Trichomonas vaginalis cathepsin D-like aspartic proteinase (Tv-CatD) is positively regulated by glucose and degrades human hemoglobin
| Autor: | Maria Inocente Mancilla-Olea, Arturo González-Robles, Jesús F. T. Miranda-Ozuna, Jaime Ortega-López, Lizbeth Sánchez-Ayala, Mar Sarai Hernández-García, Leticia Avila-González, Rossana Arroyo, Elisa E. Figueroa-Angulo, Rosa Elena Cárdenas-Guerra |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Proteases medicine.medical_treatment Protozoan Proteins Cathepsin D Biochemistry law.invention Substrate Specificity 03 medical and health sciences Hemoglobins Western blot law Zymogen medicine Trichomonas vaginalis Aspartic Acid Endopeptidases Humans chemistry.chemical_classification Cathepsin Protease medicine.diagnostic_test Cell Biology Molecular biology Recombinant Proteins 030104 developmental biology Enzyme Glucose chemistry Recombinant DNA |
| Zdroj: | The international journal of biochemistrycell biology. 97 |
| ISSN: | 1878-5875 |
| Popis: | Trichomonas vaginalis genome encodes ∼440 proteases, six of which are aspartic proteases (APs). However, only one belongs to a clan AA (EC 3.4.23.5), family A1 (pepsin A), cathepsin D-like protease. This AP is encoded by an 1113-bp gene (tv-catd), which translates into a 370-aa residues zymogen of 40.7-kDa and a theoretical pI of 4.6, generating a ∼35 kDa active enzyme after maturation (Tv-CatD). The goal of this study was to identify and analyze the effect of glucose on the expression of Tv-CatD at the transcript and protein levels, subcellular localization, and proteolytic activity. The qRT-PCR assays showed a ∼2-fold increase in tv-catd mRNA under high-glucose (HG) conditions compared to glucose-restriction (GR) conditions. We amplified, cloned, and expressed the tv-catd gene, and purified the recombinant precursor enzyme (Tv-CatDr) to generate a polyclonal antibody (anti-Tv-CatDr). Western blot (WB) and immunolocalization assays showed that glucose increases the amount of Tv-CatD in different subcellular localizations and in in vitro secretions. Additionally, Tv-CatD proteolytic activity was detected in protease-resistant extracts (PREs) using a synthetic fluorogenic peptide specific for cathepsin D/E APs at different pHs and in the presence of AP inhibitors. In a two-dimensional (2-DE) WB analysis of a PRE from parasites grown under GR and HG conditions, an anti-Tv-CatDr antibody detected a 35-kDa protein spot at pI 5.0 identified as the mature Tv-CatD form by mass spectrometry that showed proteolytic activity in 2-DE zymograms copolymerized with hemoglobin under both glucose conditions. Thus, Tv-CatD could be involved in trichomonal hemolysis. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect