Abstract
Peptidylprolyl cis-trans isomerases (PPIases) are ubiquitous proteins that catalyze the cis-trans isomerization of prolines. A number of proteins, such as Drosophila rhodopsin and the human immunodeficiency viral protein HIV-1 Gag, have been identified as endogenous substrates for PPIases. However, very little is known about the interaction of PPIases with small, disulfide-rich peptides. Marine cone snails synthesize a wide array of cysteine-rich peptides, called conotoxins, many of which contain one or more prolines or hydroxyprolines. To identify whether PPIase-associated cis-trans isomerization of these residues affects the oxidative folding of conotoxins, we identified, sequenced, and expressed three functionally active isoforms of PPIase from the venom gland of Conus novaehollandiae, and we characterized their ability to facilitate oxidative folding of conotoxins in vitro. Three conotoxins, namely mu-GIIIA, mu-SIIIA, and omega-MVIIC, derived from two distinct toxin gene families were assayed. Conus PPIase significantly increased the rate of appearance of the native form of mu-GIIIA, a peptide containing three hydroxyprolines. In contrast, the presence of PPIase had no effect on the folding of mu-SIIIA and omega-MVIIC, peptides containing no or one proline residue, respectively. We further showed that an endoplasmic reticulum-resident PPIase isoform facilitated folding of mu-GIIIA more efficiently than two cytosolic isoforms. This is the first study to demonstrate PPIase-assisted folding of conotoxins, small disulfide-rich peptides with unique structural properties.
Highlights
Predatory marine cone snails synthesize a great diversity (Ͼ50,000) of neurotoxic peptides commonly referred to as conotoxins
To identify whether peptidylprolyl cis-trans isomerase (PPIase)-associated cis-trans isomerization of these residues affects the oxidative folding of conotoxins, we identified, sequenced, and expressed three functionally active isoforms of PPIase from the venom gland of Conus novaehollandiae, and we characterized their ability to facilitate oxidative folding of conotoxins in vitro
We demonstrate that PPIase-mediated isomerization of peptidylprolyl bonds significantly affects the rate of formation of the native disulfide bonds for the -conotoxin GIIIA, a peptide with three hydroxyprolines (Hyp) and three disulfide bonds
Summary
Protein Extraction and Two-dimensional Gel Electrophoresis— Frozen venom ducts were pooled (a total of two samples pooled from three snails each), ground under liquid nitrogen, resuspended in 1 ml of cell lysis buffer (10 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 1 mM NaF, 20 mM Na4P2O7, 1% Triton-X, 10% glycerol, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 1ϫ Complete protease inhibitor mixture (Roche Applied Science), pH 7.6), and incubated for 30 min on ice. Cell lysates were centrifuged, and proteins were precipitated from the supernatants and reconstituted in rehydration buffer (8 M urea, 2% CHAPS, 0.002% bromphenol blue, 20 mM dithiothreitol, 0.5% immobilized pH gradient buffer, pH 3–11 (GE Healthcare)) at a final concentration of 1 mg/ml in preparation for isoelectric focusing (IEF). After 5 min at 10 °C, the reaction was initialized with the substrate succinyl-Ala-AlaPro-Phe-p-nitroanilide (Suc-AAPF-pNA) at a final concentration of 78 M
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