Poster Presentation 7th Modern Solid Phase Peptide Synthesis & Its Applications Symposium 2019

Insights into the structure and function of a wound healing protein (#104)

Mohadeseh Dastpeyman 1 , David Wilson 1 , Paramjit Bansal 1 , Michael Smout 1 , Alex Loukas 1 , Norelle Daly 1
  1. Centre for Biodiscovery and Molecular Development of Therapeutics, Australian Institute of Tropical Health and Medicine, James Cook University, Cairns, QLD, Australia

Granulins are a family of growth factor proteins, which contain a highly conserved cysteine motif and are involved in cell proliferation.1 The liver-fluke granulin, Ov-GRN-1, isolated from excretory/secretory product of a liver fluke, Opisthorchis viverrini, can significantly accelerate wound repair in vivo and in vitro.1However, recombinant expression of Ov-GRN-1 is challenging and leads to a low product yield, impeding its utility as a drug lead.2 To overcome this limitation, we used the “downsizing” approach and explored the structure-function relationships of the full-length protein for elucidating bioactive sequences, providing insight into folding processes and the development of novel drug leads. A series of analogues from the N-terminal and C-terminal region of Ov-GRN-1 were chemically synthesised using solid phase peptide synthesis and the structure of the analogues was studied by NMR spectroscopy and the bioactivity was assessed by in vitro cell proliferation and in vivo wound healing assays. The structural characterisation of the N-terminal truncated peptides showed that the introduction of a nonnative disulfide bond appears to stabilize the fold and allow the peptide to form a β-hairpin structure. This analogue, Ov-GRN12-35_3s, induced cell proliferation and in vivo wound healing with similar potency to the full-length protein.3NMR analysis of Ov-GRN12-35_3s indicated the presence of multiple conformations, most likely from proline isomerisation.Further investigation showed proline residues have a significant influence on the structure, activity and folding of Ov-GRN12-35_3s. The results obtained for this part of the study led to the development of a more potent analogue, GRNP4A, with improved folding yield.4The study also includes the characterization of a C-terminal granulin fragment, which albeit having no well-defined three-dimensional structure, still displayed biological activity. These results provide insight into structure-function relationships of ov-GRN-1 and might facilitate the development of a novel wound-healing agent.

  1. Dastpeyman, M., Smout, M.J., Wilson, D., Loukas, A., and Daly, N.L. Folding of granulin domains. Peptide Science, 2018, Doi:10.1002/pep1002.24062.
  2. Smout, M.J., J.P. Mulvenna, M.K. Jones, and A. Loukas. Expression, refolding and purification of Ov-GRN-1, a granulin-like growth factor from the carcinogenic liver fluke, that causes proliferation of mammalian host cells. Protein Expr. Purif. 2011; 79(2): 263-70.
  3. Bansal, P.S., Smout, M.J., Wilson, D., Cobos Caceres, C., Dastpeyman, M., Sotillo, J., Seifert, J., Brindley, P.J., Loukas, A., and Daly, N.L. Development of a potent wound healing agent based on the liver fluke
  4. Dastpeyman, M., Bansal, P.S., Wilson, D., Sotillo, J., Brindley, P.J., Loukas, A., Smout, M.J., and Daly, N.L. Structural variants of a liver fluke derived granulin peptide potently stimulate wound healing. Journal of Medicinal Chemistry, 2018 (61), 8746-8753.