Functional characterization of six aspartate (D) recombinant mojastin mutants (r-Moj): A second aspartate amino acid carboxyl to the RGD in r-Moj-D_ peptides is not sufficient to induce apoptosis of SK-Mel-28 cells.

Abstract

Disintegrins are small peptides produced in viper venom that act as integrin antagonists. When bound to integrins, disintegrins induce altered cellular behaviors, such as apoptotic induction. Disintegrins with RGDDL or RGDDM motifs induce apoptosis of normal and cancer cells. We hypothesized that a second aspartate (D) carboxyl to the RGD is sufficient to induce apoptosis. Five recombinant mojastin D mutants were produced by site-directed mutagenesis (r-Moj-DA, r-Moj-DG, r-Moj-DL, r-Moj-DN, and r-Moj-DV). Stable αv integrin knockdown and shRNA scrambled control SK-Mel-28 cell lines were produced to test a second hypothesis: r-Moj-D_ peptides bind to αv integrin. Only r-Moj-DL, r-Moj-DM, and r-Moj-DN induced apoptosis of SK-Mel-28 cells (at 29.4%, 25.6%, and 36.2%, respectively). Apoptotic induction was significantly reduced in SK-Mel-28 cells with a stable αv integrin knockdown (to 2%, 17%, and 2%, respectively), but not in SK-Mel-28 cells with a stable scrambled shRNA. All six r-Moj-D_ peptides inhibited cell proliferation; ranging from 49.56% (r-Moj-DN) to 75.6% (r-Moj-DA). Cell proliferation inhibition by r-Moj-D_ peptides was significantly reduced in SK-Mel-28 cells with a stable αv integrin knockdown. All six r-Moj-D_ peptides inhibited SK-Mel-28 cell migration at high levels (69% to 100%). As a consequence, rac-1 mRNA expression levels were significantly reduced as early as 1 h after treatment, suggesting that rac-1 is involved in the cell migration activity of SK-Mel-28.

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)

“…Our research group have demonstrated that substituting one or two of the amino acids immediately carboxyl of the RGD of r-Moj resulted in the elimination of platelet aggregation inhibition for the r-Moj-DM mutant (Table 2), the introduction of apoptotic inducing activity for r-Moj-DM (Ramos et al, 2016) and r-Moj-MP (Fig. 3A), and activity potency differences for mutants produced for the Ramos et al (2016) study and the current one.…”

“…Expression and purification of r-Moj-WL, r-Moj-WM, r-Moj-WP, r-Moj-WN, r-Moj-MN, r-Moj-MP, and r-Moj-NM peptides were performed with a method described earlier (Ramos et al, 2016). …”

“…Targeted mechanisms include activation or repression of genes involved in cell death and survival, activation of apoptosis, angiogenesis inhibition, cell division inhibition, and metastasis inhibition. Several investigators have shown that disintegrins have the ability to induce cellular responses that target the mechanisms listed above (McBride et al, 2016; Ramos et al, 2016; Lucena et al 2014, 2015; Ribeiro et al, 2014; Carey et al, 2012; Selistre-de-Araujo et al, 2010; Minea et al, 2010; Seoane et al, 2010; Ren et al, 2006; Alimenti et al, 2004; Brassard et al, 1999; Wu et al, 2003; Hong et al, 2003; Zhou et al, 1999; Yeh et al, 1998). …”

“…The truncated recombinant mojastin cDNA (r-Moj-WN) was cloned from a Mohave rattlesnake venom gland (Soto et al, 2007). In a previous article, we produced and tested six r-Moj peptides with a second aspartate immediately carboxyl to their RGD (Ramos et al, 2016). Our research demonstrated that a second aspartate is not sufficient to induce apoptosis of SK-Mel-28 cells.…”

“…Our research demonstrated that a second aspartate is not sufficient to induce apoptosis of SK-Mel-28 cells. However, r-Moj-D_ disintegrins activated anti-cancer activities by antagonizing the αv integrin subunit (Ramos et al, 2016). Furthermore, we demonstrated that 40 genes were differentially expressed in SK-Mel-28 cells after r-Moj-DM treatment (McBride et al, 2016).…”