Claudio A. M. Sampaio

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 double headed serine proteinase inhibitor — human plasma kallikrein and elastase inhibitor — from Boophilus microplus larvae

Preying on cattle, the hard tick Boophilus microplus causes heavy economical losses to Brazil. Tick proteins are a good target to be used as tools for tick control. Serine protease inhibitors from B. microplus larvae (BmTI) were preliminarily characterized. One-week-old larvae were the source of a 2% protein solution in 5 mM Tris-HCl, 20 mM NaCl, pH 7.4. The inhibitors were purified by affinity chromatography on trypsin-Sepharose, and ion-exchange chromatography on Resource Q column, and they separated in two major active peaks, corresponding to 10-kDa and 18-kDa proteins (BmTI-B and BmTI-A, respectively). Both purified proteins inhibited trypsin with Ki of 0.3 and 3.0 nM, respectively, but only the 18-kDa protein inhibited elastase (Ki 1.4 nM) and plasma kallikrein (Ki 120 nM). BmTI-A did not change prothrombin time (PT) and thrombin time (TT), but it increased activated partial thromboplastin time (APTT) was dose-dependent. The partial amino acid sequence indicated that BmTI-A belongs to the bovine pancreatic trypsin inhibitor (BPTI)-Kunitz type inhibitor family. These inhibitors (by their properties) play a role in the feeding process of the tick. Development of antibodies against these proteins may be used to impair the normal feeding and consequently, the parasite would be no longer viable.

BmTI antigens induce a bovine protective immune response against Boophilus microplus tick.

Boophilus microplus trypsin inhibitors (BmTIs) present in larvae were preliminarily characterized as active proteins, approximately 10-18 kDa, by SDS-PAGE. BmTIs showed trypsin inhibitory activity on reverse zymography containing gelatin (0.03%) and also inhibited others serine proteinases (human neutrophil elastase and human plasma kallikrein). Bos indicus, Nelore breed calves, previously sensitized with BmTIs and challenged with tick larvae (20,000 larvae/animal), showed 72.8% efficacy to interfere in tick development with 69.7% and 71.3% reduction of both tick number and egg weight, respectively. Cattle BmTls antiserum titer was approximately 1:8000. The maximum level of BmTls antibody production was detected 40 days after the first immunization by ELISA. Our preliminary results suggest that B. microplus serine proteinase inhibitors may play a role in the tick larvae fixation and feeding processes. Therefore, the development of antibodies against BmTIs might impair the normal parasitism.

A prothrombin activator serine protease from the Lonomia obliqua caterpillar venom (Lopap) biochemical characterization.

Lonomia obliqua venom causes a severe consumptive coagulopathy, which can lead to a hemorrhagic syndrome. The crude bristles extract displays a procoagulant activity due to a Factor X and to a prothrombin activating activity. Here, we describe a 69 kDa prothrombin activator serine protease purified from L. obliqua caterpillar bristle extract using gel filtration (Sephadex G 75) and HPLC (C(4) column). The purified protein was able to activate prothrombin in a dose-dependent manner, and calcium ions increased this activity. The prothrombin-derived fluorogenic peptide (Abz-YQTFFNPRTGSQ-EDDnp) had its main cleavage site at the Arg-Thr bond. The kinetic parameters obtained for this substrate were Kmapp of 4.5 microM, kcat of 5.32 s(-1), and a kcat/Kmapp of 1.2 x 10(6) M(-1) s(-1). The prothrombin fragments generated by the purified enzyme corresponded to the molecular masses of prethrombin 2, fragment 1, fragment 2, and thrombin as seen in SDS-PAGE. The thrombin generated was able to clot purified fibrinogen. The partial amino acid sequence of the purified protein, named Lopap (L. obliqua prothrombin activator protease), showed no similarity to any known prothrombin activator.

The BIR domain of IAP-like protein 2 is conformationally unstable: implications for caspase inhibition.

Several IAP (inhibitor of apoptosis) proteins regulate cell fate decisions, and the X-linked IAP (XIAP) does so in part by inhibiting caspases, proteases that execute the apoptotic pathway. A tissue-specific homologue of XIAP, known as ILP2 (IAP-like protein 2), has previously been implicated in the control of apoptosis in the testis by direct inhibition of caspase 9. In examining this protein we found that the putative caspase 9 interaction domain is a surprisingly weak inhibitor and is also conformationally unstable. Comparison with the equivalent domain in XIAP demonstrated that the instability is due to the lack of a linker segment N-terminal to the inhibitory BIR (baculovirus IAP repeat) domain. Fusion of a 9-residue linker from XIAP to the N-terminus of ILP2 restored tight caspase 9 inhibition, dramatically increased conformational stability and allowed crystallization of the ILP2 BIR domain in a form strikingly similar to the XIAP third BIR domain. We conclude that ILP2 is an unstable protein, and cannot inhibit caspase 9 in a physiological way on its own. We speculate that ILP2 requires assistance from unidentified cellular factors to be an effective inhibitor of apoptosis in vivo.

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.

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.

Basic proteinases from Bothrops moojeni (caissaca) venom—I. Isolation and activity of two serine proteinases, MSP 1 and MSP 2, on synthetic substrates and on platelet aggregation

Two serine proteinases, MSP 1 and MSP 2, were isolated from Bothrops moojeni venom by chromatographies on Sephadex G-100, DEAE-Sephacel (pH 7.5) and SP-Sephadex C-50 (pH 7.5). Both enzymes are basic glycoproteins. On sodium dodecyl sulfate-polyacrylamide electrophoresis, MSP 1 presented two close protein bands corresponding to the mol. wts of 34,000 and 32,500. MSP 2 behaved as a single-chain protein with a mol. wt of 38,000. Specific esterolytic activities of MSP 1 and MSP 2 on alpha-N-tosyl-L-arginine methyl ester (TAME) are 33 mumol min-1 mg-1 and 184 mumol min-1 mg-1, respectively. The most sensitive substrates for the amidolytic activity of both proteinases were the thrombin substrate D-Phe-pipecolyl(Pip)-Arg-4-nitroanilide(Nan) and the glandular kallikrein substrate D-Val-Leu-Arg-Nan. MSP 1, in a concentration of 10(-8) M, causes platelet aggregation in platelet-rich plasma and washed platelets. It also enhances the ADP-induced platelet aggregation. Prostaglandin E1 (PGE1), phenylmethylsulfonyl fluoride (PMSF) and ethylenediamine tetracetic acid (EDTA) abolished completely the aggregation induced by MSP 1. Torresea cearensis trypsin inhibitor (TCTI) inhibited both amidolytic (Ki = 1.96 x 10(-7) M) and platelet-aggregating (Ki = 1.66 x 10(-7) M) activities of MSP 1. The esterolytic activity of MSP 1 and MSP 2 was completely abolished by PMSF, only partially by soybean trypsin inhibitor (SBTI) and benzamidine and not affected by Trasylol. MSP 2 was also inhibited by TCTI (Ki = 0.7 x 10(-7) M).