Natterins, a new class of proteins with kininogenase activity characterized from Thalassophryne nattereri fish venom.

A novel family of proteins with kininogenase activity and unique primary structure was characterized using combined pharmacological, proteomic and transcriptomic approaches of Thalassophryne nattereri fish venom. The major venom components were isolated and submitted to bioassays corresponding to its main effects: nociception and edema. These activities were mostly located in one fraction (MS3), which was further fractionated. The isolated protein, named natterin, was able to induce edema, nociception and cleave human kininogen and kininogen-derived synthetic peptides, releasing kallidin (Lys-bradykinin). The enzymatic digestion was inhibited by kallikrein inhibitors as Trasylol and TKI. Natterin N-terminal peptide showed no similarity with already known proteins present in databanks. Primary structure of natterin was obtained by a transcriptomic approach using a representative cDNA library constructed from T. nattereri venom glands. Several expressed sequence tags (ESTs) were obtained and processed by bioinformatics revealing a major group (18%) of related sequences unknown to gene or protein sequence databases. This group included sequences showing the N-terminus of isolated natterin and was named Natterin family. Analysis of this family allowed us to identify five related sequences, which we called natterin 1-4 and P. Natterin 1 and 2 sequences include the N-terminus of the isolated natterin. Furthermore, internal peptides of natterin 1-3 were found in major spots of whole venom submitted to mass spectrometry/2DGE. Similarly to the ESTs, the complete sequences of natterins did not show any significant similarity with already described tissue kallikreins, kininogenases or any proteinase, all being entirely new. These data present a new task for the knowledge of the action of kininogenases and may help in understanding the mechanisms of T. nattereri fish envenoming, which is an important medical problem in North and Northeast of Brazil.

Natterins, a new class of proteins with kininogenase activity characterized from Thalassophryne nattereri fish venom

A novel family of proteins with kininogenase activity and unique primary structure was characterized using combined pharmacological, proteomic and transcriptomic approaches of Thalassophryne nattereri fish venom. The major venom components were isolated and submitted to bioassays corresponding to its main effects: nociception and edema. These activities were mostly located in one fraction (MS3), which was further fractionated. The isolated protein, named natterin, was able to induce edema, nociception and cleave human kininogen and kininogen-derived synthetic peptides, releasing kallidin (Lys-bradykinin). The enzymatic digestion was inhibited by kallikrein inhibitors as Trasylol and TKI. Natterin N-terminal peptide showed no similarity with already known proteins present in databanks. Primary structure of natterin was obtained by a transcriptomic approach using a representative cDNA library constructed from T. nattereri venom glands. Several expressed sequence tags (ESTs) were obtained and processed by bioinformatics revealing a major group (18%) of related sequences unknown to gene or protein sequence databases. This group included sequences showing the N-terminus of isolated natterin and was named Natterin family. Analysis of this family allowed us to identify five related sequences, which we called natterin 1-4 and P. Natterin 1 and 2 sequences include the N-terminus of the isolated natterin. Furthermore, internal peptides of natterin 1-3 were found in major spots of whole venom submitted to mass spectrometry/2DGE. Similarly to the ESTs, the complete sequences of natterins did not show any significant similarity with already described tissue kallikreins, kininogenases or any proteinase, all being entirely new. These data present a new task for the knowledge of the action of kininogenases and may help in understanding the mechanisms of T. nattereri fish envenoming, which is an important medical problem in North and Northeast of Brazil.

ZapA, a possible virulence factor from Proteus mirabilis exhibits broad protease substrate specificity

The opportunistic bacterium Proteus mirabilis secretes a metalloprotease, ZapA, considered to be one of its virulence factors due to its IgA-degrading activity. However, the substrate specificity of this enzyme has not yet been fully characterized. In the present study we used fluorescent peptides derived from bioactive peptides and the oxidized ß-chain of insulin to determine the enzyme specificity. The bradykinin- and dynorphin-derived peptides were cleaved at the single bonds Phe-Ser and Phe-Leu, with catalytic efficiencies of 291 and 13 mM/s, respectively. Besides confirming already published cleavage sites, a novel cleavage site was determined for the ß-chain of insulin (Val-Asn). Both the natural and the recombinant enzyme displayed the same broad specificity, demonstrated by the presence of hydrophobic, hydrophilic, charged and uncharged amino acid residues at the scissile bonds. Native IgA, however, was resistant to hydrolysis by ZapA

ZapA, a possible virulence factor from Proteus mirabilis exhibits broad protease substrate specificity.

The opportunistic bacterium Proteus mirabilis secretes a metalloprotease, ZapA, considered to be one of its virulence factors due to its IgA-degrading activity. However, the substrate specificity of this enzyme has not yet been fully characterized. In the present study we used fluorescent peptides derived from bioactive peptides and the oxidized beta-chain of insulin to determine the enzyme specificity. The bradykinin- and dynorphin-derived peptides were cleaved at the single bonds Phe-Ser and Phe-Leu, with catalytic efficiencies of 291 and 13 mM/s, respectively. Besides confirming already published cleavage sites, a novel cleavage site was determined for the beta-chain of insulin (Val-Asn). Both the natural and the recombinant enzyme displayed the same broad specificity, demonstrated by the presence of hydrophobic, hydrophilic, charged and uncharged amino acid residues at the scissile bonds. Native IgA, however, was resistant to hydrolysis by ZapA.

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.

Free ATP inhibits thimet oligopeptidase (EC 3.4.24.15) activity, induces autophosphorylation in vitro, and controls oligopeptide degradation in macrophage.

The fate of the proteasome-generated peptides depends upon the cytosolic peptidases whose activities ought to be regulated. One of the most important oligopeptide-degrading and -binding proteins in the cytosol is the thimet oligopeptidase (EC 3.4.24.15), ubiquitously found in mammalian tissues. To date, there is no indication whether thimet oligopeptidase activities are physiologically regulated. Here, we present evidences suggesting that the concentration of unbound ATP in the cytosol regulates the thimet oligopeptidase activities both, in vitro and ex vivo. To perform these studies two oligopeptides were used: a quenched fluorescent peptide, which is susceptible to thimet oligopeptidase degradation, and the ovalbumin257-264 (MHC class I ovalbumin epitope), which displays high affinity to the thimet oligopeptidase without being degraded. We also showed that the thimet oligopeptidase undergoes autophosphorylation by ATP, a modification that does not affect the peptidase activity. The autophosphorylation is abolished in the presence of the thimet oligopeptidase substrates, as well as by the effect of a site directed inhibitor of this enzyme, and by the substitution of Glu474 for Asp at the metallo-peptidase motif. Altogether, the results presented here suggest that Zn2+ at the active center of the thimet oligopeptidase is the target for the ATP binding, leading to the inhibition of the enzyme activity, and inducing autophosphorylation. These effects, which depend upon the concentration of the unbound ATP, may help to explain the fate of the proteasomal-generated oligopeptides in the cytosol.

Development of an operational substrate for ZapA, a metalloprotease secreted by the bacterium Proteus mirabilis.

The protease ZapA, secreted by Proteus mirabilis, has been considered to be a virulence factor of this opportunistic bacterium. The control of its expression requires the use of an appropriate methodology, which until now has not been developed. The present study focused on the replacement of azocasein with fluorogenic substrates, and on the definition of enzyme specificity. Eight fluorogenic substrates were tested, and the peptide Abz-Ala-Phe-Arg-Ser-Ala-Ala-Gln-EDDnp was found to be the most convenient for use as an operational substrate for ZapA. A single peptide bond (Arg-Ser) was cleaved with a Km of 4.6 microM, a k cat of 1.73 s-1, and a catalytic efficiency of 376 (mM s)-1. Another good substrate for ZapA was peptide 6 (Abz-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Gln-EDDnp) which was cleaved at a single bond (Phe-Ser) with a Km of 13.6 microM, a k cat of 3.96 s-1 and a catalytic efficiency of 291 (mM s)-1. The properties of the amino acids flanking the scissile bonds were also evaluated, and no clear requirement for the amino acid residue at P1 was found, although the enzyme seems to have a preference for a hydrophobic residue at P2.

Development of an operational substrate for ZapA, a metalloprotease secreted by the bacterium Proteus mirabilis

The protease ZapA, secreted by Proteus mirabilis, has been considered to be a virulence factor of this opportunistic bacterium. The control of its expression requires the use of an appropriate methodology, which until now has not been developed. The present study focused on the replacement of azocasein with fluorogenic substrates, and on the definition of enzyme specificity. Eight fluorogenic substrates were tested, and the peptide Abz-Ala-Phe-Arg-Ser-Ala-Ala-Gln-EDDnp was found to be the most convenient for use as an operational substrate for ZapA. A single peptide bond (Arg-Ser) was cleaved with a Km of 4.6 µM, a k cat of 1.73 s-1, and a catalytic efficiency of 376 (mM s)-1. Another good substrate for ZapA was peptide 6 (Abz-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Gln-EDDnp) which was cleaved at a single bond (Phe-Ser) with a Km of 13.6 µM, a k cat of 3.96 s-1 and a catalytic efficiency of 291 (mM s)-1. The properties of the amino acids flanking the scissile bonds were also evaluated, and no clear requirement for the amino acid residue at P1 was found, although the enzyme seems to have a preference for a hydrophobic residue at P2.

Molecular and immunochemical evidences demonstrate that endooligopeptidase A is the predominant cytosolic oligopeptidase of rabbit brain.

Oligopeptidases are tissue endopeptidases that do not attack proteins and are likely to be involved in the maturation and degradation of peptide hormones and neuropeptides. The rabbit brain endooligopeptidase A and the rat testes soluble metallopeptidase (EC 3.4.24.15) are thiol-activated oligopeptidases which are able to generate enkephalin from a number of opioid peptides and to inactivate bradykinin and neurotensin by hydrolyzing the same peptide bonds. A monospecific antibody raised against the purified rabbit brain endooligopeptidase A allowed the identification of a 2. 3 kb cDNA coding for a truncated enzyme of 512 amino acids, displaying the same enzymatic features as endooligopeptidase A. In spite of all efforts, employing several strategies, the full-length cDNA could not be cloned until now. The analysis of the deduced amino acid sequence showed no similarity to the rat testes metalloendopeptidase sequence, except for the presence of the typical metalloprotease consensus sequence [HEXXH]. The antibody raised against recombinant endooligopeptidase A specifically inhibited its own activity and reduced the thiol-activated oligopeptidase activity of rabbit brain cytosol to less than 30%. Analysis of the endooligopeptidase A tissue distribution indicated that this enzyme is mainly expressed in the CNS, whereas the soluble metallo EC 3.4.24.15 is mainly expressed in peripheral tissues.

Probing the specificity of cysteine proteinases at subsites remote from the active site: analysis of P4, P3, P2' and P3' variations in extended substrates.

We have determined the kinetic parameters for the hydrolysis by papain, cathepsin B and cathepsin L of internally quenched fluorescent peptides derived from the lead peptides Abz-AAFRSAQ-EDDnp [in which Abz and EDDnp stand for o-aminobenzoic acid and N-(2,4-dinitrophenyl)ethylenediamine respectively], to map the specificity of S(4) and S(3) subsites, and Abz-AFRSAAQ-EDDnp, to identify the specificity of S(2)' and S(3)'. Abz and EDDnp were the fluorescent quencher pair. These two series of peptides were cleaved at the Arg-Ser bond and systematic modifications at P(4), P(3), P(2)' and P(3)' were made. The S(4) to S(2)' subsites had a significant influence on the hydrolytic efficiencies of the three enzymes. Only papain activity was observed to be dependent on S(3)', indicating that its binding site is larger than those of cathepsins B and L. Hydrophobic amino acids were accepted at S(4), S(3), S(2)' and S(3)' of the three enzymes. The best substrates for cathepsins L and B had Trp and Asn at P(2)' respectively; variations at this position were less accepted by these enzymes. The best substrates for papain were peptides containing Trp, Tyr or Asn at P(3)'. Basic residues at P(3) and P(4) were well accepted by cathepsin L and papain. We also explored the susceptibility of substrates Abz-AFRSXAQ-EDDnp, modified at P(2)' (X), to human cathepsin B mutants from which one or two occluding loop contacts had been removed. The modifications at His(111) (H111A) and His(110) (H110A) of cathepsin B led to an increase in k(cat) values of one or two orders of magnitude. The hydrolytic efficiencies of these cathepsin B mutants became closer to those of papain or cathepsin L.

Thimet oligopeptidase (EC 3.4.24.15), a novel protein on the route of MHC class I antigen presentation.

The initial processing of antigens leading to major histocompatibility complex (MHC) class I antigenic peptides is carried out by the proteasome. However, how the final epitopes are generated and protected from degradation by cytosolic peptidases remains unknown. Coincidentally, peptides associated with the MHC class I molecules range from 8 to 13 amino acid residues, similarly to the optimum substrate size required for the cytosolic thimet oligopeptidase. Here we have investigated the putative intracellular function of thimet oligopeptidase related to antigen presentation. Using a well-characterized antigen-presenting cell system, we were able to demonstrate either inhibition or stimulation of CD8 T cell proliferation and cytotoxicity, manipulating intracellular thimet oligopeptidase levels with its specific inhibitor cFP-Ala-Ala-Tyr-pAb or loading the enzyme itself into the antigen-presenting cells. Our results suggest that thimet oligopeptidase should take an important function in the pathway of antigen presentation via MHC class I through a mechanism yet unknown.

Thimet oligopeptidase and the stability of MHC class I epitopes in macrophage cytosol.

In this study we investigated the fate of a class of proteasome-generated oligopeptides, exposing them to the crude cytosol of macrophages or to the purified recombinant thimet oligopeptidase. Among the proteasome products of known sequences are MHC class I epitopes, 13 of which were randomly chosen to be used as putative substrates. Surprisingly, our results clearly showed that the majority of the peptides were poorly or not degraded, either by the purified enzyme or by the crude macrophage cytosol. The peptides, which were resistant to hydrolysis, displayed high affinity for the thimet oligopeptidase as competitive inhibitors. Regardless of the fact that our data do not allow prediction of whether or not a specific peptide would be degraded, it seems very likely that the structural features, which rule out the stability of the MHC class I peptides in the cytosol, may have implications in an optimized repertoire selection for antigen presentation.

Neuropeptide specificity and inhibition of recombinant isoforms of the endopeptidase 3.4.24.16 family: comparison with the related recombinant endopeptidase 3.4.24.15.

Endopeptidase EC 3.4.24.16 (EP24.16c, neurolysin) and thimet oligopeptidase EC 3.4.24.15 are close related members of a large family of metalloproteases. Besides their cytosolic and membrane bound form, endopeptidase EC 3.4.24.16 appears to be present in the inner membrane of the mitochondria (EP24.16m). We have overexpressed two porcine EP24.16 isoforms in E. coli and purified the recombinant proteins to homogeneity. We show here that these peptidases hydrolyse a series of neuropeptides with similar rates and at sites reminiscent of those elicited by classically purified human brain EP24.16c. All neuropeptides, except neurotensin, were similarly cleaved by recombinant endopeptidase 3.4.24.15 (EP24.15, thimet oligopeptidase), another zinc-containing metalloenzyme structurally related to EP24.16. These two EP24.16 isoforms were drastically inhibited by Pro-Ile and dithiothreitol and remained unaffected by a specific carboalkyl inhibitor (CFP-AAY-pAb) directed toward the related EP24.15. The present purification procedure of EP24.16 should allow to establish, by mutagenesis analysis, the mechanistic properties of the enzyme.

Major increase in endopeptidase activity of human cathepsin B upon removal of occluding loop contacts.

The main feature distinguishing cathepsin B from other cysteine proteases of the papain family is the presence of a large insertion loop, termed the occluding loop, which occupies the S' subsites of the enzyme. The loop is held in place mainly by two contacts with the rest of the enzyme, involving residues His110 and Arg116 on the loop that form salt bridges with Asp22 and Asp224, respectively. The influence of this loop on the endopeptidase activity of cathepsin B has been investigated using site-directed mutagenesis and internally quenched fluorogenic (IQF) substrates. Wild-type cathepsin B displays poor activity against the substrates Abz-AFRSAAQ-EDDnp and Abz-QVVAGA-EDDnp as compared to cathepsin L and papain. Appreciable increases in kcat/KM were observed for cathepsin B containing the single mutations D22A, H110A, R116A, and D224A. The highest activity however is observed for mutants where both loop to enzyme contacts are disrupted. For the triple-mutant D22A/H110A/R116A, an optimum kcat/KM value of 12 x 10(5) M-1 s-1 was obtained for hydrolysis of Abz-AFRSAAQ-EDDnp, which corresponds to a 600-fold increase relative to wild-type cathepsin B and approaches the level of activity observed with cathepsin L or papain. By comparison, the mutations have little effect on the hydrolysis of Cbz-FR-MCA. The influence of the mutations on the pH dependency of activity also indicates that the complexity of pH activity profiles normally observed for cathepsin B is related to the presence of the occluding loop. The major increase in endopeptidase activity is attributed to an increase in loop "flexibility" and suggests that the occluding loop might move when an endopeptidase substrate binds to the enzyme. The possible contribution of these interactions in regulating endopeptidase activity and the implications for cathepsin B activity in physiological or pathological conditions are discussed.

Design of kallidin-releasing tissue kallikrein inhibitors based on the specificities of the enzyme's binding subsites.

The tissue kallikrein inhibitors reported in the present work were derived by selectively replacing residues in Nalpha-substituted arginine- or phenylalanine-pNA (where pNA is p-nitroanilide), and in peptide substrates for these enzymes. Phenylacetyl-Arg-pNA was found to be an efficient inhibitor of human tissue kallikrein (Ki 0.4 microM) and was neither a substrate nor an inhibitor of plasma kallikrein. The peptide inhibitors having phenylalanine as the P1 residue behaved as specific inhibitors for kallidin-releasing tissue kallikreins, while plasma kallikrein showed high affinity for inhibitors containing (p-nitro)phenylalanine at the same position. The Ki value of the most potent inhibitor developed, Abz-Phe-Arg-Arg-Pro-Arg-EDDnp [where Abz is o-aminobenzoyl and EDDnp is N-(2,4-dinitrophenyl)-ethylenediamine], was 0.08 microM for human tissue kallikrein. Progress curve analyses of the inhibition of human tissue kallikrein by benzoyl-Arg-pNA and phenylacetyl-Phe-Ser-Arg-EDDnp indicated a single-step mechanism for reversible formation of the enzyme-inhibitor complex.

Pharmacological characterization of novel tissue kallikrein inhibitors in vivo.

In this study we have investigated the effect of novel tissue kallikreins on the plasma protein exudation induced by porcine pancreatic kallikrein (PPK) in the rabbit skin in vivo. The tissue kallikrein inhibitors here described were synthesized based on analogues of peptide substrates for tissue kallikreins. The intradermal injection of PPK and rabbit urinary kallikrein, but not of rabbit plasma kallikrein, significantly increased the microvascular permeability leading to local oedema formation in the rabbit skin. At the dose of 3-200 nmol/site, the intradermal co-administration of the tissue kallikrein inhibitors Bz-F-F-S-R-EDDnp (Ki = 0.1 microM; ESP5), PAC-F-S-R-EDDnp (Ki = 0.7 microM; ESP6), Bz-F-F-A-P-R-NH2 (Ki = 7.8 microM; ESP8), PAC-F-F-R-P-R-NH2 (Ki = 0.3 microM; ESP9) and Bz-F-F-S-R-NH2 (Ki = 0.3 microM; ESP11) dose-dependently inhibited the plasma protein exudation induced by PPK. The most potent compound was ESP6 (IC25 = 7.8 nmol/site) followed by ESP5 (IC25 = 14.2 nmol/site), ESP8 (IC25 = 25 nmol/site), ESP9 (IC25 = 30 nmol/site) and ESP11 (IC25 = 50.4 nmol/site). The compounds Bz-F-F-R-P-R-NH2 (Ki = 0.5 microM; ESP1), Bz-F-F-pNa (Ki = 0.4 microM; ESP3), Bz-F(NH2)-F-R-P-R-NH2 (Ki = 1.1 microM; ESP7) and Bz-F-F-S-P-R-NH2 (Ki = 4.6 microM; ESP10) had no significant effect on the PPK-induced plasma protein exudation in doses up to 200 nmol/site. ESP6 also inhibited the PPK-induced plasma protein exudation when administered systemically. This compound may constitute a useful tool to further investigate both the physiological and pathological role of tissue kallikreins.

Determinants of the unusual cleavage specificity of lysyl-bradykinin-releasing kallikreins.

Kinetic data for the hydrolysis by human tissue kallikrein of fluorogenic peptides with o-aminobenzoyl-Phe-Arg (Abz-FR) as the acyl group and different leaving groups demonstrate that interactions with the S'1, S'2 and S'3 subsites are important for cleavage efficiency. In addition, studies on the hydrolysis of fluorogenic peptides with the human kininogen sequence spanning the scissile Met-Lys bond [Abz-M-I-S-L-M-K-R-P-N-(2,4-dinitrophenyl)ethylenediamine] and analogues with different residues at positions P'1, P'2 and P'3 showed that (a) the presence of a proline residue at P'3 and the interactions with the tissue kallikrein-binding sites S2 to S'2 are determinants of Met-Lys bond cleavage and (b) residues P3, P4 and/or P5 arc important for cleavage efficiency. The substitution of phenylalanine for methionine or arginine in substrates with scissile Met-Lys or Arg-Xaa bonds demonstrated that lysyl-bradykinin-releasing tissue kallikreins also have a primary specificity for phenylalanine. The replacement of arginine by phenylalanine in (D)P-F-R-p-nitroanilide (pNA) produced an efficient and specific chromogenic substrate, (D)P-F-F-pNA, for the lysyl-bradykinin-releasing tissue kallikreins as it is resistant to plasma kallikrein and other arginine hydrolases.