Determining role of the Tryptophan carboxyl of the RGD binding motif of r-Moj in inducing Sk-Mel-28 cellular proliferation inhibition

Integrins are transmembrane proteins that play a key role in cell matrix adhesion. They also provide cellular signaling function, and are able to regulate certain cellular behaviors such as: shape change, motility, and cell proliferation. Disintegrins are found in certain types of snake venom, and are small peptides that contain an integrin-binding motif consisting of: arginine, glycine and aspartate (RGD). The two amino acids (c-terminal to the RGD sequence) provide specificity to the disintegrin peptide. In previous research we showed that the wild type recombinant version of the disintegrin mojastin (r-Moj-WN) inhibited cell proliferation of the human melanoma cell line, SK-Mel-28.  We investigated the effects of tryptophan to determine if the tryptophan carboxyl of the RGD is able to induce inhibition of cellular proliferation. We hypothesized that the presence of tryptophan may be sufficient to inhibit cell proliferation.  We tested three mojastin recombinant mutants: r-Moj-WM, r-Moj-WP, and r-Moj-MN. Fifty-thousand Sk-mel-28 cells were treated with 5µM of recombinant peptide for 24 hr. Cell proliferation inhibition was tested indirectly using a WST-1 assay. Our results demonstrated that both r-Moj-WP and r-Moj-MN peptides inhibited cell proliferation by 47% and 44.7%, respectively.  However, the r-Moj-WM mutant failed to inhibit cell proliferation, significantly. Data suggest that tryptophan alone may not be sufficient to inhibit cell proliferation. It is possible that the combination of different amino acids carboxyl of the RGD are responsible for proliferation inhibition.