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.
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.
Seoane
Agustin I
AI
Biological Sciences Department, San José State University, One Washington Square, San José, CA 95192-0100, USA.
Tran
Victoria L
VL
Sanchez
Elda E
EE
White
Stephanie A
SA
Choi
Jason L
JL
Gaytán
Brandon
B
Chavez
Natalie
N
Reyes
Steven R
SR
Ramos
Carla J
CJ
Tran
Luan H
LH
Lucena
Sara E
SE
Sugarek
Maria
M
Perez
John C
JC
Mandal
Stephanie A
SA
Ghorab
Shervin
S
Rodriguez-Acosta
Alexis
A
Fung
Branden K
BK
Soto
Julio G
JG
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
D017931:DNA Primers; D019483:Disintegrins; C554078:mojastin protein, Crotalus scutulatus
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