease in platelet count and inhibition of collagen-induced platelet aggregation were observed, as well as leukocyte infiltration in the lungs. In addition, we observed congestion and hemorrhage in renal glomeruli and necrosis in renal distal tubules. These data support the hypothesis that Lopap contributes to the clinical syndrome caused by human contact with L. obliqua, most probably through prothrombin activation, resulting in a consumption coagulopathy.

Insularinase A, a prothrombin activator from Bothrops insularis venom, is a metalloprotease derived from a gene encoding protease and disintegrin domains

Abstract The first low-molecular-mass metalloprotease presenting prothrombin activating activity was purified from Bothrops insularis venom and named insularinase A. It is a single-chain protease with a molecular mass of 22 639 Da. cDNA sequence analysis revealed that the disintegrin domain of the precursor protein is post-translationally processed, producing the mature insularinase A. Analysis of its deduced amino acid sequence showed a high similarity with several fibrin(ogen)olytic metalloproteases and only a moderate similarity with prothrombin activators. However, SDS-PAGE of prothrombin after activation by insularinase A showed fragment patterns similar to those generated by group A prothrombin activators, which convert prothrombin into meizothrombin independently of the prothrombinase complex. In addition, insularinase A activates factor X and hydrolyses fibrinogen and fibrin. Chelating agents fully inhibit all insularinase A activities. Insularinase A induced neither detachment nor apoptosis of human endothelial cells and was also not able to trigger an endothelial proinflammatory cell response. Nitric oxide and prostacyclin levels released by endothelial cells were significantly increased after treatment with insularinase A. Our results show that, although its primary structure is related to class P-I fibrin(ogen)olytic metalloproteases, insularinase A is functionally similar to group A prothrombin activators.

Primary sequence determination of a Kunitz inhibitor isolated from Delonix regia seeds

A serine proteinase inhibitor was purified from Delonix regia seeds a Leguminosae tree of the Caesalpinioideae subfamily. The inhibitor named DrTI, inactivated trypsin and human plasma kallikrein with K(i )values 2.19x10(-8) M and 5.25 nM, respectively. Its analysis by SDS-PAGE 10-20% showed that the inhibitor is a protein with a single polypeptide chain of M(r) 22 h Da. The primary sequence of the inhibitor was determined by Edman degradation, thus indicating that it contained 185 amino acids and showed that it belongs to the Kunitz type family; however, its reactive site did not contain Arg or Lys at the putative reactive site (position 63, SbTI numbering) or it was displaced when compared to other Kunitz-type inhibitors.