The effect of leader peptide mutations on the biological function of bovine myostatin gene

The growth of muscle fibers can be negatively regulated by bovine myostatin. The first two exons of myostatin gene code for the N-propeptide and its third exon codes for the C-polypeptide. Myostatin is secreted as a latent configuration formed by dimerization of two matured C peptides non-covalently linked with the N terminal pro-peptide. Pro-peptide has two distinct functions in guiding protein folding and regulating biological activity of myostatin. When the structure of the leader peptide is altered via mutations resulting in more tight binding with the mature peptide, myostatin function is inhibited, resulting in the changes of P21 and CDK2 expression levels which are relatedto the regulation of cell cycle. In the present study, the coding region of bMSTN (bovine myostatin) gene was amplified and mutated (A224C and G938A) through fusion PCR, and the mutated bMSTN gene (bMSTN-mut) was inserted in frame into the pEF1a-IRES-DsRed-Express2 vector and transfected into bovine fibroblast cells. The expression levels of bMSTN-mut, P21 and CDK2 (cyclin dependent kinase 2) were examined with qPCR and Western-blotting. Changes in cell cycle after transfection were also analyzed with flow cytometry. The results indicated that leader peptide mutation resulted in down-regulation of P21 expression levels and up-regulation of CDK2 expression levels. The flow cytometry results showed that the proportion of cells in the G0/G1-phase was lower and that of cells in the S-phase was higher in bMSTN-mut transfected group than that in the control group. The proliferation rate of bMSTN-mut transfected cells was also significantly higher than that of the control cells. In conclusion, the studies have shown that the pEF1a-IRES-DsRed-Express2–bMSTN-mut recombinant plasmid could effectively promote the proliferation of bovine fibroblast cells. The site-directed mutagenesis of bMSTN gene leader peptide and in vitro expression in bovine fibroblast cells could be helpful to further the studies of bMSTN in regulating bovine muscle cell growth and development.