Functional analysis of a recombinant PIII-SVMP, GST-acocostatin; an apoptotic inducer of HUVEC and HeLa, but not SK-Mel-28 cells.
Disintegrins and disintegrin-like peptides interact with integrins and interfere with cell-cell and cell-matrix interactions. A disintegrin-like snake venom gene, Acocostatin was cloned from the venom gland mRNA of Agkistrodon contortrix contortrix. Acocostatin belongs to the PIII-SVMP subfamily of disintegrin-like peptides. The recombinant acocostatin peptide was produced and purified as GST-fusion. The GST-acocostatin peptide, at 44 μg/mL, inhibited platelet aggregation by 30% in PRP and 18% in whole blood. In addition GST-acocostatin, at 220 μg/mL, inhibited SK-Mel-28 cell migration by 48%, but did not inhibit T24 cell migration. The GST-acocostatin peptide ability to induce apoptosis on HUVEC, HeLa, and SK-Mel-28 cells was determined using Annexin V-FITC and chromatin fragmentation assays after 24 h of treatment. At 5 μM GST-acocostatin peptide, 19.68%+/- 3.09 of treated HUVEC, and 35.86% +/- 2.05 of treated HeLa cells were in early apoptosis. The GST-acocostatin peptide also caused chromatin fragmentation of HUVEC and HeLa cells as determined by fluorescent microscopy and Hoechst staining. The GST-acocostatin peptide failed to induce apoptosis of SK-Mel-28 cells. We characterized the HUVEC, HeLa, and T24 integrin expression by flow cytometry, as the first step in determining GST-acocostatin binding specificity. Our results indicate that HUVEC express αv, αvβ3, αvβ5, α6, β1, and β3 integrin receptors. HeLa cells express α1, α2, α6, αv, αvβ5, and β1 integrin receptors. T24 cells express α1, α3, α6, αv, αvβ3, αvβ5, β1, β3, and β6 integrin receptors.
Biological Sciences Department, San José State University, One Washington Square, San José, CA 95192-0100, USA.
Takele
T
Teklemariam
Agustin I
AI
Seoane
Carla J
CJ
Ramos
Elda E
EE
Sanchez
Sara E
SE
Lucena
John C
JC
Perez
Stephanie A
SA
Mandal
Julio G
JG
Soto
D017836:Agkistrodon; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D002843:Chromatin / Q000378:metabolism; D019483:Disintegrins / Q000235:genetics*; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D010455:Peptides / Q000235:genetics* / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects; D011994:Recombinant Proteins / Q000235:genetics* / Q000494:pharmacology; D012910:Snake Venoms / Q000235:genetics*; D014471:Umbilical Veins / Q000166:cytology
D002843:Chromatin; D019483:Disintegrins; D016023:Integrins; D010455:Peptides; D011994:Recombinant Proteins; D012910:Snake Venoms; D005982:Glutathione Transferase; D016650:Fluorescein-5-isothiocyanate
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