Folding of Truncated Granulin Peptides

Abstract

Granulins are a family of unique protein growth factors which are found in a range of species and have several bioactivities that include cell proliferation and wound healing. They typically contain six disulfide bonds, but the sequences, structures and bioactivities vary significantly. We have previously shown that an N-terminally truncated version of a granulin from the human liver fluke, Opisthorchis viverrini, can fold independently into a “mini-granulin” structure and has potent wound healing properties in vivo. The incorporation of a non-native third disulfide bond, with respect to the full-length granulin module, was critical for the formation of regular secondary structure in the liver fluke derived peptide. By contrast, this third disulfide bond is not required for a carp granulin-1 truncated peptide to fold independently. This distinction led us to explore granulins from the zebrafish model organism. Here we show that the mini-granulin fold occurs in a naturally occurring paragranulin (half-domain) from zebrafish, and is also present in a truncated form of a full-length zebrafish granulin, suggesting this structure might be a common property in either naturally occurring or engineered N-terminally truncated granulins and the carp granulin-1 folding is an anomaly. The in vitro folding yield is significantly higher in the naturally occurring paragranulin, but only the truncated zebrafish granulin peptide promoted the proliferation of fibroblasts consistent with a growth factor function, and therefore the function of the paragranulin remains unknown. These findings provide insight into the folding and evolution of granulin domains and might be useful in the elucidation of the structural features important for bioactivity to aid the design of more potent and stable analogues for the development of novel wound healing agents.

Structural analysis of a U-superfamily conotoxin containing a mini-granulin fold: Insights into key features that distinguish between the ICK and granulin folds

“…The human skin normal fibroblast cell line, 1BR.3.GN, obtained from the European Collection of Authenticated Cell Cultures was confirmed to be mycoplasma free and cultured to test effects of TxVIIB on cell proliferation on a xCELLigence SP system (ACEA Bioscience) as described previously ( 18 ). A plant extract, which previously shown enhanced cell proliferation (unpublished results), was used as a positive control in the assay, serum-free media was used as a negative control.…”

“…The granulin framework is widespread throughout nature and contains a unique pattern of 12 cysteine residues, which form a laddered arrangement of six disulfide bonds and stacking of β-hairpins ( 17 ). A naturally occurring half-module also exists with six cysteine residues arranged in a CysI-CysIII, CysII-CysV, and CysIV-CysVI connectivity ( 18 ). The association of the ICK connectivity with the granulin structural motif was initially identified in conotoxin MiXXVIIA (G2-superfamily), where the peptide displayed similar structure to the N-terminal region of a human granulin module but contained three-disulfide bonds equivalent to the ICK connectivity ( 16 ).…”

Identification of an inter-cysteine loop potentially involved in the activity of Opisthorchis viverrini-granulin-1

“…GRN-L2 resulted in a 13.6% and 13.7% increase (P < 0.01) in cell growth compared to the negative control peptide when applied to cells at concentrations of 200 nmol/L and 1 μmol/L respectively. A 21-residue tropomyosin fragment was used as a negative control; tropomyosin fragments have previously been shown to have no cell proliferation effects [12,13,22]. Importantly, this peptide was produced in the laboratory using the same conditions as the Ov-GRN 12-35_3s fragments.…”

“…Although Ov-GRN-1 has potent wound-healing properties, low yields in recombinant expression have limited its development as a potential wound-healing agent [11], and prompted studies aimed at producing smaller versions with synthetic methods. A truncated form (GRN 12-35_3s ) corresponding to residues 12-35 was produced using solid-phase peptide synthesis and folded into a relatively well-defined structure containing a β-hairpin with three disulfide bonds [12]. This truncated form of Ov-GRN-1 had similar potency to the full-length protein in an in vivo wound healing assay [12] and contains two native disulfide bonds and one non-native disulfide bond, based on the full-length granulin disulfide connectivity [13].…”

“…A truncated form (GRN 12-35_3s ) corresponding to residues 12-35 was produced using solid-phase peptide synthesis and folded into a relatively well-defined structure containing a β-hairpin with three disulfide bonds [12]. This truncated form of Ov-GRN-1 had similar potency to the full-length protein in an in vivo wound healing assay [12] and contains two native disulfide bonds and one non-native disulfide bond, based on the full-length granulin disulfide connectivity [13].…”