Development of a Potent Wound Healing Agent Based on the Liver Fluke Granulin Structural Fold

Abstract

Granulins are a family of protein growth factors that are involved in cell proliferation. An orthologue of granulin from the human parasitic liver fluke Opisthorchis viverrini, known as Ov-GRN-1, induces angiogenesis and accelerates wound repair. Recombinant Ov-GRN-1 production is complex and poses an obstacle for clinical development. To identify the bioactive region(s) of Ov-GRN-1, four truncated N-terminal analogues were synthesized and characterized structurally using NMR spectroscopy. Peptides that contained only two native disulfide bonds lack the characteristic granulin β-hairpin structure. Remarkably, the introduction of a non-native disulfide bond was critical for formation of β-hairpin structure. Despite this structural difference, both two and three disulfide-bonded peptides drove proliferation of a human cholangiocyte cell line and demonstrated potent wound healing in mice. Peptides derived from Ov-GRN-1 are leads for novel wound healing therapeutics, as they are likely less immunogenic than the full-length protein and more convenient to produce.

Conotoxin Φ‐MiXXVIIA from the Superfamily G2 Employs a Novel Cysteine Framework that Mimics Granulin and Displays Anti‐Apoptotic Activity

“…[17] Using the real-time cell monitor xCELLigence system [18] we showed substantial additional cell proliferation in full nutrient media ( Figure 3A)ofthe human liver cholangiocyte cell line H69 [16] when treated with synthetic F-MiXXVIIA (EC 50 17.85 mm,F igure 3B). [20] Substantial proliferation was not observed, how-ever,alack of cell death proportional to F-MiXXVIIA concentration was noted (EC 50 2.2 mm,F igure 3C). While F-MiXXVIIA induced cell proliferation, it is significantly less potent than O. viverinni liver fluke granulin, [16] which has ap roliferative EC 50 of 11.5 nm on the same H69 cell line.…”

“…[15,19,23] Nevertheless,t he proliferative capacity of F-MiXXVIIA on cholangiocytes seen at low micromolar concentrations is comparable to several O. viverinni granulin N-terminal 8-35 amino acid peptides we have produced for other studies [20] and to that of human granulin Fonb reast cancer cell lines. [15,19,23] Nevertheless,t he proliferative capacity of F-MiXXVIIA on cholangiocytes seen at low micromolar concentrations is comparable to several O. viverinni granulin N-terminal 8-35 amino acid peptides we have produced for other studies [20] and to that of human granulin Fonb reast cancer cell lines.…”

Folding of granulin domains

“…The parasitic liver fluke granulin, Ov ‐GRN‐1, isolated from the excretory/secretory (ES) products of the carcinogenic flatworm liver fluke Opisthorchis viverrini has significant sequence differences to human granulins . The full‐length Ov ‐GRN‐1 structure has not yet been determined due to the difficulty in generating sufficient quantities of recombinant refolded protein .…”

“…Granulins were initially identified in the granules of human leukocytes and are now known to occur in a wide range of organisms with examples in particular species of oyster, insects, parasites, ascidians, mollusks, and plants . They are involved in several physiological functions and disease processes such as cell growth modulatory effects, wound repair, inflammation, and tumor growth . We have recently shown that a granulin from a liver fluke, and peptides engineered based on this protein, have potent wound healing properties in a mouse model .…”

“…They are involved in several physiological functions and disease processes such as cell growth modulatory effects, wound repair, inflammation, and tumor growth . We have recently shown that a granulin from a liver fluke, and peptides engineered based on this protein, have potent wound healing properties in a mouse model . The peptides in particular have significant potential in the development of new therapeutics for wound healing in conditions such as diabetes, which is often associated with chronic wounds.…”