Surface plasmon resonance kinetic analysis on the binding of MSV to myostatin, ActRIIB to myostatin and myostatin to itself.

$<p>Immobilized onto the flow-cell. Rate constants of association and dissociation interactions (k_a and k_d) and the equilibrium dissociation constants (K_D) of Myostatin, ActRIIB and MSV interactions are shown.</p

Alternative splicing of sheep myostatin pre-mRNA and translation of MSV mRNA into protein.

<p>(A) A representative Northern blot identified canonical myostatin (Mstn) and MSV mRNAs in poly(A)⁺ RNA isolated from sheep skeletal muscle using a radiolabeled probe complementary to exon 1 & 2 sequence of sheep myostatin (nt 1–621). (B) Schematic representation of alternative splicing of the myostatin gene. Genomic structure, splicing of canonical myostatin and MSV mRNAs are shown as determined by RT-PCR amplification and sequencing. The sheep myostatin gene has a cryptic third intron sequence (Int 3, 1011 bp) located 21 bp downstream of the intron 2/exon 3 boundary, thereby removing the coding sequence of the canonical mature myostatin protein. Alternate splicing creates a new ORF (966 bp) by appending a novel C-terminal coding sequence (exon 3b, 198 bp) to a truncated propeptide coding sequence of myostatin (exon 1 & 2 and 3a) in the MSV transcript. Open boxes show 5′ and 3′ untranslated regions, filled boxes represent translated sequences. Also shown are exons (Ex), introns (Int), translation start (ATG) and stop (TGA, TAA) sites, and the size of each transcript. Location of the 11 bp deletion in exon 3 identified in Belgian Blue cattle is also indicated. (C) Tissue-specific mRNA expression of MSV and myostatin was analyzed in <i>biceps femoris</i> (Biceps), <i>quadriceps</i> (Quad.) and <i>semitendinosus</i> (Semit.) muscles, and heart, liver, brain, kidney, testicle, ovary, gut, skin and aorta tissues of three months old sheep using RT-PCR. Actin was used as a positive control for each tissue sample. NTC is a no template PCR control. (D) Multiple polypeptide sequence alignment of the predicted C-terminus of MSV in sheep, cattle, pig and dolphin. A consensus proteolytic cleavage site [(K/R)-(X)_n-(K/R)↓ where n = 0, 2, 4, 6 and X is any amino acid except cysteine at aa 271–274] has been identified for precursor convertases. A dotted line indicates the location of the putative cleavage site. The scale shows the positions of the amino acid residues in the MSV polypeptide sequence. The unshaded background highlights residues that are different from the consensus sequence. An <i>in-silico</i> predicted secondary structure of mature sheep MSV is also shown. (E) Schematic representation of the known and proposed proteolytic processing of canonical myostatin and MSV precursors, respectively. The location of the secretion signal peptide and the C-terminal cleavage sites are indicated. Grey filling shows the novel C-terminus of the MSV precursor. Black bars denote the location of polypeptide sequences used to raise MSV-specific polyclonal antibodies (MSVab and MSVab65). (F) Detection of MSV-immunoreactive proteins in <i>semitendinosus</i> muscles of sheep and cattle and its absence in <i>gastrocnemius</i> muscles of mouse and rat (20 µg of total protein per lane) using an anti-MSVab in Western immunoblotting. Recombinant peptide (Recomb.) corresponds to a polypeptide for the C-terminal 65 amino acids (11.9 kDa) of sheep MSV. Molecular weights of a protein marker are also indicated.</p

Functional analysis of MSV.

<p>(A) Detection of full length MSV mRNA in a stable MSV over-expressing (MSV-line) and an empty vector stably transfected C₂C₁₂ myoblast line (Control-line) using RT-PCR. GAPDH was used as a positive control for each sample. NTC is a no template PCR control. (B) Effect of endogenous over-expression of MSV on the proliferation of C₂C₁₂ myoblasts. Proliferation of the MSV- and Control-line was determined at 0, 24, 48 and 72 h using the WST-1 cell proliferation reagent (**P<0.01, ***P<0.001, n = 8). (C) Effect of rMSV on the proliferation of C₂C₁₂ myoblasts. C₂C₁₂ myoblasts were treated with increasing concentrations of rMSV for 48 h, and cell replication was determined using the WST-1 cell proliferation reagent (*P<0.05, **P<0.01, ***P<0.001, n = 8). (D) Effect of rMSV on the proliferation of sheep myoblasts. Sheep myoblasts were treated with increasing concentrations of rMSV for 48 h, and cell replication was determined using the WST-1 cell proliferation reagent (***P<0.001, n = 8). (E) The abundance of CDK2 protein in nuclear and cytoplasmic fractions of the MSV- and Control-line during proliferation (**P<0.01, n = 3). The abundance of actin and SP-1 proteins was used as cytoplasmic and nuclear loading controls, respectively. (F) The abundance of Cyclin E protein in nuclear and cytoplasmic fractions of the MSV- and Control-line during proliferation (**P<0.01, **P<0.001, n = 3). The abundance of actin and SP-1 proteins was used as cytoplasmic and nuclear loading controls, respectively. (G) The abundance of Myf5, MyoD, Myogenin, MRF4, Pax7 and MEF2 proteins was determined using Western immunoblotting in proliferating myoblasts of the MSV- and Control-line (*P<0.05, n = 3).</p