A novel trypsin Kazal-type inhibitor from Aedes aegypti with thrombin coagulant inhibitory activity

Kazal-type inhibitors play several important roles in invertebrates, such as anticoagulant, vasodilator and antimicrobial activities. Putative Kazal-type inhibitors were described in several insect transcriptomes. In this paper we characterized for the first time a Kazal unique domain trypsin inhibitor from the Aedes aegypti mosquito. Previously, analyses of sialotranscriptome of A. aegypti showed the potential presence of a Kazal-type serine protease inhibitor, in female salivary glands, carcass and also in whole male, which we named AaTI (A. aegypti trypsin inhibitor). AaTI sequence showed amino acid sequence similarity with insect thrombin inhibitors, serine protease inhibitor from Litopenaeus vannamei hemocytes and tryptase inhibitor from leech Hirudo medicinalis (LDTI). In this work we expressed, purified and characterized the recombinant AaTI (rAaTI). Molecular weight of purified rAaTI was 7 kDa rAaTI presented dissociation constant (K(i)) of 0.15 and 3.8 nM toward trypsin and plasmin, respectively, and it weakly inhibited thrombin amidolytic activity. The rAaTI was also able to prolong prothrombin time, activated partial thromboplastin time and thrombin time. AaTI transcription was confirmed in A. aegypti female salivary gland and gut 3 h and 24 h after blood feeding, suggesting that this molecule can act as anticoagulant during the feeding and digestive processes. Its transcription in larvae and pupae suggested that AaTI may also play other functions during the mosquito`s development. (C) 2010 Elsevier Masson SAS. All rights reserved.

Biochemical and functional properties of a thrombin-like enzyme isolated from Bothrops pauloensis snake venom

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Molecular characterization of BjussuSP-I, a new thrombin-like enzyme with procoagulant and kallikrein-like activity isolated from Bothrops jararacussu snake venom

A thrombin-like enzyme, named BjussuSP-I, isolated from Bothrops jararacussu snake venom, is an acidic single-chain glycoprotein with M-r = 61,000, pI similar to 3.8 and 6% sugar. BjussuSP-I shows high proteolytic activity upon synthetic substrates, such as S-2238 and S-2288. It also shows procoagulant and kallikrein-like activity, but is unable to act on platelets and plasmin. These activities are inhibited by specific inhibitors of this class of enzymes. The complete cDNA sequence of BjussuSP-I with 696 bp encodes open reading frames of 232 amino acid residues, which conserve the common domains of thrombin-like serine proteases. BjussuSP-I shows a high structural homology with other thrombin-like enzymes from snake venoms where common amino acid residues are identified as those corresponding to the catalytic site and subsites S1, S2 and S3 already reported. In this study, we also demonstrated the importance of N-linked glycans, to improve thrombin-like activity of BjussuSP-I toxin. (c) 2007 Elsevier Masson SAS. All rights reserved.

Expression of protease nexin-1 and plasminogen activators during follicular growth and the periovulatory period in cattle

Extracellular matrix remodeling occurs during ovarian follicular development, mediated by plasminogen activators (PAs) and PA inhibitors including protease nexin-1 (PN-1). In the present study we measured expression/activity of the PA system in bovine follicles at different stages of development by timed collection of ovaries during the first follicular wave and during the periovulatory period, and in follicles collected from an abattoir. The abundance of mRNA encoding PN-1, tissue-type PA (tPA), urokinase (uPA) and PA inhibitor-1 (PAI-1) were initially upregulated by human chorionic gonadotropin (hCG) in bovine preovulatory follicular wall homogenates. PN-1, PAI-1 and tPA mRNA expression then decreased near the expected time of ovulation, whereas uPA mRNA levels remained high. PN-1 concentration in follicular fluid (FF) decreased and reached the lowest level at the time of ovulation, whereas plasmin activity in FF increased significantly after hCG. Follicles collected from the abattoir were classified as non-atretic, early-atretic or atretic based on FF estradiol and progesterone content: PN-1 protein levels in FF were significantly higher in non-atretic than in atretic follicles, and plasmin activity was correspondingly higher in the atretic follicles. No changes in PN-1 levels in FF were observed during the growth of pre-deviation follicles early in a follicular wave. These results indicate that PN-1 may be involved in the process of atresia in non-ovulatory dominant follicles and the prevention of precocious proteolysis in periovulatory follicles.

Biochemical and enzymatic characterization of BpMP-I, a fibrinogenolytic metalloproteinase isolated from Bothropoides pauloensis snake venom

Snake Venom Metalloproteinases (SVMPs) are the most abundant components present in Viperidae venom. They are important in the induction of systemic alterations and local tissue damage after envenomation. In the present study, a metalloproteinase named BpMPI was isolated from Bothropoides pauloensis snake venom and its biochemical and enzymatic characteristics were determined. BpMPI was purified in two chromatography steps on ion exchange CM-Sepharose Fast flow and Sephacryl S-300. This protease was homogeneous on SOS-PAGE and showed a single chain polypeptide of 20 kDa under non reducing conditions. The partial amino acid sequence of the enzyme showed high similarity with other SVMPs enzymes from snake venoms. BpMPI showed proteolytic activity upon azocasein and bovine fibrinogen and was inhibited by EDTA, 1,10 phenanthroline and beta-mercaptoethanol. Moreover, this enzyme showed stability at neutral and alkaline pH and it was inactivated at high temperatures. BpMPI was able to hydrolyze glandular and tissue kallikrein substrates, but was unable to act upon factor Xa and plasmin substrates. The enzyme did not induce local hemorrhage in the dorsal region of mice even at high doses. Taken together, our data showed that BpMP-I is in fact a fibrinogenolytic metalloproteinase and a non hemorrhagic enzyme. (C) 2011 Elsevier Inc. All rights reserved.

OmpL1 Is an Extracellular Matrix- and Plasminogen-Interacting Protein of Leptospira spp.

Leptospirosis is a zoonosis with multisystem involvement caused by pathogenic strains of the genus Leptospira. OmpL1 is an outer membrane protein of Leptospira spp. that is expressed during infection. In this work, we investigated novel features of this protein. We describe that OmpL1 is a novel leptospiral extracellular matrix (ECM)-binding protein and a plasminogen (PLG) receptor. The recombinant protein was expressed in Escherichia coli BL21(DE3) Star/pLysS as inclusion bodies, refolded, and purified by metal-chelating chromatography. The protein presented a typical beta-strand secondary structure, as evaluated by circular dichroism spectroscopy. The recombinant protein reacted with antibodies in serum samples from convalescent leptospirosis patients with a high specificity compared to serum samples from individuals with unrelated diseases. These data strengthen the usefulness of OmpL1 as a diagnostic marker of leptospirosis. The characterization of the immunogenicity of recombinant OmpL1 in inoculated BALB/c mice showed that the protein has the capacity to elicit humoral and cellular immune responses, as denoted by high antibody titers and the proliferation of lymphocytes. We demonstrate that OmpL1 has the ability to mediate attachment to laminin and plasma fibronectin, with KD (equilibrium dissociation constant) values of 2,099.93 +/- 871.03 nM and 1,239.23 +/- 506.85 nM, respectively. OmpL1 is also a PLG receptor, with a KD of 368.63 +/- 121.23 nM, capable of generating enzymatically active plasmin. This is the first report that shows and characterizes OmpL1 as an ECM-interacting and a PLG-binding protein of Leptospira spp. that may play a role in bacterial pathogenesis when expressed during infection.

Lsa30, a novel adhesin of Leptospira interrogans binds human plasminogen and the complement regulator C4bp

Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Surface proteins have the potential to promote several activities, including adhesion. This work aimed to study the leptospiral coding sequence (CDS) LIC11087, genome annotated as hypothetical outer membrane protein. The LIC11087 gene was cloned and expressed in Escherichia coil BL21 (DE3) strain by using the expression vector pAE. The recombinant protein tagged with N-terminal 6XHis was purified by metal-charged chromatography and characterized by circular dichroism (CD) spectroscopy. The recombinant protein has the ability to mediate attachment to the extracellular matrix (ECM) components, laminin and plasma fibronectin, and was named Lsa30 (Leptospiral surface adhesin of 30 kDa). Lsa30 binds to laminin and to plasma fibronectin in a dose-dependent and saturable manner, with dissociation equilibrium constants (K-D) of 292 +/- 24 nM and 157 +/- 35 nM, respectively. Moreover, the Lsa30 is a plasminogen (PLC) receptor, capable of generating plasmin, in the presence of activator. This protein may interfere with the complement cascade by interacting with C4bp regulator. The Lsa30 is probably a new surface protein of Leptospira as revealed by immunofluorescence assays with living organisms and the reactivity with antibodies present in serum samples of experimentally infected hamsters. Thus, Lsa30 is a novel versatile protein that may play a role in mediating adhesion and may help pathogenic Leptospira to overcome tissue barriers and to escape the immune system. (C) 2012 Elsevier Ltd. All rights reserved.

Methionine-induced hyperhomocysteinemia reverts fibrinolytic pathway activation in a murine model of acute promyelocytic leukemia

Increased fibrinolysis is an important component of acute promyelocytic leukemia (APL) bleeding diathesis. APL blasts overexpress annexin II (ANXII), a receptor for tissue plasminogen activator (tPA), and plasminogen, thereby increasing plasmin generation. Previous studies suggested that ANXII plays a pivotal role in APL coagulopathy. ANXII binding to tPA can be inhibited by homocysteine and hyperhomocysteinemia can be induced by L-methionine supplementation. In the present study, we used an APL mouse model to study ANXII function and the effects of hyperhomocysteinemia in vivo. Leukemic cells expressed higher ANXII and tPA plasma levels (11.95 ng/mL in leukemic vs 10.74 ng/mL in wild-type; P = .004). In leukemic mice, administration of L-methionine significantly increased homocysteine levels (49.0 mu mol/mL and < 6.0 mu mol/mL in the treated and nontreated groups, respectively) and reduced tPA levels to baseline concentrations. The latter were also decreased after infusion of the LCKLSL peptide, a competitor for the ANXII tPA-binding site (11.07 ng/mL; P = .001). We also expressed and purified the p36 component of ANXII in Pichia methanolica. The infusion of p36 in wild-type mice increased tPA and thrombin-antithrombin levels, and the latter was reversed by L-methionine administration. The results of the present study demonstrate the relevance of ANXII in vivo and suggest that methionine-induced hyperhomocysteinemia may reverse hyperfibrinolysis in APL. (Blood. 2012;120(1):207-213)

Features of two proteins of Leptospira interrogans with potential role in host-pathogen interactions

Background: Leptospirosis is considered a re-emerging infectious disease caused by pathogenic spirochaetes of the genus Leptospira. Pathogenic leptospires have the ability to survive and disseminate to multiple organs after penetrating the host. Leptospires were shown to express surface proteins that interact with the extracellular matrix (ECM) and to plasminogen (PLG). This study examined the interaction of two putative leptospiral proteins with laminin, collagen Type I, collagen Type IV, cellular fibronectin, plasma fibronectin, PLG, factor H and C4bp. Results: We show that two leptospiral proteins encoded by LIC11834 and LIC12253 genes interact with laminin in a dose - dependent and saturable mode, with dissociation equilibrium constants (K-D) of 367.5 and 415.4 nM, respectively. These proteins were named Lsa33 and Lsa25 (Leptospiral surface adhesin) for LIC11834 and LIC12253, respectively. Metaperiodate - treated laminin reduced Lsa25 - laminin interaction, suggesting that sugar moieties of this ligand participate in this interaction. The Lsa33 is also PLG - binding receptor, with a K-D of 23.53 nM, capable of generating plasmin in the presence of an activator. Although in a weak manner, both proteins interact with C4bp, a regulator of complement classical route. In silico analysis together with proteinase K and immunoflorescence data suggest that these proteins might be surface exposed. Moreover, the recombinant proteins partially inhibited leptospiral adherence to immobilized laminin and PLG. Conclusions: We believe that these multifunctional proteins have the potential to participate in the interaction of leptospires to hosts by mediating adhesion and by helping the bacteria to escape the immune system and to overcome tissue barriers. To our knowledge, Lsa33 is the first leptospiral protein described to date with the capability of binding laminin, PLG and C4bp in vitro.

Biochemical characterization and comparative analysis of two distinct serine proteases from Bothrops pirajai snake venom

This study reports the isolation and biochemical characterization of two different serine proteases from Bothrops pirajai snake venom, thus providing a comparative analysis of the enzymes. The isolation process consisted of three consecutive chromatographic steps (Sephacryl S-200, Benzamidine Sepharose and C2/C18), resulting in two serine proteases, named BpirSP27 and BpirSP41 after their molecular masses by mass spectrometry (27,121 and 40,639 Da, respectively). Estimation by SDS-PAGE under denaturing conditions showed that, when deglycosylated with PNGase F, BpirSP27 and BpirSP41 had their molecular masses reduced by approximately 15 and 42%, respectively. Both are acidic enzymes, with pI of approximately 4.7 for BpirSP27 and 3.7 for BpirSP41, and their N-terminal amino acid sequences showed 57% identity to each other, with high similarity to the sequences of other snake venom serine proteases (SVSPs). The enzymes showed different actions on bovine fibrinogen, with BpirSP27 acting preferentially on the B beta chain and BpirSP41 on both A alpha and B beta chains. The two serine proteases were also able to degrade fibrin and blood clots in vitro depending on the doses and incubation periods, with higher results for BpirSP41. Both enzymes coagulated the human plasma in a dose-dependent manner, and BpirSP41 showed a higher coagulant potential, with minimum coagulant dose (MCD) of similar to 3.5 mu g versus 20 mu g for BpirSP27. The enzymes were capable of hydrolyzing different chromogenic substrates, including S-2238 for thrombin-like enzymes, but only BpirSP27 acted on the substrate S-2251 for plasmin. They also showed high stability against variations of temperature and pH, but their activities were significantly reduced after preincubation with Cu2+ ion and specific serine protease inhibitors. In addition. BpirSP27 induced aggregation of washed platelets to a greater extent than BpirSP41. The results showed significant structural and functional differences between B. pirajai serine proteases, providing interesting insights into the structure-function relationship of SVSPs. (C) 2012 Elsevier Masson SAS. All rights reserved.

“Features of two proteins of Leptospira interrogans with potential role in host-pathogen interactions”

Abstract Background Leptospirosis is considered a re-emerging infectious disease caused by pathogenic spirochaetes of the genus Leptospira. Pathogenic leptospires have the ability to survive and disseminate to multiple organs after penetrating the host. Leptospires were shown to express surface proteins that interact with the extracellular matrix (ECM) and to plasminogen (PLG). This study examined the interaction of two putative leptospiral proteins with laminin, collagen Type I, collagen Type IV, cellular fibronectin, plasma fibronectin, PLG, factor H and C4bp. Results We show that two leptospiral proteins encoded by LIC11834 and LIC12253 genes interact with laminin in a dose - dependent and saturable mode, with dissociation equilibrium constants (KD) of 367.5 and 415.4 nM, respectively. These proteins were named Lsa33 and Lsa25 (Leptospiral surface adhesin) for LIC11834 and LIC12253, respectively. Metaperiodate - treated laminin reduced Lsa25 - laminin interaction, suggesting that sugar moieties of this ligand participate in this interaction. The Lsa33 is also PLG - binding receptor, with a KD of 23.53 nM, capable of generating plasmin in the presence of an activator. Although in a weak manner, both proteins interact with C4bp, a regulator of complement classical route. In silico analysis together with proteinase K and immunoflorescence data suggest that these proteins might be surface exposed. Moreover, the recombinant proteins partially inhibited leptospiral adherence to immobilized laminin and PLG. Conclusions We believe that these multifunctional proteins have the potential to participate in the interaction of leptospires to hosts by mediating adhesion and by helping the bacteria to escape the immune system and to overcome tissue barriers. To our knowledge, Lsa33 is the first leptospiral protein described to date with the capability of binding laminin, PLG and C4bp in vitro.

Interaction of leptospira elongation factor tu with plasminogen and complement factor h: a metabolic leptospiral protein with moonlighting activities

The elongation factor Tu (EF-Tu), an abundant bacterial protein involved in protein synthesis, has been shown to display moonlighting activities. Known to perform more than one function at different times or in different places, it is found in several subcellular locations in a single organism, and may serve as a virulence factor in a range of important human pathogens. Here we demonstrate that Leptospira EF-Tu is surface-exposed and performs additional roles as a cell-surface receptor for host plasma proteins. It binds plasminogen in a dose-dependent manner, and lysine residues are critical for this interaction. Bound plasminogen is converted to active plasmin, which, in turn, is able to cleave the natural substrates C3b and fibrinogen. Leptospira EF-Tu also acquires the complement regulator Factor H (FH). FH bound to immobilized EF-Tu displays cofactor activity, mediating C3b degradation by Factor I (FI). In this manner, EF-Tu may contribute to leptospiral tissue invasion and complement inactivation. To our knowledge, this is the first description of a leptospiral protein exhibiting moonlighting activities

Bifidobacterium - Human Host Interaction: Role of Human Plasminogen

Bifidobacterium is an important genus of the human gastrointestinal microbiota, affecting several host physiological features. Despite the numerous Bifidobacterium related health-promoting activities, there is still a dearth of information about the molecular mechanisms at the basis of the interaction between this microorganism and the host. Bacterial surface associated proteins may play an important role in this interaction because of their ability to intervene with host molecules, as recently reported for the host protein plasminogen. Plasminogen is the zymogen of the trypsin-like serine protease plasmin, an enzyme with a broad substrate specificity. Aim of this thesis is to deepen the knowledge about the interaction between Bifidobacterium and the human plasminogen system and its role in the Bifidobacterium-host interaction process. As a bifidobacterial model, B. animalis subsp. lactis BI07 has been used because of its large usage in dairy and pharmaceutical preparations. We started from the molecular characterization of the interaction between plasminogen and one bifidobacterial plasminogen receptor, DnaK, a cell wall protein showing high affinity for plasminogen, and went on with the study of the impact of intestinal environmental factors, such as bile salts and inflammation, on the plasminogen-mediated Bifidobacterium-host interaction. According to our in vitro findings, by enhancing the activation of the bifidobacterial bound plasminogen to plasmin, the host inflammatory response results in the decrease of the bifidobacterial adhesion to the host enterocytes, favouring bacterial migration to the luminal compartment. Conversely, in the absence of inflammation, plasminogen acts as a molecular bridge between host enterocytes and bifidobacteria, enhancing Bifidobacterium adhesion. Furthermore, adaptation to physiological concentrations of bile salts enhances the capability of this microorganism to interact with the host plasminogen system. The host plasminogen system thus represents an important and flexible tool used by bifidobacteria in the cross-talk with the host.

Purificazione di plasminogeno con membrane di affinita'

Alcune patologie dell’occhio come la retinopatia diabetica, il pucker maculare, il distacco della retina possono essere curate con un intervento di vitrectomia. I rischi associati all’intervento potrebbero essere superati ricorrendo alla vitrectomia enzimatica con plasmina in associazione o in sostituzione della vitrectomia convenzionale. Inoltre, l’uso di plasmina autologa eviterebbe problemi di rigetto. La plasmina si ottiene attivando il plasminogeno con enzimi quali l’attivatore tissutale (tPA) e l’urochinasi ( uPA ) . La purificazione del plasminogeno dal sangue avviene normalmente attraverso cromatografia di affinità con resina. Tuttavia, le membrane di affinità costituiscono un supporto ideale per questa applicazione poiché possono essere facilmente impaccate prima dell’intervento, permettendo la realizzazione di un dispositivo monouso che fornisce un processo rapido ed economico. Obiettivo di questo lavoro è la preparazione di membrane di affinità per la purificazione del plasminogeno utilizzando L-lisina come ligando di affinità. Per questo scopo sono state usate membrane in cellulosa rigenerata ad attivazione epossidica, modificate con due diversi protocolli per l’immobilizzazione di L-lisina. La densità ligando è stata misurata mediante un saggio colorimetrico che usa l’acido arancio 7 come indicatore. La resa di immobilizzazione è stata studiata in funzione del tempo di reazione e della concentrazione di L-lisina. Le membrane ottimizzate sono state caratterizzate con esperimenti dinamici usando siero bovino e umano, i risultati sono stati confrontati con quelli ottenuti in esperimenti paralleli condotti con una resina commerciale di affinità con L-lisina. Durante gli esperimenti con siero, le frazioni provenienti da ogni fase cromatografica sono state raccolte e analizzate con HPLC ed elettroforesi SDS-PAGE. In particolare, l’elettroforesi dei campioni eluiti presenta una banda del plasminogeno ben definita indicando che le membrane di affinità con L-lisina sono adatte alla purificazione del plasminogeno. Inoltre, è emerso che le membrane hanno maggiore produttività della resina commerciale di riferimento.

Prozessierung des humanen GARP–Rezeptors und die physiologischen Auswirkungen

GARP (Glycoprotein A Repetitions Predominant) ist ein Oberflächenrezeptor auf regulatorischen T–Zellen (TRegs), der den latenten TGF–β (Transforming Growth Factor–β) bindet. Ein Funktionsverlust von T Regs hat gravierende Autoimmunerkrankungen wie das Immunodysregulation Polyendocrinopathy Enteropathy X–linked Syndrome (IPEX), Multiple Sklerose (MS) oder Rheumatoide Arthritis (RA) zur Folge. GARP stellt über eine Erhöhung der Aktivierbarkeit von TGF–β den regulatorischen Phänotyp von TRegs sicher und inhibiert die Ausbreitung von autoreaktiven TH17 Zellen.rn In dieser Arbeit stand die Regulation von GARP selbst im Mittelpunkt. Es konnte gezeigt werden, dass es sich innerhalb der kiefertragenden Vertebraten um ein strikt konserviertes Protein handelt. Datenbankanalysen machten deutlich, dass es zuerst in basalen Knochenfischen zusammen mit anderen Komponenten der adaptiven Immunantwort auftritt. Ein 3D–Modell, welches über Homologiemodellierung erstellt wurde, gab Aufschluss über die Struktur des Rezeptors und mögliche intramolekulare Disulfidbrücken. Für in vitro Versuche wurde eine lösliche Variante von GARP durch einen Austausch der Transmembrandomäne durch C–terminale Meprin α Domänen konstruiert. Diese Variante wurde in der eukaryotischen Zellkultur zuverlässig in den Überstand sezerniert und konnte chromatografisch gereinigt werden. Mit diesem rekombinanten GARP wurden Prozessierungsversuche mit Autoimmunpathogenese assoziierten Proteasen durchgeführt. Dabei zeigte sich, dass die Serinproteasen Trypsin, Neutrophile Elastase und Plasmin, sowie die Metalloprotease MMP2 in der Lage sind, GARP vollständig zu degradieren. In TGF–β sensitiven Proliferationsuntersuchungen stellte sich heraus, dass die entstandenen Fragmente immer noch in der Lage waren die Aktivierbarkeit von TGF–β zu erhöhen. Neben der Degradierung durch die oben genannten Proteasen konnte ebenfalls beobachtet werden, dass MMP9 und Ovastacin in der Lage sind GARP spezifisch zu schneiden. Ovastacin mRNA wurde in dieser Arbeit das erste Mal außerhalb der Oocyte, in T–Zellen beschrieben. Mit GARP wurde zudem das zweite Proteinsubstrat, neben dem Zona Pellucida Protein 2 identifiziert. Das durch MMP9 erzeugte N–terminale Fragment besitzt zwar die Eigenschaft, an TGF–β zu binden, kann aber die Aktivierbarkeit von TGF–β nicht mehr wie das intakte GARP erleichtern. rnDiese Arbeit zeigte, dass GARP durch Proteolyse reguliert wird, wobei die entstehenden Fragmente unterschiedlichen Einfluss auf die Aktivierbarkeit von TGF–β haben. Dieses Wissen bildet die Grundlage für weitere Untersuchungen im translationalen Forschungsbereich, um die gewonnenen Erkenntnisse zur Immunmodulation in der Therapie verschiedener Krankheiten einsetzen zu können.rn