Participación Juvenil en la escuela

The initial indicator for successful refolding of myostatin is the presence of a dimer in non-reducing conditions. However, this is not a guarantee that the native structure has been obtained. To confirm refolding has produced the native structure, activity must be established; this is not possible for MstnPP because the protein has no known measurable activity. In vivo, mature myostatin is produced via cleavage of the myostatin precursor protein by furin convertase. Therefore, production of the growth factor and subsequent activity measurements was carried out using commercially available furin convertase enzyme (Jin, Dunn et al. 2004; Funkenstein and Rebhan 2007).

3.4.1 Furin digest

Furin digest was carried out according to published methods and the manufacturer’s guidelines initially, with subsequent modifications as detailed in Chapter 2.

Analysis of myostatin furin cleavage by reducing (R) SDS-PAGE shows reduction of the precursor band (1) at 50 kDa and concomitant appearance of bands at 37 (2) and 12 (3) kDa (Fig. 3.8a). These represent the propeptide region and the mature growth factor region monomer respectively. In the absence of reducing agent, the growth factor monomer is replaced with a dimer band at 24 kDa (4), indicating successful refolding of the growth factor during the MstnPP refolding protocol. Undigested MstnPP dimer is also visible (5).

To confirm SDS-PAGE results, Western blotting with antibodies against both the myostatin growth factor (anti-MSTN) and the N-terminal His-tag (anti-HIS) was performed. The anti-MSTN antibody recognises a short region at the C-terminus of the growth factor (349-364) and will therefore bind to both the precursor and the growth factor, but not the propeptide region. The anti-HIS antibody will bind to the His-tagged precursor and propeptide, but not the digested growth factor.

Figure 3.8 Furin digest of MstnPP.

a. SDS-PAGE and Western blotting using antibodies against myostatin (anti-MSTN) and the His-tag (anti-HIS) in reducing (R) and non-reducing (NR) conditions. Bands are as follows: 1, undigested MstnPP monomer; 2, propeptide; 3, growth factor monomer; 4, growth factor dimer; 5, undigested MstnPP dimer; 6, unknown 70 kDa band.

b. The anti-MSTN epitope (blue) modeled in the myostatin growth factor monomer (i) and dimer (ii).

Western blots confirm SDS-PAGE results of successful furin digestion and the production of propeptide and growth factor regions. The anti-MSTN Western shows smearing, and a reduction in band intensities, in the non-reducing (NR) lane. One explanation for the former may be an increase in globularity of the disulphide-linked dimer, causing altered movement through the gel. Transfer onto the nitrocellulose membrane may also allow increased mobility. A reduction in band intensity may be due to reduced accessibility of the antibody to the antigenic region in the dimer. This

suggestion is supported by observation of the myostatin crystal structure (Cash, Rejon

et al. 2009) which shows that the epitope region (blue) is partially hidden in the dimer (Fig. 3.8b (ii)) compared to the monomer (Fig. 3.8b (i)). As expected, the anti-HIS Western blot shows propeptide bands and a small amount of undigested MstnPP only; an absence of smearing and propeptide dimerisation suggest that the propeptide does not form disulphide bonds.

An intriguing result is the presence of a band at approximately 70 kDa in non- reducing conditions (6), seen in both the Coomassie-stained gel and the anti-MSTN Western blot. The 125 kDa MstnPP dimer is present although faint, suggesting formation of a novel complex. By size, possibilities for the 70 kDa species are the growth factor dimer plus one propeptide (24 + 37 = 61 kDa), precursor plus growth factor monomer (50 + 12 = 62 kDa) with dimerisation between the two growth factor domains, or two propeptide domains (37 + 37 = 74 kDa). Previous research into the latent myostatin complex expressed in a mammalian system presented non-reducing SDS-PAGE and anti-myostatin Western blot data that also shows a band of approximately 70 kDa (Wolfman, McPherron et al. 2003). Although antibodies against both the propeptide and C-terminus detected this band, it does not appear in the anti-HIS Western blot shown here (Fig. 3.8a). This may be due to blocking of the His-tag during complex formation, or cleavage of the His-tag, which was often observed in the absence of Tev protease. Alternatively, the 70 kDa band has been suggested to represent misfolded protein (Wolfman, McPherron et al. 2003), also a possibility as there was often a proportion of undigested precursor protein remaining after furin digest (Chapter 3), which was not removed by the addition of extra furin convertase.

In either situation, these results show production of the growth factor dimer and successful processing of MstnPP by furin, supporting the conclusion that refolding was successful.

3.4.2 Activity assays

The standard assay for myostatin growth factor activity is an ability to inhibit the proliferation of C2C12 mouse myoblasts (Thomas, Langley et al. 2000; Taylor, Bhasin et al. 2001; Joulia, Bernardi et al. 2003) with cell proliferation routinely

measured using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole)-based assay.The yellow MTT salt is reduced to purple formazan in the mitochondria of living cells; absorbance is directly correlated to cell number. Original MTT-assay kits require washing, harvesting and solubilization steps prior to spectrophotometric analysis and the use of phenol-red free media to prevent interference of the indicator with measurements. In contrast, the WST-1 reagant (Roche) is a modified form of MTT that produces a water-soluble formazan product. No extra steps are necessary and phenol-red containing media can be used.

Figure 3.9 Activity of the myostatin growth factor by C2C12 proliferation assays.

Relative absorbance (450-630 nm) is proportional to cell growth using the WST-1 reagant (Roche). Bars are labelled are as follows: Heat, heat-treated furin digest; Acid, acid-treated furin digest; MstnPP, undigested MstnPP; Latent complex, untreated furin-digested MstnPP. Cells only and buffer only bars indicate negative controls. Effective concentration of growth factor used was 10 μg/mL within the latent complex; concentration of all other proteins was equivalent to that of the latent complex when the growth factor was 10 μg/mL. * P < 0.05 by Student’s T-test.

Following furin cleavage, the latent complex was either heat-treated (80°C for 5 minutes) (Funkenstein and Rebhan 2007) or acid-treated (1 M HCl to pH 2.5 and incubation on ice for 1 hour at 4 °C followed by neutralization with 1 M NaOH to pH 7.5) (Lawrence, Pircher et al. 1985), methods shown to be effective previously (Lawrence, Pircher et al. 1985), and added to the media of C2C12 myoblasts. The

concentration of the growth factor (10 μg/mL) was estimated by comparing band intensity of furin-digested samples to BSA standards after reducing SDS-PAGE. Both heat- and acid-treated digests were able to significantly inhibit the proliferation of mouse C2C12 myoblasts when compared to all controls (Fig. 3.9), which were cells only, cells plus the furin digest buffer only, the undigested precursor protein and untreated latent complex. Protein concentrations for the latter two matched that of the total furin digest that represented 10 μg/mL of growth factor. This concentration is greater than that seen in other published reports (Berry, Thomas et al. 2002) indicating reduced activity for the refolded myostatin growth factor produced here. Although one possibility is that a proportion of the myostatin growth factor is not correctly folded, the most likely explanation is incomplete dissociation of the latent complex during acid- and heat-treatment, as suggested in Chapter 4 (Fig. 4.10).

4

Biochemical and biophysical analysis of the myostatin

precursor protein and latent complex

The TGF-β propeptide region and latent complex play important roles in the production and regulation of the mature protein. The propeptide region of the precursor protein has been suggested to have a chaperone-like function in the correct folding of the growth factor as the myostatin growth factor is unable to fold in vitro in its absence (Jin, Dunn et al. 2004). After cleavage and latent complex formation the propeptide has a role in export and stability, likely through interactions with the extracellular matrix, and post-secretion, is a potent inhibitor of the growth factor. Therefore, investigation into the structure and function of the precursor, the propeptide and the latent complex is paramount for an understanding of the biology of all TGF-β growth factors, and for myostatin, will provide important information for therapeutic intervention. However, little investigation into the precursor proteins or latent complexes of the TGF-β family has been carried out to date.

This chapter describes the biochemical and biophysical analysis of MstnPP, the processed latent complex, and the propeptide domain within these. The presented results provide novel information that has implications for the understanding of myostatin processing and regulation in vivo.