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Site-specific effects of diselenide bridges on the oxidative folding of a cystine knot peptide, omega-selenoconotoxin GVIA.

ABSTRACT: Structural and functional studies of small, disulfide-rich peptides depend on their efficient chemical synthesis and folding. A large group of peptides derived from animals and plants contains the Cys pattern C-C-CC-C-C that forms the inhibitory cystine knot (ICK) or knottin motif. Here we report the effect of site-specific incorporation of pairs of selenocysteine residues on oxidative folding and the functional activity of omega-conotoxin GVIA, a well-characterized ICK-motif peptidic antagonist of voltage-gated calcium channels. Three selenoconotoxin GVIA analogues were chemically synthesized; all three folded significantly faster in the glutathione-based buffer compared to wild-type GVIA. One analogue, GVIA[C8U,C19U], exhibited significantly higher folding yields. A recently described NMR-based method was used for mapping the disulfide connectivities in the three selenoconotoxin analogues. The diselenide-directed oxidative folding of selenoconotoxins was predominantly driven by amino acid residue loop sizes formed by the resulting diselenide and disulfide cross-links. Both in vivo and in vitro activities of the analogues were assessed; the block of N-type calcium channels was comparable among the analogues and wild-type GVIA, suggesting that the diselenide replacement did not affect the bioactive conformation. Thus, diselenide substitution may facilitate oxidative folding of pharmacologically diverse ICK peptides. The diselenide replacement has been successfully applied to a growing number of bioactive peptides, including alpha-, mu-, and omega-conotoxins, suggesting that the integrated oxidative folding of selenopeptides described here may prove to be a general approach for efficient synthesis of diverse classes of disulfide-rich peptides.

SUBMITTER: Gowd KH

PROVIDER: S-EPMC2867246 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Site-specific effects of diselenide bridges on the oxidative folding of a cystine knot peptide, omega-selenoconotoxin GVIA.

Gowd Konkallu Hanumae KH Yarotskyy Viktor V Elmslie Keith S KS Skalicky Jack J JJ Olivera Baldomero M BM Bulaj Grzegorz G

Biochemistry 20100301 12

Structural and functional studies of small, disulfide-rich peptides depend on their efficient chemical synthesis and folding. A large group of peptides derived from animals and plants contains the Cys pattern C-C-CC-C-C that forms the inhibitory cystine knot (ICK) or knottin motif. Here we report the effect of site-specific incorporation of pairs of selenocysteine residues on oxidative folding and the functional activity of omega-conotoxin GVIA, a well-characterized ICK-motif peptidic antagonist ...[more]

PMID: 20175537

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Project description:A divalent knottin containing two separate integrin binding epitopes (RGD) in the adjacent loops, 3-4A, was recently developed and reported in our previous publication. In the current study, 3-4A was radiofluorinated with a 4-nitrophenyl 2-(18)F-fluoropropinate ((18)F-NFP) group and the resulting divalent positron emission tomography (PET) probe, (18)F-FP-3-4A, was evaluated as a novel imaging probe to detect integrin αvβ3 positive tumors in living animals. Knottin 3-4A was synthesized by solid phase peptide synthesis, folded, and site-specifically conjugated with (18/19)F-NFP to produce the fluorinated peptide (18/19)F-fluoropropinate-3-4A ((18/19)F-FP-3-4A). The stability of (18)F-FP-3-4A was tested in both phosphate buffered saline (PBS) buffer and mouse serum. Cell uptake assays of the radiolabeled peptides were performed using U87MG cells. In addition, small animal PET imaging and biodistribution studies of (18)F-FP-3-4A were performed in U87MG tumor-bearing mice. The receptor targeting specificity of the radiolabeled peptide was also verified by coinjecting the probe with a blocking peptide cyclo(RGDyK). Our study showed that (18)F-FP-3-4A exhibited excellent stability in PBS buffer (pH 7.4) and mouse serum. Small animal PET imaging and biodistribution data revealed that (18)F-FP-3-4A exhibited rapid and good tumor uptake (3.76 ± 0.59% ID/g and 2.22 ± 0.62% ID/g at 0.5 and 1 h, respectively). (18)F-FP-3-4A was rapidly cleared from the normal tissues, resulting in excellent tumor-to-normal tissue contrasts. For example, liver uptake was only 0.39 ± 0.07% ID/g and the tumor to liver ratio was 5.69 at 1 h p.i. Furthermore, coinjection of cyclo(RGDyK) with (18)F-FP-3-4A significantly inhibited tumor uptake (0.41 ± 0.12 vs 1.02 ± 0.19% ID/g at 2.5 h) in U87MG xenograft models, demonstrating specific accumulation of the probe in the tumor. In summary, the divalent probe (18)F-FP-3-4A is characterized by rapid and high tumor uptake and excellent tumor-to-normal tissue ratios. (18)F-FP-3-4A is a highly promising knottin based PET probe for translating into clinical imaging of tumor angiogenesis.

Dataset Information

Site-specific effects of diselenide bridges on the oxidative folding of a cystine knot peptide, omega-selenoconotoxin GVIA.

Publications

Site-specific effects of diselenide bridges on the oxidative folding of a cystine knot peptide, omega-selenoconotoxin GVIA.

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