Parameters affecting in vitro oxidation/folding of maurotoxin, a four-disulphide-bridged scorpion toxin

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 -GIIIA, -SIIIA, and -MVIIC, derived from two distinct toxin gene families were assayed. Conus PPIase significantly increased the rate of appearance of the native form of -GIIIA, a peptide containing three hydroxyprolines. In contrast, the presence of PPIase had no effect on the folding of -SIIIA and -MVIIC, peptides containing no or one proline residue, respectively. We further showed that an endoplasmic reticulum-resident PPIase isoform facilitated folding of -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.

Section: Conotoxin Folding Studies-to Investigate the Role Of Conusmentioning

confidence: 82%

Section: The Nucleotide Sequence(s) Reported In This Paper Has Been Smentioning

confidence: 99%

Section: The Nucleotide Sequence(s) Reported In This Paper Has Been Smentioning

confidence: 99%

See 1 more Smart Citation

Identification of Conus Peptidylprolyl Cis-Trans Isomerases (PPIases) and Assessment of Their Role in the Oxidative Folding of Conotoxins

Safavi-Hemami

Bułaj

Olivera

et al. 2010

“…Interestingly, bovine PDI also facilitates folding of cyclotides. This is not unexpected, since bovine PDI has also been shown to facilitate folding of other nonbovine disulfide-rich peptides, including examples from cone snails and scorpions (39,40). The final yield of correctly folded cyclic peptide in the presence of OaPDI was about 4-fold greater than that of the linear peptide, although the folding rate was about 3-fold greater for linear kalata B1.…”

Section: Reducedmentioning

confidence: 99%

A Novel Plant Protein-disulfide Isomerase Involved in the Oxidative Folding of Cystine Knot Defense Proteins

Gruber

Cemazar

Clark

et al. 2007

127

113

We have isolated a protein-disulfide isomerase (PDI) from Oldenlandia affinis (OaPDI), a coffee family (Rubiaceae) plant that accumulates knotted circular proteins called cyclotides. The novel plant PDI appears to be involved in the biosynthesis of cyclotides, since it co-expresses and interacts with the cyclotide precursor protein Oak1. OaPDI exhibits similar isomerase activity but greater chaperone activity than human PDI. Since domain c of OaPDI is predicted to have a neutral pI, we conclude that this domain does not have to be acidic in nature for PDI to be a functional chaperone. Its redox potential of ؊157 ؎ 4 mV supports a role as a functional oxidoreductase in the plant. The mechanism of enzyme-assisted folding of plant cyclotides was investigated by comparing the folding of kalata B1 derivatives in the presence and absence of OaPDI. OaPDI dramatically enhanced the correct oxidative folding of kalata B1 at physiological pH. A detailed investigation of folding intermediates suggested that disulfide isomerization is an important role of the new plant PDI and is an essential step in the production of insecticidal cyclotides.Protein-disulfide isomerase (PDI 3 ; EC 5.3.4.1) is an oxidoreductase enzyme that belongs to the thioredoxin superfamily (1). It has a major role in oxidative folding of polypeptides in the endoplasmic reticulum (ER) of eukaryotic cells and functions as an ER chaperone (2). The exact mechanism of action of PDI is not clear, but it is believed to bind polypeptides through hydrophobic interactions and forms (oxidizes), breaks (reduces), and/or shuffles (isomerizes) disulfide bonds in substrate molecules via a dithiol-disulfide exchange between its active-site CXXC motif and the substrate polypeptide (3).Cyclotides are small disulfide-rich peptides found in plants of the coffee (Rubiaceae) and violet (Violaceae) families (4). They are typically about 30 amino acids in length and have the unique structural features of a cyclic backbone and a knotted arrangement of three-disulfide bonds, referred to as the cyclic cystine knot motif (5). Their compact cyclic cystine knot motif makes them exceptionally resistant to thermal, chemical, or enzymatic degradation (6). Cyclotides exhibit a range of biological activities, including anti-bacterial, cytotoxic, and anti-human immunodeficiency virus activities (7), but their natural function is as plant defense molecules (8, 9). Kalata B1, from the Rubiaceae species Oldenlandia affinis, was the first cyclotide discovered (10), although its macrocyclic structure was not delineated until 1995 (11). So far, the sequences of nearly 100 cyclotides have been reported, and it has been suggested that they may surpass the well known plant defensins in number and diversity (12, 13). Their unique structural framework, range of bioactivities, and sequence diversity make them interesting targets for pharmaceutical applications (14).Cyclotides have a characteristic surface-exposed patch of hydrophobic residues that accounts for their late elution on reverse-phase HPLC ...

“…The complete chemical synthesis of a toxin with only D-amino acids has been seldom reported. In one pioneering work, Di Luccio et al (42) reported the chemical synthesis of D-maurotoxin, a four-disulfide-bridged toxin active on potassium channels. Interestingly, the kinetic features of the in vitro oxidation/ folding of D-maurotoxin are indistinguishable from those of L-maurotoxin.…”

Section: Discussionmentioning

confidence: 99%

d-Maurocalcine, a Pharmacologically Inert Efficient Cell-penetrating Peptide Analogue

Poillot

Dridi

Bichraoui

et al. 2010

Self Cite

Maurocalcine has been the first demonstrated animal toxin acting as a cell-penetrating peptide. Although it possesses competitive advantages, its use as a cell-penetrating peptide (CPP) requires that analogues be developed that lack its characteristic pharmacological activity on ryanodine-sensitive calcium channels without affecting its cell-penetrating and vector efficiencies. Here, we present the synthesis, three-dimensional 1 H NMR structure, and activity of D-maurocalcine. We demonstrate that it possesses all of the desired features for an excellent CPP: preserved structure, lack of pharmacological action, conserved vector properties, and absence of cell toxicity. This is the first report of a folded/oxidized animal toxin in its D-diastereomer conformation for use as a CPP. The protease resistance of this new peptide analogue, combined with its efficient cell penetration at concentrations devoid of cell toxicity, suggests that D-maurocalcine should be an excellent vector for in vivo applications.