Functional analysis of four single (RGDWL, RGDWM, RGDWP, RGDMN) and two double (RGDNM, RGDMP) mutants: The importance of methionine (M) in the functional potency of recombinant mojastin (r-Moj)

Abstract

We have demonstrated in previous studies that a single amino acid change can alter the activity of the recombinant disintegrin r-Moj. In this study, four r-Moj recombinants containing single mutations (r-Moj-WL, r-Moj-WM, r-Moj-WP, r-Moj-MN) and two containing double mutations (r-Moj-MP and r-Moj-NM) at the binding loop were produced, purified, and tested. All r-Moj-W_, r-Moj-M_, and r-Moj-NM mutant peptides inhibited platelet aggregation at higher potency than r-Moj-D_ mutants. Five of the seven r-Moj peptides inhibited angiogenesis at different levels. Two of the mutant peptides with a methionine at the second position carboxyl of the RGD (r-Moj-WM and r-Moj-NM) were the strongest angiogenesis inhibitors, with r-Moj-WM being the most potent. Recombinant r-Moj-MP and r-Moj-WN failed to inhibit angiogenesis. Only the r-Moj-MP mutant peptide induced apoptosis of SK-Mel-28 cells significantly (p = 0.001). This was confirmed by chromatin condensation. Proliferation of SK-Mel-28 cells was inhibited at high levels (>70%) by all r-Moj mutant peptides. Recombinant r-Moj-MN and r-Moj-WN failed to inhibit cell migration significantly (p > 0.5). Recombinant r-Moj-NM was the strongest cell migration inhibitor (98% ± 0.69), followed by r-Moj-MP (80% ± 2.87), and r-Moj-WM (61.8% ± 5.45). The lowest inhibitor was r-Moj-WL (50% ± 12.16). Our functional data suggest that the most potent r-Moj disintegrins contain a methionine in the first or the second position carboxyl to the RGD.

Keywords: Angiogenesis; Apoptosis; Binding-loop mutants; Cell migration; Cell proliferation; Melanoma cell line; Mojastin; Recombinant disintegrin.