scholarly journals The TvLEGU-1, a Legumain-Like Cysteine Proteinase, Plays a Key Role inTrichomonas vaginalisCytoadherence

2013 ◽  
Vol 2013 ◽  
pp. 1-18 ◽  
Author(s):  
Francisco Javier Rendón-Gandarilla ◽  
Lucero de los Angeles Ramón-Luing ◽  
Jaime Ortega-López ◽  
Ivone Rosa de Andrade ◽  
Marlene Benchimol ◽  
...  
Keyword(s):  
Proteolytic Activity ◽  
Virulence Factor ◽  
Trichomonas Vaginalis ◽  
Potential Role ◽  
Polyclonal Antibodies ◽  
Cysteine Proteinase ◽  
Two Dimensional ◽  
Cell Binding ◽  
Vaginal Secretions ◽  
Michael Acceptor

The goal of this paper was to characterize aTrichomonas vaginaliscysteine proteinase (CP) legumain-1 (TvLEGU-1) and determine its potential role as a virulence factor duringT. vaginalisinfection. A 30-kDa band, which migrates in three protein spots (pI~6.3, ~6.5, and ~6.7) with a different type and level of phosphorylation, was identified as TvLEGU-1 by one- and two-dimensional Western blot (WB) assays, using a protease-rich trichomonad extract and polyclonal antibodies produced against the recombinant TvLEGU-1 (anti-TvLEGU-1r). Its identification was confirmed by mass spectrometry. Immunofluorescence, cell binding, and WB assays showed that TvLEGU-1 is upregulated by iron at the protein level, localized on the trichomonad surface and in lysosomes and Golgi complex, bound to the surface of HeLa cells, and was found in vaginal secretions. Additionally, the IgG and Fab fractions of the anti-TvLEGU-1r antibody inhibited trichomonal cytoadherence up to 45%. Moreover, the Aza-Peptidyl Michael Acceptor that inhibited legumain proteolytic activity in live parasites also reduced levels of trichomonal cytoadherence up to 80%. In conclusion, our data show that the proteolytic activity of TvLEGU-1 is necessary for trichomonal adherence. Thus, TvLEGU-1 is a novel virulence factor upregulated by iron. This is the first report that a legumain-like CP plays a role in a pathogen cytoadherence.

Trichomonas vaginalis: characterization of a 39-kDa cysteine proteinase found in patient vaginal secretions

2004 ◽  
Vol 107 (3-4) ◽  
pp. 125-135 ◽  
Author(s):  
Rodolfo Hernández-Gutiérrez ◽  
Leticia Avila-González ◽  
Jaime Ortega-López ◽  
Fernando Cruz-Talonia ◽  
Guillermo Gómez-Gutierrez ◽  
...  
Keyword(s):  
Trichomonas Vaginalis ◽  
Cysteine Proteinase ◽  
Vaginal Secretions

Location of the cell-binding domain of CP65, a 65kDa cysteine proteinase involved in Trichomonas vaginalis cytotoxicity

2006 ◽  
Vol 38 (12) ◽  
pp. 2114-2127 ◽  
Author(s):  
Eduardo Solano-González ◽  
María Elizbeth Alvarez-Sánchez ◽  
Leticia Avila-González ◽  
Victor Hugo Rodríguez-Vargas ◽  
Rossana Arroyo ◽  
...  
Keyword(s):  
Trichomonas Vaginalis ◽  
Cysteine Proteinase ◽  
Binding Domain ◽  
Cell Binding ◽  
Cell Binding Domain

scholarly journals CP30, a Cysteine Proteinase Involved inTrichomonas vaginalis Cytoadherence

2000 ◽  
Vol 68 (9) ◽  
pp. 4907-4912 ◽  
Author(s):  
M. Remedios Mendoza-López ◽  
Cecilia Becerril-Garcia ◽  
Loriz V. Fattel-Facenda ◽  
Leticia Avila-Gonzalez ◽  
Martha E. Ruíz-Tachiquín ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Hela Cell ◽  
Trichomonas Vaginalis ◽  
Cysteine Proteinase ◽  
Collagen Iv ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Cell Monolayers ◽  
Thiol Proteinase

ABSTRACT We describe here the participation of a Trichomonas vaginalis 30-kDa proteinase (CP30) with affinity to the HeLa cell surface in attachment of this parasite to host epithelial cells. The CP30 band is a cysteine proteinase because its activity was inhibited by E-64, a thiol proteinase inhibitor. In two-dimensional substrate gel electrophoresis of total extracts of the trichomonad isolate CNCD 147, three spots with proteolytic activity were detected in the 30-kDa region, in the pI range from 4.5 to 5.5. Two of the spots (pI 4.5 and 5.0) bound to the surfaces of fixed HeLa cells corresponding to the CP30 band. The immunoglobulin G fraction of the rabbit anti-CP30 antiserum that recognized a 30-kDa band by Western blotting and immunoprecipitated CP30 specifically inhibited trichomonal cytoadherence to HeLa cell monolayers in a concentration-dependent manner and reacted with CP30 at the parasite surface. CP30 degraded proteins found on the female urogenital tract, including fibronectin, collagen IV, and hemoglobin. Interestingly, CP30 digested fibronectin and collagen IV only at pH levels between 4.5 and 5.0. Moreover, trichomonosis patients whose diagnosis was confirmed by in vitro culture possessed antibody to CP30 in both sera and vaginal washes, and CP30 activity was found in vaginal washes. Our results suggest that surface CP30 is a cysteine proteinase necessary for trichomonal adherence to human epithelial cells.

Vulvovaginitis in Premenarcheal Girls: Clinical Features and Diagnostic Evaluation

PEDIATRICS ◽  
1982 ◽  
Vol 70 (2) ◽  
pp. 193-198 ◽  
Author(s):  
Jan E. Paradise ◽  
Joseph M. Campos ◽  
Harvey M. Friedman ◽  
Gertrude Frishmuth
Keyword(s):  
Chlamydia Trachomatis ◽  
Trichomonas Vaginalis ◽  
Vaginal Discharge ◽  
Matched Control ◽  
Convincing Evidence ◽  
Control Group ◽  
Common Cause ◽  
Matched Control Group ◽  
Vaginal Secretions ◽  
Prepubertal Girls

Fifty-four premenarcheal patients (median age 5.8 years) with symptoms or signs of vulvovaginitis were studied, and the results of cultures of vaginal secretions were compared with those from an age-matched control group. Vaginal discharge was found on examination in 24 of 42 patients with a complaint of discharge, and in two of 12 patients without a complaint of discharge. Convincing evidence of bacterial or monilial infection was found in 14 of the 26 patients with discharge on examination, but in none of the 28 patients without discharge (P < .001). In the latter group pinworm infestation was present in one patient. Moniliasis occurred exclusively in girls who were pubertal (P < .001). Four patients were found to have gonorrhea. No patient appeared to have symptoms or signs caused by Bacteroides sp, Chlamydia trachomatis, viruses, or Trichomonas vaginalis. Noninfectious causes were identified in four patients with and 13 without discharge (P < .025); the most common cause was poor hygiene, implicated in six patients. Bubble bath use was implicated in only one patient. In 22 patients, no specific cause could be identified. All patients with poor hygiene as the only cause, and most with no demonstrable etiology, recovered after being advised to institute improved perineal hygiene. Patients with vaginal discharge are likely to have specific infections, and therefore cultures should be taken, in particular for Neisseria gonorrhoeae. Genital pruritus in prepubertal girls has little or no etiologic specificity, but in pubertal girls with vaginal discharge it suggests the presence of monilial vaginitis.

Glucose-restriction increasesTrichomonas vaginaliscellular damage towards HeLa cells and proteolytic activity of cysteine proteinases (CPs), such as TvCP2

Parasitology ◽  
2019 ◽  
Vol 146 (9) ◽  
pp. 1156-1166 ◽  
Author(s):  
Jesús F. T. Miranda-Ozuna ◽  
Luis Alberto Rivera-Rivas ◽  
Rosa Elena Cárdenas-Guerra ◽  
Mar Sarai Hernández-García ◽  
Sarahí Rodríguez-Cruz ◽  
...  
Keyword(s):  
Proteolytic Activity ◽  
Hela Cells ◽  
Trichomonas Vaginalis ◽  
Host Cells ◽  
Cellular Damage ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Cell Monolayers ◽  
Glucose Restriction ◽  
Effect Of Glucose

AbstractTrichomonas vaginalisinduces cellular damage to the host cells (cytotoxicity) through the proteolytic activity of multiple proteinases of the cysteine type (CPs). Some CPs are modulated by environmental factors such as iron, zinc, polyamines, etc. Thus, the goal of this study was to assess the effect of glucose onT. vaginaliscytotoxicity, proteolytic activity and the particular role of TvCP2 (TVAG_057000) during cellular damage. Cytotoxicity assays showed that glucose-restriction (GR) promotes the highest HeLa cell monolayers destruction (~95%) by trichomonads compared to those grown under high glucose (~44%) condition. Zymography and Western blot using different primary antibodies showed that GR increased the proteolytic activity, amount and secretion of certain CPs, including TvCP2. We further characterized the effect of glucose on TvCP2. TvCP2 increases in GR, localized in vesicles close to the plasma membrane and on the surface ofT. vaginalis. Furthermore, pretreatment of GR-trichomonads with an anti-TvCP2r polyclonal antibody specifically reduced the levels of cytotoxicity and apoptosis induction to HeLa cells in a concentration-dependent manner. In conclusion, our data show that GR, as a nutritional stress condition, promotes trichomonal cytotoxicity to the host cells, increases trichomonad proteolytic activity and amount of CPs, such as TvCP2 involved in cellular damage.

scholarly journals Glycosylation of procathepsin L does not account for species molecular-mass differences and is not required for proteolytic activity

1989 ◽  
Vol 262 (3) ◽  
pp. 931-938 ◽  
Author(s):  
S M Smith ◽  
S E Kane ◽  
S Gal ◽  
R W Mason ◽  
M M Gottesman
Keyword(s):  
Molecular Mass ◽  
Proteolytic Activity ◽  
Cysteine Proteinase ◽  
Cathepsin L ◽  
Enzymic Activity ◽  
A431 Cells ◽  
Molecular Masses ◽  
Procathepsin L ◽  
Endoglycosidase H

Cathepsin L is a major lysosomal cysteine proteinase in mouse and human cells. Despite similar predicted molecular masses, procathepsin L in these two species migrates on SDS/polyacrylamide gels with apparent molecular masses of 39 kDa and 42 kDa respectively. To determine if glycosylation differences account for this discrepancy, and to ascertain whether glycosylation is essential for enzymic activity, mouse and human procathepsins L were expressed at high concentrations in mouse NIH 3T3 cells or in human A431 cells after DNA-mediated transfection of cloned DNAs for these enzymes. In pulse-chase studies, human procathepsin L transfectants synthesized and secreted large amounts of enzymically active 42 kDa proenzyme and processed it into 34 kDa and 26 kDa intracellular peptides, a pattern of secretion and processing similar to that seen with endogenous or transfected mouse procathepsin L. Both translation of cloned procathepsin L cDNAs in vitro and Endoglycosidase H treatment of 39 kDa mouse and 42 kDa human procathepsin L resulted in non-glycosylated proteins 2 kDa lower in molecular mass than the untreated proteins for both species. This suggests that glycosylation differences are not responsible for the molecular-mass disparity between the two species. Moreover, Endoglycosidase H-treated mouse enzyme retained full proteolytic activity, indicating that glycosylation of cathepsin L is not essential for enzymic function.

Isolation and ultrastructural localization of a soluble protein from Ophiostoma ulmi

1990 ◽  
Vol 68 (11) ◽  
pp. 2517-2524 ◽  
Author(s):  
R. S. Jeng ◽  
A. M. Svircev
Keyword(s):  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein A ◽  
Leading Edge ◽  
Ultrastructural Localization ◽  
Immunogold Labelling ◽  
Two Dimensional ◽  
Thin Sections ◽  
Ophiostoma Ulmi ◽  
Gold Label

Two-dimensional polyacrylamide gel electrophoresis was used to identify and isolate a soluble polypeptide, the QP1 protein, which is characteristic of the vegetative hyphae of nonaggressive isolate Q412 of Ophiostoma ulmi. Individual QP1 spots were excised from 16 two-dimensional gels. Polypeptides were eluted from the gel spots by electroelution and lyophilized. The protein was injected into rabbits for the production of polyclonal antibodies. Antiserum specificity was tested by transferring polypeptides from a two-dimensional gel onto nitrocellulose and treating with QP1 serum. The resulting immunoblot contained a single spot that corresponded in shape and location to that of the QP1 polypeptide. Thin sections of fungal mycelia, from nonaggressive isolate Q412 and the aggressive isolate VA of O. ulmi, were treated with QP1 antibodies and protein A – gold. The gold label was localized in thin sections over conidial and hyphal cell walls of the nonaggressive isolate. The aggressive isolate was nonreactive. Mycelia from nonaggressive isolates Q412 and Q311 and aggressive isolates VA and CESS16K of O. ulmi were grown on solid medium, treated with QP1 antibodies, labelled with protein A – gold, and prepared for scanning electron microscopy. The gold-labelled QP1 polypeptide was detected on the leading edge of a small number of hyphae from nonaggressive isolates Q412 and Q311. Key words: immunogold labelling, Ophiostoma ulmi, soluble proteins.

PLATELETS OF A PATIENT LACKING GLYCOPROTEINS lib AND Ilia AGGREGATE TO HIGH CONCENTRATIONS OF THROMBIN OR COLLAGEN

1987 ◽  
Author(s):  
J L McGregor ◽  
L McGregor ◽  
M Hans ◽  
A Sayegh ◽  
M C Trzeeiak ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Polyclonal Antibodies ◽  
Two Dimensional ◽  
Binding Studies ◽  
Western Blots ◽  
Glycoprotein Complex ◽  
Sds Page ◽  
Bleeding Episodes ◽  
High Concentrations ◽  
Induced Aggregation

The aim of this study was to investigate the platelets of a patient having bleeding episodes that began in infancy. The patient’s platelets in citrated-PRP did not aggregate when stimulated with ADP (5 and 10 uM), collagen (2.5 ug/ml), or sodium arachidonate (1 uM). However, washed patient platelets, in the presence of 2mM calcium, aggregated and secreted when stimulated with high concentrations of thrombin (0.36, 0.72 and lU/ml) or collagen (2, 4, 10 ug/ml). Monoclonal antibodies (Mab) LYP18 (directed against the IIb-IIIa glycoprotein complex) and LYP8 (anti-thrombospondin) inhibited thrombin and collagen induced aggregation of control but not the patient platelets. Patient thrombin -stimulated platelets did not bind 125I-labelled fibrinogen (40 to 320 ug/ml). Moreover, stimulating the washed patient's platelets with ADP (10-100 uM), in the presence of fibrinogen (2mg/ml), did not result in aggregation. Binding studies using Mab 125I-LYP2 (directed against the IIb-IIIa glycoprotein complex) showed the absence of the complex on the patient's platelets. The absence of the IIb-IIIa complex on the patient's platelets was also observed using crossed immunoelectro -phoresis and Mab 125I-LYP2 or 125I-LYP18. Individual glycoproteins (lib or Ilia) were not detected on silver stained two-dimensional (non-reduced/reduced) SDS-PAGE. Moreover, Western blots of |he patients platelets used in combination with anti-PLA or anti-LEK polyclonal antibodies failed to detect the presence of these two glycoproteins. These results indicate that this patient has Glanzmann's thrombasthenia or a variant of this disease. Moreover, this study shows that platelets lacking the IIb-IIIa glycoprotein complex can aggregate in responseto collagen or thrombin in the presence of physiological concentrations of calcium.

Sign in / Sign up

Export Citation Format

Share Document