Determining the Importance of Aspartate and Methionine Amino Acids Carboxyl of the RGD Motif in Recombinant Mojastin Disintegrins in Apoptotic Induction

Integrins are cell surface receptors that play an important role in apoptotic induction. Disintegrins are small peptides, from viper venom, containing a binding motif composed of the amino acids arginine (R), glycine (G) and aspartate (D). This tripeptide motif has a high affinity to the integrin-binding site. Previous research has shown that recombinant mojastin (r-Moj) disintegrins with the amino acids D and M immediately carboxyl to the RGD motif induce apoptosis of a human melanoma cell line (SK-Mel-28). I am testing two hypotheses. First hypothesis: recombinant disintegrins with a D amino acid following the RGD (RGDD) will induce apoptosis of SK-Mel-28 cells. Second hypothesis: recombinant disintegrins with an M amino acid following the RGD (RGD_M) will induce apoptosis of SK-Mel-28 cells. Five mojastin mutants were obtained (r-Moj-DA, r-Moj-DG, r-Moj-DV, r-Moj-NM, and r-Moj-WM) to test these hypotheses by site directed mutagenesis. Chromatin fragmentation was used to determine if r-Moj-DA, r-Moj-NM, and r-Moj-WM induce apoptosis of SK-Mel-28. For this method, SK-MEL-28 cells were grown on chambered slides and treated with 5 µM of the peptides for 24 hr. Treated cells were stained with Hoechst and visualized under a Zeiss fluorescent microscope. Preliminary results indicated that r-Moj-DA, r-Moj-NM, and r-Moj-WM induced apoptosis. Chromatin fragmentation will be performed with r-Moj-DG and r-Moj-DV. TUNEL assays will be performed with all the recombinant peptides to measure the number of cells undergoing apoptosis after treatment. This project was funded by NSF-REU grant DBI 1004350.