Trypanosoma cruzi heparin-binding proteins present a flagellar membrane localization and serine proteinase activity

Parasitology ◽  
2012 ◽  
Vol 140 (2) ◽  
pp. 171-180 ◽  
Author(s):  
F. O. R. OLIVEIRA-JR ◽  
C. R. ALVES ◽  
F. S. SILVA ◽  
L. M. C. CÔRTES ◽  
L. TOMA ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Host Cell ◽  
Binding Proteins ◽  
Proteinase Inhibitors ◽  
Serine Proteinase ◽  
Biochemical Properties ◽  
Heparin Binding ◽  
Flagellar Membrane ◽  
The Stability ◽  
Ph Range

SUMMARYHeparin-binding proteins (HBPs) play a key role in Trypanosoma cruzi-host cell interactions. HBPs recognize heparan sulfate (HS) at the host cell surface and are able to induce the cytoadherence and invasion of this parasite. Herein, we analysed the biochemical properties of the HBPs and also evaluated the expression and subcellular localization of HBPs in T. cruzi trypomastigotes. A flow cytometry analysis revealed that HBPs are highly expressed at the surface of trypomastigotes, and their peculiar localization mainly at the flagellar membrane, which is known as an important signalling domain, may enhance their binding to HS and elicit the parasite invasion. The plasmon surface resonance results demonstrated the stability of HBPs and their affinity to HS and heparin. Additionally, gelatinolytic activities of 70 kDa, 65·8 kDa and 59 kDa HBPs over a broad pH range (5·5–8·0) were revealed using a zymography assay. These proteolytic activities were sensitive to serine proteinase inhibitors, such as aprotinin and phenylmethylsulfonyl fluoride, suggesting that HBPs have the properties of trypsin-like proteinases.

Trypanosoma cruzi heparin-binding proteins and the nature of the host cell heparan sulfate-binding domain

2008 ◽  
Vol 44 (4) ◽  
pp. 329-338 ◽  
Author(s):  
Francisco Odencio Rodrigues de Oliveira ◽  
Carlos Roberto Alves ◽  
Cláudia Magalhães Calvet ◽  
Leny Toma ◽  
Rodrigo Ippolito Bouças ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Heparan Sulfate ◽  
Host Cell ◽  
Binding Proteins ◽  
Binding Domain ◽  
Heparin Binding

Trypanosoma cruzi heparin-binding proteins mediate the adherence of epimastigotes to the midgut epithelial cells of Rhodnius prolixus

Parasitology ◽  
2012 ◽  
Vol 139 (6) ◽  
pp. 735-743 ◽  
Author(s):  
F. O. R. OLIVEIRA ◽  
C. R. ALVES ◽  
F. SOUZA-SILVA ◽  
C. M. CALVET ◽  
L. M. C. CÔRTES ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Trypanosoma Cruzi ◽  
Mammalian Cells ◽  
Binding Proteins ◽  
Rhodnius Prolixus ◽  
Insect Vector ◽  
Heparin Binding ◽  
Molecular Masses ◽  
Pre Treatment

SUMMARYHeparin-binding proteins (HBPs) have been demonstrated in both infective forms of Trypanosoma cruzi and are involved in the recognition and invasion of mammalian cells. In this study, we evaluated the potential biological function of these proteins during the parasite-vector interaction. HBPs, with molecular masses of 65·8 kDa and 59 kDa, were isolated from epimastigotes by heparin affinity chromatography and identified by biotin-conjugated sulfated glycosaminoglycans (GAGs). Surface plasmon resonance biosensor analysis demonstrated stable receptor-ligand binding based on the association and dissociation values. Pre-incubation of epimastigotes with GAGs led to an inhibition of parasite binding to immobilized heparin. Competition assays were performed to evaluate the role of the HBP-GAG interaction in the recognition and adhesion of epimastigotes to midgut epithelial cells of Rhodnius prolixus. Epithelial cells pre-incubated with HBPs yielded a 3·8-fold inhibition in the adhesion of epimastigotes. The pre-treatment of epimastigotes with heparin, heparan sulfate and chondroitin sulfate significantly inhibited parasite adhesion to midgut epithelial cells, which was confirmed by scanning electron microscopy. We provide evidence that heparin-binding proteins are found on the surface of T. cruzi epimastigotes and demonstrate their key role in the recognition of sulfated GAGs on the surface of midgut epithelial cells of the insect vector.

scholarly journals Neutralization of heparin activity by neutrophil lactoferrin

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 421-428
Author(s):  
HF Wu ◽  
RL Lundblad ◽  
FC Church
Keyword(s):  
Defense Mechanisms ◽  
Biochemical Characterization ◽  
Proteinase Inhibitors ◽  
Inflammatory Diseases ◽  
Anticoagulant Activity ◽  
Serine Proteinase ◽  
Heparin Binding ◽  
Necrosis Factor Alpha ◽  
Platelet Factor ◽  
Thrombin Inhibition

Lactoferrin is a prominent component of neutrophil secondary granules, and its blood concentration is increased in certain inflammatory diseases. In contrast to the well-described biochemical characterization of lactoferrin as an iron-binding protein, its physiologic role in the regulation of inflammation and other host defense mechanisms is unclear. In this report, we provide evidence that lactoferrin has a potent heparin-neutralizing activity during thrombin inhibition by the serine proteinase inhibitors (serpins) antithrombin and heparin co-factor II. Activated neutrophil supernatant, which contains lactoferrin and other heparin-binding proteins, could neutralize the heparin-dependent antithrombin-thrombin inhibition reaction. The addition of lactoferrin to plasma corrected the heparin- induced prolongation of blood plasma coagulation as measured by the activated partial thromboplastin time (aPTT). Treatment of whole blood with specific inflammatory mediators, fMLP, lipopolysaccharide (LPS), and tumor necrosis factor-alpha (TNF-alpha) increased the concentration of both plasma lactoferrin and platelet factor 4 while inhibiting the blood anticoagulant activity of heparin as measured by the aPTT. These results suggest that the prothrombotic sequelae of some inflammatory processes may be partly due to various agonists that release neutrophil lactoferrin, which can then neutralize glycosaminoglycan-dependent serpin-thrombin inhibition reactions.

Leishmania (Viannia) braziliensis: insights on subcellular distribution and biochemical properties of heparin-binding proteins

Parasitology ◽  
2011 ◽  
Vol 139 (2) ◽  
pp. 200-207 ◽  
Author(s):  
LUZIA MONTEIRO DE CASTRO CÔRTES ◽  
MIRIAN CLAUDIA DE SOUZA PEREIRA ◽  
FRANCISCO ODÊNCIO RODRIGUES DE OLIVEIRA ◽  
SUZANA CORTE-REAL ◽  
FRANKLIN SOUZA DA SILVA ◽  
...  
Keyword(s):  
Subcellular Distribution ◽  
Binding Proteins ◽  
Biochemical Properties ◽  
Public Health Issue ◽  
Major Protein ◽  
Dependent Manner ◽  
Heparin Binding ◽  
Spr Biosensor ◽  
Sds Page ◽  
Vector Borne

SUMMARYLeishmaniasis is a vector-borne disease and an important public health issue. Glycosaminoglycan ligands inLeishmaniaparasites are potential targets for new strategies to control this disease. We report the subcellular distribution of heparin-binding proteins (HBPs) inLeishmania (Viannia) braziliensisand specific biochemical characteristics ofL. (V.) braziliensisHBPs. Promastigotes were fractionated, and flagella and membrane samples were applied to HiTrap Heparin affinity chromatography columns. Heparin-bound fractions from flagella and membrane samples were designated HBP Ffand HBP Mf, respectively. Fraction HBP Ffpresented a higher concentration of HBPs relative to HBP Mf, and SDS-PAGE analyses showed 2 major protein bands in both fractions (65 and 55 kDa). The 65 kDa band showed gelatinolytic activity and was sensitive to inhibition by 1,10-phenanthroline. The localization of HBPs on the promastigote surfaces was confirmed using surface plasmon resonance (SPR) biosensor analysis by binding the parasites to a heparin-coated sensor chip; that was inhibited in a dose-dependent manner by pre-incubating the parasites with variable concentrations of heparin, thus indicating distinct heparin-binding capacities for the two fractions. In conclusion, protein fractions isolated from either the flagella or membranes ofL. (V.) braziliensispromastigotes have characteristics of metallo-proteinases and are able to bind to glycosaminoglycans.

scholarly journals Host Cell Invasion by TRYPANOSOMA cRUZI Is Potentiated by Activation of Bradykinin B2 Receptors

2000 ◽  
Vol 192 (9) ◽  
pp. 1289-1300 ◽  
Author(s):  
Julio Scharfstein ◽  
Veronica Schmitz ◽  
Veronica Morandi ◽  
Marcia M. A. Capella ◽  
Ana Paula C. A. Lima ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Host Cell ◽  
Cho Cells ◽  
Plasma Membranes ◽  
Proteinase Inhibitors ◽  
Umbilical Vein ◽  
Cysteine Proteinase ◽  
Chinese Hamster ◽  
Free Calcium ◽  
Umbilical Vein Endothelial Cells

The parasitic protozoan Trypanosoma cruzi employs multiple molecular strategies to invade a broad range of nonphagocytic cells. Here we demonstrate that the invasion of human primary umbilical vein endothelial cells (HUVECs) or Chinese hamster ovary (CHO) cells overexpressing the B2 type of bradykinin receptor (CHO-B2R) by tissue culture trypomastigotes is subtly modulated by the combined activities of kininogens, kininogenases, and kinin-degrading peptidases. The presence of captopril, an inhibitor of bradykinin degradation by kininase II, drastically potentiated parasitic invasion of HUVECs and CHO-B2R, but not of mock-transfected CHO cells, whereas the B2R antagonist HOE 140 or monoclonal antibody MBK3 to bradykinin blocked these effects. Invasion competence correlated with the parasites' ability to liberate the short-lived kinins from cell-bound kininogen and to elicit vigorous intracellular free calcium ([Ca2+]i) transients through B2R. Invasion was impaired by membrane-permeable cysteine proteinase inhibitors such as Z-(SBz)Cys-Phe-CHN2 but not by the hydrophilic inhibitor 1-trans-epoxysuccinyl-l-leucyl-amido-(4-guanidino) butane or cystatin C, suggesting that kinin release is confined to secluded spaces formed by juxtaposition of host cell and parasite plasma membranes. Analysis of trypomastigote transfectants expressing various cysteine proteinase isoforms showed that invasion competence is linked to the kinin releasing activity of cruzipain, herein proposed as a factor of virulence in Chagas' disease.

scholarly journals Neutralization of heparin activity by neutrophil lactoferrin

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 421-428 ◽  
Author(s):  
HF Wu ◽  
RL Lundblad ◽  
FC Church
Keyword(s):  
Defense Mechanisms ◽  
Biochemical Characterization ◽  
Proteinase Inhibitors ◽  
Inflammatory Diseases ◽  
Anticoagulant Activity ◽  
Serine Proteinase ◽  
Heparin Binding ◽  
Necrosis Factor Alpha ◽  
Platelet Factor ◽  
Thrombin Inhibition

Abstract Lactoferrin is a prominent component of neutrophil secondary granules, and its blood concentration is increased in certain inflammatory diseases. In contrast to the well-described biochemical characterization of lactoferrin as an iron-binding protein, its physiologic role in the regulation of inflammation and other host defense mechanisms is unclear. In this report, we provide evidence that lactoferrin has a potent heparin-neutralizing activity during thrombin inhibition by the serine proteinase inhibitors (serpins) antithrombin and heparin co-factor II. Activated neutrophil supernatant, which contains lactoferrin and other heparin-binding proteins, could neutralize the heparin-dependent antithrombin-thrombin inhibition reaction. The addition of lactoferrin to plasma corrected the heparin- induced prolongation of blood plasma coagulation as measured by the activated partial thromboplastin time (aPTT). Treatment of whole blood with specific inflammatory mediators, fMLP, lipopolysaccharide (LPS), and tumor necrosis factor-alpha (TNF-alpha) increased the concentration of both plasma lactoferrin and platelet factor 4 while inhibiting the blood anticoagulant activity of heparin as measured by the aPTT. These results suggest that the prothrombotic sequelae of some inflammatory processes may be partly due to various agonists that release neutrophil lactoferrin, which can then neutralize glycosaminoglycan-dependent serpin-thrombin inhibition reactions.

scholarly journals Covalent binding of proteinases in their reaction with α2-macroglobulin

1980 ◽  
Vol 187 (3) ◽  
pp. 695-701 ◽  
Author(s):  
G S Salvesen ◽  
A J Barrett
Keyword(s):  
Active Site ◽  
Proteinase Inhibitors ◽  
Covalent Binding ◽  
Sodium Dodecyl ◽  
Serine Proteinase ◽  
Covalent Bond ◽  
Leucocyte Elastase ◽  
Α2 Macroglobulin ◽  
Covalent Bond Formation ◽  
The Stability

Although it is known that most of the plasma proteinase inhibitors form complexes with proteinases that are not dissociated by SDS (sodium dodecyl sulphate), there has been disagreement as to whether this is true for alpha 2M (alpha 2-macroglobulin). We have examined the stability to SDS with reduction of complexes between alpha 2M and several 125I-labelled proteinases (trypsin, plasmin, leucocyte elastase, pancreatic elastase and papain) by gel electrophoresis. For each enzyme, some molecules were separated from the denatured alpha 2M chains, but amounts ranging from 8.3% (papain) to 61.2% (trypsin) were bound with a stability indicative of a covalent link. Proteolytic activity was essential for the covalent binding to occur, and the proteinase molecules became attached to the larger of the two proteolytic derivatives (apparent mol.wt. 111 000) of the alpha 2M subunit. We take this to mean that cleavage of the proteinase-susceptible site sometimes leads to covalent-bond formation between alpha 2M and proteinase. Whatever the nature of this bond, it does not involve the active site of the proteinase, as bound serine-proteinase molecules retain the ability to react with the active-site-directed reagent [3H]Dip-F (di-isopropyl phosphorofluoridate). Our conclusion is that the ability to form covalent links is not essential for the inhibitory capacity of alpha 2M. It may, however, help to stabilize the complexes against dissociation or proteolysis.

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