scholarly journals Characterization of the Cell Adhesion Site ofTrypanosoma cruzi Metacyclic Stage Surface Glycoprotein gp82

2000 ◽  
Vol 68 (2) ◽  
pp. 478-484 ◽  
Author(s):  
Patricio M. Manque ◽  
Daniel Eichinger ◽  
Maria A. Juliano ◽  
Luiz Juliano ◽  
Jorge E. Araya ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Adhesion ◽  
Recombinant Proteins ◽  
Synthetic Peptides ◽  
Surface Glycoprotein ◽  
Target Cells ◽  
Cell Binding ◽  
Parasite Invasion ◽  
Central Domain ◽  
Adhesion Site

ABSTRACT The surface glycoprotein gp82, expressed in the insect-stage metacyclic trypomastigotes of Trypanosoma cruzi, has been implicated in mammalian cell invasion. Here we have characterized the cell adhesion site of gp82 by using recombinant proteins and synthetic peptides based on gp82. The recombinant protein Del-4/8, lacking 65 amino acids of gp82 central domain (at positions 257 to 321), was virtually devoid of cell-binding activity and lacked the ability to inhibit parasite invasion, in contrast to J18, the construct containing the full-length gp82 sequence (amino acids 1 to 516). Constructs with shorter deletions, i.e., Del-4 (deleted from 257 to 271) and Del-8 (deleted from 293 to 321), bound to target cells to a significantly lesser degree than did J18. The sites deleted in recombinant proteins Del-4 and Del-8 contained acidic amino acids critical for cell adhesion. Thus, the cell-binding capacity of protein Del-E/D, lacking the glutamic acid (259/260) and aspartic acid (303/304) pairs, was negligible, as was its capacity to inhibit parasite internalization. Of a set of synthetic peptides spanning the gp82 central domain, a 22-mer hybrid peptide, p4/8, formed by two noncontiguous sequences (at positions 257 to 273 and 302 to 306) and containing the four acidic residues, competed with the binding of J18 protein to target cells and significantly inhibited (∼60%) the penetration of parasites. This peptide, generated by the juxtaposition of sequences that are separated by a hydrophobic stretch in the linear molecule, appears to be mimicking a conformation-dependent cell-binding site of gp82. Experiments of antibody competition with a set of 20-mer overlapping peptides mapped the epitope for 3F6, a monoclonal antibody directed to gp82 that inhibits parasite invasion, to the sequence represented by peptide p3 (244 to 263), which has a partial overlap with the cell adhesion site.

scholarly journals Inhibition of platelet adhesion to fibronectin, fibrinogen, and von Willebrand factor substrates by a synthetic tetrapeptide derived from the cell-binding domain of fibronectin

Blood ◽  
1985 ◽  
Vol 66 (4) ◽  
pp. 946-952 ◽  
Author(s):  
DM Haverstick ◽  
JF Cowan ◽  
KM Yamada ◽  
SA Santoro
Keyword(s):  
Amino Acids ◽  
Von Willebrand Factor ◽  
Synthetic Peptides ◽  
Dependent Manner ◽  
Cell Binding ◽  
Binding Region ◽  
Concentration Dependent Manner ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Adhesion Of Platelets

The role in platelet function of the cell-binding region of fibronectin was explored by the use of synthetic peptides. The prototypical peptide gly-arg-gly-asp-ser was capable of inhibiting thrombin-induced platelet aggregation without altering the degree of platelet activation as judged by the secretion of 14C-serotonin. The peptide also effectively inhibited, in a concentration-dependent manner, the binding of radiolabeled fibronectin to platelets and the adhesion of platelets to fibronectin substrates. The smallest peptide from the cell-binding region of fibronectin which retained full activity was arg-gly-asp-ser. Transposition of amino acids or conservative substitutions of amino acids within this short sequence resulted in inactive peptides. Peptides containing the arg-gly-asp-ser sequence were also capable of inhibiting the adhesion of platelets to fibrinogen and von Willebrand factor substrates. Examination of the entire panel of synthetic peptides for ability to inhibit adhesion to fibrinogen or von Willebrand factor substrates revealed the same structure-function relationships that had been determined in the studies with fibronectin.

scholarly journals Inhibition of platelet adhesion to fibronectin, fibrinogen, and von Willebrand factor substrates by a synthetic tetrapeptide derived from the cell-binding domain of fibronectin

Blood ◽  
1985 ◽  
Vol 66 (4) ◽  
pp. 946-952 ◽  
Author(s):  
DM Haverstick ◽  
JF Cowan ◽  
KM Yamada ◽  
SA Santoro
Keyword(s):  
Amino Acids ◽  
Von Willebrand Factor ◽  
Synthetic Peptides ◽  
Dependent Manner ◽  
Cell Binding ◽  
Binding Region ◽  
Concentration Dependent Manner ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Adhesion Of Platelets

Abstract The role in platelet function of the cell-binding region of fibronectin was explored by the use of synthetic peptides. The prototypical peptide gly-arg-gly-asp-ser was capable of inhibiting thrombin-induced platelet aggregation without altering the degree of platelet activation as judged by the secretion of 14C-serotonin. The peptide also effectively inhibited, in a concentration-dependent manner, the binding of radiolabeled fibronectin to platelets and the adhesion of platelets to fibronectin substrates. The smallest peptide from the cell-binding region of fibronectin which retained full activity was arg-gly-asp-ser. Transposition of amino acids or conservative substitutions of amino acids within this short sequence resulted in inactive peptides. Peptides containing the arg-gly-asp-ser sequence were also capable of inhibiting the adhesion of platelets to fibrinogen and von Willebrand factor substrates. Examination of the entire panel of synthetic peptides for ability to inhibit adhesion to fibrinogen or von Willebrand factor substrates revealed the same structure-function relationships that had been determined in the studies with fibronectin.

scholarly journals Central domain of a potyvirus VPg is involved in the interaction with the host translation initiation factor eIF4E and the viral protein HcPro

2007 ◽  
Vol 88 (3) ◽  
pp. 1029-1033 ◽  
Author(s):  
G. Roudet-Tavert ◽  
T. Michon ◽  
J. Walter ◽  
T. Delaunay ◽  
E. Redondo ◽  
...  
Keyword(s):  
Amino Acids ◽  
Recombinant Proteins ◽  
Translation Initiation ◽  
Viral Protein ◽  
Binding Protein ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Biological Functions ◽  
Central Domain

Using recombinant proteins produced in bacteria or in infected plants, interactions between the VPg and HcPro of Lettuce mosaic potyvirus (LMV) and between LMV VPg and the lettuce translation initiation factor 4E, the cap-binding protein (eIF4E), were demonstrated in vitro. Interaction with eIF4E and HcPro both involved the same VPg central domain. The structure of this domain in the VPg context was predicted to include an amphiphilic α-helix, with the amino acids related to biological functions in various potyviruses exposed at the hydrophilic side.

Antimyotoxic Activity of Synthetic Peptides Derived from Bothrops atrox Snake Gamma Phospholipase A2 Inhibitor Selected by Virtual Screening

2019 ◽  
Vol 19 (22) ◽  
pp. 1952-1961 ◽  
Author(s):  
J.C. Sobrinho ◽  
A.F. Francisco ◽  
R. Simões-Silva ◽  
A.M. Kayano ◽  
J.J. Alfonso Ruiz Diaz ◽  
...  
Keyword(s):  
Amino Acids ◽  
Molecular Docking ◽  
Synthetic Peptides ◽  
Cell Damage ◽  
Neutralization Assay ◽  
Phospholipase Activity ◽  
Phospholipases A2 ◽  
Catalytically Active ◽  
Bothrops Atrox

Background: Several studies have aimed to identify molecules that inhibit the toxic actions of snake venom phospholipases A2 (PLA2s). Studies carried out with PLA2 inhibitors (PLIs) have been shown to be efficient in this assignment. Objective: This work aimed to analyze the interaction of peptides derived from Bothrops atrox PLIγ (atPLIγ) with a PLA2 and to evaluate the ability of these peptides to reduce phospholipase and myotoxic activities. Methods: Peptides were subjected to molecular docking with a homologous Lys49 PLA2 from B. atrox venom modeled by homology. Phospholipase activity neutralization assay was performed with BthTX-II and different ratios of the peptides. A catalytically active and an inactive PLA2 were purified from the B. atrox venom and used together in the in vitro myotoxic activity neutralization experiments with the peptides. Results: The peptides interacted with amino acids near the PLA2 hydrophobic channel and the loop that would be bound to calcium in Asp49 PLA2. They were able to reduce phospholipase activity and peptides DFCHNV and ATHEE reached the highest reduction levels, being these two peptides the best that also interacted in the in silico experiments. The peptides reduced the myotubes cell damage with a highlight for the DFCHNV peptide, which reduced by about 65%. It has been suggested that myotoxic activity reduction is related to the sites occupied in the PLA2 structure, which could corroborate the results observed in molecular docking. Conclusion: This study should contribute to the investigation of the potential of PLIs to inhibit the toxic effects of PLA2s.

scholarly journals A molecularly defined skin test reagent for the diagnosis of bovine tuberculosis compatible with vaccination against Johne’s Disease

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sonya Middleton ◽  
Sabine Steinbach ◽  
Michael Coad ◽  
Kevina McGill ◽  
Colm Brady ◽  
...  
Keyword(s):  
Skin Test ◽  
Recombinant Proteins ◽  
Bovine Tuberculosis ◽  
Synthetic Peptides ◽  
Blood Test ◽  
High Specificity ◽  
Active Components ◽  
Interferon Gamma Release Assays ◽  
Skin Responses

AbstractTuberculin Purified Protein Derivatives (PPDs) exhibit multiple limitations: they are crude extracts from mycobacterial cultures with largely unknown active components; their production depends on culture of mycobacteria requiring expensive BCL3 production facilities; and their potency depends on the technically demanding guinea pig assay. To overcome these limitations, we developed a molecularly defined tuberculin (MDT) by adding further antigens to our prototype reagent composed of ESAT-6, CFP-10 and Rv3615c (DIVA skin test, DST). In vitro screening using PBMC from infected and uninfected cattle shortlisted four antigens from a literature-based list of 18 to formulate the MDT. These four antigens plus the previously identified Rv3020c protein, produced as recombinant proteins or overlapping synthetic peptides, were formulated together with the three DST antigens into the MDT to test cattle experimentally and naturally infected with M. bovis, uninfected cattle and MAP vaccinated calves. We demonstrated significant increases in MDT-induced skin responses compared to DST in infected animals, whilst maintaining high specificity in unvaccinated or MAP vaccinated calves. Further, MDT can also be applied in in vitro blood-based interferon-gamma release assays. Thus, MDT promises to be a robust diagnostic skin and blood test reagent overcoming some of the limitations of PPDs and warrants full validation.

scholarly journals Interaction of wild-type signal sequences and their charged variants with model and natural membranes

1993 ◽  
Vol 293 (1) ◽  
pp. 43-49 ◽  
Author(s):  
N M Rao ◽  
R Nagaraj
Keyword(s):  
Amino Acids ◽  
Synthetic Peptides ◽  
Model Membranes ◽  
Signal Peptides ◽  
Wild Type ◽  
Signal Sequences ◽  
Hydrophobic Region ◽  
Phospholipases A2 ◽  
Charged Amino Acids

The interaction of synthetic peptides corresponding to wild-type signal sequences, and their mutants having charged amino acids in the hydrophobic region, with model and natural membranes has been studied. At high peptide concentrations, i.e. low lipid/peptide ratios, the signal peptides cause release of carboxyfluorescein (CF) from model membranes with lipid compositions corresponding to those of translocation-competent as well as translocation-incompetent membranes. Interestingly, mutant sequences, which were non-functional in vivo, caused considerable release of CF compared with the wild-type sequences. Both wild-type and mutant signal sequences perturb model membranes even at lipid/peptide ratios of 1000:1, as indicated by the activities of phospholipases A2, C and D. These studies indicate that such mutant signals are non-functional not because of their inability to interact with membranes, but due to defective targeting to the membrane. The signal peptides inhibit phospholipase C activity in microsomes, uncouple oxidative phosphorylation in mitochondria and increase K+ efflux from erythrocytes, and one of the mutant sequences is a potent degranulator of the mast cells. Both wild-type and mutant signal sequences have the ability to perturb vesicles of various lipid compositions. With respect to natural membranes, the peptides do not show any bias towards translocation-competent membranes.

scholarly journals Mapping of a cell-binding domain in the cell adhesion molecule gp80 of Dictyostelium discoideum.

1988 ◽  
Vol 107 (5) ◽  
pp. 1835-1843 ◽  
Author(s):  
R K Kamboj ◽  
L M Wong ◽  
T Y Lam ◽  
C H Siu
Keyword(s):  
Cell Adhesion ◽  
Dictyostelium Discoideum ◽  
Fusion Proteins ◽  
Binding Activity ◽  
Binding Domain ◽  
Globular Domain ◽  
Cell Binding ◽  
Homophilic Interaction ◽  
A Cell ◽  
Cell Binding Domain

At the aggregation stage of Dictyostelium discoideum development, a cell surface glycoprotein of Mr 80,000 (gp80) has been found to mediate the EDTA-resistant type of cell-cell adhesion via homophilic interaction (Siu, C.-H., A. Cho, and A. H. C. Choi. 1987. J. Cell Biol. 105:2523-2533). To investigate the structure-function relationships of gp80, we have isolated full length cDNA clones for gp80 and determined the DNA sequence. The deduced structure of gp80 showed three major domains. An amino-terminal globular domain composed of the bulk of the protein is supported by a short stalk region, which is followed by a membrane anchor at the carboxy terminus. Structural analysis suggested that the cell-binding domain of gp80 resides within the globular domain near the amino terminus. To investigate the relationship of the cell-binding activity to this region of the polypeptide, three protein A/gp80 (PA80) gene fusions were constructed using the expression vector pRIT2T. These PA80 fusion proteins were assayed for their ability to bind to aggregation stage cells. Binding of 125I-labeled fusion proteins PA80I (containing the Val123 to Ile514 fragment of gp80) and PA80II (Val123 to Ala258) was dosage dependent and could be inhibited by precoating cells with the cell cohesion-blocking mAb 80L5C4. On the other hand, there was no appreciable binding of PA80III (Ile174 to Ile514) to cells. Reassociation of cells was significantly inhibited in the presence of PA80I or PA80II. In addition, 125I-labeled PA80II exhibited homophilic interaction with immobilized PA80I, PA80II, or gp80. The results of these studies lead to the mapping of a cell-binding domain in the region between Val123 and Leu173 of gp80 and provide direct evidence that the cell-binding activity of gp80 resides in the protein moiety.

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