title: The mojastin mutant Moj-DM induces apoptosis of the human melanoma SK-Mel-28, but not the mutant Moj-NN nor the non-mutated recombinant Moj-WN.
abstract: In this study, three recombinant mojastin peptides (Moj-WN, Moj-NN, and Moj-DM) were produced and compared functionally. Recombinant Moj peptides were purified as GST-fusions. GST-Moj-WN and GST-Moj-NN inhibited ADP-induced platelet aggregation in platelet rich plasma. The GST-Moj-WN had an IC(50) of 160nM, while the GST-Moj-NN had an IC(50) of 493nM. The GST-Moj-DM did not inhibit platelet aggregation. All three GST-Moj peptides inhibited SK-Mel-28 cell adhesion to fibronectin. The GST-Moj-WN inhibited the binding of SK-Mel-28 cells to fibronectin with an IC(50) of 11nM, followed by the GST-Moj-NN (IC(50) of 28nM), and the GST-Moj-DM (IC(50) of 46nM). The GST-Moj peptides' ability to induce apoptosis on SK-Mel-28 cells was determined using Annexin-V-FITC and nuclear fragmentation assays. Cells were incubated with 5muM GST-Moj peptides for 24h. At 5microM GST-Moj-DM peptide, 13.56%+/-2.08 of treated SK-Mel-28 cells were in early apoptosis. The GST-Moj-DM peptide also caused nuclear fragmentation as determined by fluorescent microscopy and Hoechst staining. The GST-Moj-WN and GST-Moj-NN peptides failed to induce apoptosis. We characterized the SK-Mel-28 integrin expression, as the first step in determining r-Moj binding specificity. Our results indicate that SK-Mel-28 cells express alphavbeta3, alphav, alpha6, beta1, and beta3 integrin receptors.
authors.affiliation: Biological Sciences Department, San José State University, One Washington Square, San José, CA 95192-0100, USA.
authors.forename: Agustin I
authors.initials: AI
authors.lastname: Seoane
authors.forename: Victoria L
authors.initials: VL
authors.lastname: Tran
authors.forename: Elda E
authors.initials: EE
authors.lastname: Sanchez
authors.forename: Stephanie A
authors.initials: SA
authors.lastname: White
authors.forename: Jason L
authors.initials: JL
authors.lastname: Choi
authors.forename: Brandon
authors.initials: B
authors.lastname: Gaytán
authors.forename: Natalie
authors.initials: N
authors.lastname: Chavez
authors.forename: Steven R
authors.initials: SR
authors.lastname: Reyes
authors.forename: Carla J
authors.initials: CJ
authors.lastname: Ramos
authors.forename: Luan H
authors.initials: LH
authors.lastname: Tran
authors.forename: Sara E
authors.initials: SE
authors.lastname: Lucena
authors.forename: Maria
authors.initials: M
authors.lastname: Sugarek
authors.forename: John C
authors.initials: JC
authors.lastname: Perez
authors.forename: Stephanie A
authors.initials: SA
authors.lastname: Mandal
authors.forename: Shervin
authors.initials: S
authors.lastname: Ghorab
authors.forename: Alexis
authors.initials: A
authors.lastname: Rodriguez-Acosta
authors.forename: Branden K
authors.initials: BK
authors.lastname: Fung
authors.forename: Julio G
authors.initials: JG
authors.lastname: Soto
mesh_terms: D000595:Amino Acid Sequence; D017209:Apoptosis; D001483:Base Sequence; D045744:Cell Line, Tumor; D017931:DNA Primers; D019483:Disintegrins / Q000235:genetics*; D006801:Humans; D020128:Inhibitory Concentration 50; D008545:Melanoma / Q000473:pathology*; D008969:Molecular Sequence Data; D009154:Mutation; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
chemical_list: D017931:DNA Primers; D019483:Disintegrins; C554078:mojastin protein, Crotalus scutulatus
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