Research Article: Maltose binding protein-fusion enhances the bioactivity of truncated forms of pig myostatin propeptide produced in E. coli

Date Published: April 3, 2017

Publisher: Public Library of Science

Author(s): Sang Beum Lee, Sung Kwon Park, Yong Soo Kim, Sabato D’Auria.

http://doi.org/10.1371/journal.pone.0174956

Abstract

Myostatin (MSTN) is a potent negative regulator of skeletal muscle growth. MSTN propeptide (MSTNpro) inhibits MSTN binding to its receptor through complex formation with MSTN, implying that MSTNpro can be a useful agent to improve skeletal muscle growth in meat-producing animals. Four different truncated forms of pig MSTNpro containing N-terminal maltose binding protein (MBP) as a fusion partner were expressed in E. coli, and purified by the combination of affinity chromatography and gel filtration. The MSTN-inhibitory capacities of these proteins were examined in an in vitro gene reporter assay. A MBP-fused, truncated MSTNpro containing residues 42–175 (MBP-Pro42-175) exhibited the same MSTN-inhibitory potency as the full sequence MSTNpro. Truncated MSTNpro proteins containing either residues 42–115 (MBP-Pro42-115) or 42–98 (MBP-Pro42-98) also exhibited MSTN-inhibitory capacity even though the potencies were significantly lower than that of full sequence MSTNpro. In pull-down assays, MBP-Pro42-175, MBP-Pro42-115, and MBP-Pro42-98 demonstrated their binding to MSTN. MBP was removed from the truncated MSTNpro proteins by incubation with factor Xa to examine the potential role of MBP on MSTN-inhibitory capacity of those proteins. Removal of MBP from MBP-Pro42-175 and MBP-Pro42-98 resulted in 20-fold decrease in MSTN-inhibitory capacity of Pro42-175 and abolition of MSTN-inhibitory capacity of Pro42-98, indicating that MBP as fusion partner enhanced the MSTN-inhibitory capacity of those truncated MSTNpro proteins. In summary, this study shows that MBP is a very useful fusion partner in enhancing MSTN-inhibitory potency of truncated forms of MSTNpro proteins, and MBP-fused pig MSTNpro consisting of amino acid residues 42–175 is sufficient to maintain the full MSTN-inhibitory capacity.

Partial Text

Myostatin (MSTN) is a member of the transforming growth factor-β (TGF-β) superfamily and negatively regulates skeletal muscle growth and development with little effect on other tissues [1–3]. Similar to other TGF-β family member proteins, MSTN is translated as a precursor protein (preproMSTN) consisting of a signal sequence, an N-terminal propeptide domain (MSTNpro) and a C-terminal mature (active) domain [2, 4]. Upon entering into the endoplasmic reticulum, signal peptide is removed from preproMSTN, then proMSTN forms a disulfide-linked homodimer and is proteolytically processed at a conserved Arg-Arg-X-Arg site by the furin family of proprotein convertases to generate MSTNpro and mature MSTN [5, 6]. After the cleavage, two MSTNpros make a non-covalent complex formation with disulfide-linked homodimer of mature MSTN in a latent/inactive state to suppress MSTN binding to its receptor [6–8]. Administration or overexpression of MSTNpro has been shown to enhance skeletal muscle growth in laboratory animal species [4, 5, 9–14], supporting the inhibitory role of MSTNpro on MSTN activity.

The current study shows that a MBP-fused pig MSTNpro consisting of amino acid residues 42–175 is sufficient to maintain the full MSTN-inhibitory capacity. Our recent study also demonstrated that MBP-fused flatfish MSTN1pro domain consisting of residues 45–100 had a full MSTN-inhibitory capacity [19]. Similarly, it was reported that a GST-fused human MSTNpro consisting of amino acid residues 42–218 had the full MSTN-inhibitory capacity [16]. These studies together indicate that full sequence of MSTNpro may not be required for a full MSTN-inhibitory capacity. It has, however, been unknown whether a fusion partner could play a role in modulating MSTN-inhibitory capacity of the truncated forms of MSTNpro, and the current results show that MBP fusion enhances MSTN-inhibitory capacity of the truncated forms of MSTNpro. The MSTN-inhibitory capacity of MBP-removed MSTNpro domain containing residues 42–175 was almost 20-fold lower as compared to MBP-fused MSTNPro domain containing residues 42–175, and MBP-removed MSTNPro domain containing residues 42–98 showed little MSTN-inhibitory capacity. Of note, our current and previous results [20, 27] showed that MBP alone did not bind MSTN to suppress its activity. Notably, a GST-fused human MSTNpro consisting of residues 42–98 showed no MSTN-inhibitory capacity [16], while we observed MSTN-inhibitory capacity of MBP-fused MSTNPro domain containing residues 42–98, indicating that the enhancement of MSTN-inhibitory capacity is more likely to be confined to MBP rather than to other fusion partners. Even though it is well known that MBP is an effective solubility enhancer as a fusion partner [28, 29], as far as we know, this is the first demonstration of biological activity enhancement of a passenger protein by MBP fusion. With regard to the mechanism of solubility enhancement by MBP, it has been proposed that MBP transiently binds folding intermediates or aggregation-prone passenger protein, keeping the passenger protein in a soluble state until either spontaneous or chaperon-mediated folding can occur [29]. The mechanisms of how MBP enhances the MSTN-inhibitory capacity of truncated forms of pig MSTNpro is not known currently. Based on the proposed mechanism of solubility enhancement by MBP, it is, however, speculated that MBP fusion possibly stabilized the folding state of the Pro42-98, keeping Pro42-98 from the formation of homogeneous or heterogeneous soluble aggregates. In fact, our data (Fig 7) indicated the heterogeneous soluble aggregate formation upon removal of MBP. To clearly explain the mechanisms of how MBP enhances the MSTN-inhibitory capacity of truncated forms of pig MSTNpro, future studies, such as molecular modeling or determination of 3D structure, are warranted.

 

Source:

http://doi.org/10.1371/journal.pone.0174956

 

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