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).
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.
Biological Sciences Department, San José State University, One Washington Square, San José, CA 95192-0100, United States.
Daniel A
DA
Gutierrez
Biological Sciences Department, San José State University, One Washington Square, San José, CA 95192-0100, United States.
Ana S
AS
Aranda
Biological Sciences Department, San José State University, One Washington Square, San José, CA 95192-0100, United States.
David A R
DA
Carrillo
Biological Sciences Department, San José State University, One Washington Square, San José, CA 95192-0100, United States.
Melissa A
MA
Koshlaychuk
National Natural Toxins Research Center, Texas A&M University, Kingsville, TX 78363, United States.
Elda E
EE
Sanchez
National Natural Toxins Research Center, Texas A&M University, Kingsville, TX 78363, United States.
Sara E
SE
Lucena
Biological Sciences Department, San José State University, One Washington Square, San José, CA 95192-0100, United States. Electronic address: Julio.Soto@sjsu.edu.
Julio G
JG
Soto
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D019483:Disintegrins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity*; D006801:Humans; D008715:Methionine / Q000378:metabolism*; D009154:Mutation; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity*; D017386:Sequence Homology, Amino Acid
D019483:Disintegrins; D010975:Platelet Aggregation Inhibitors; D011994:Recombinant Proteins; C554078:mojastin protein, Crotalus scutulatus; D008715:Methionine
Angiogenesis; Apoptosis; Binding-loop mutants; Cell migration; Cell proliferation; Melanoma cell line; Mojastin; Recombinant disintegrin