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

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 -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.

Differential gene expression in Pyropia columbina (Bangiales, Rhodophyta) under natural hydration and desiccation conditions

“…These proteins catalyze the cis-trans isomerisation of prolines (C 5 H 9 NO 2 ), and have been described in the processes of cellular signalling (with a calmodulin-binding domain), regulation of gene transcription, and acting as chaperones and folding catalysts. Specifically, in the gastropod Conus novaehollandiae these proteins facilitate the oxidative folding of several neurotoxic peptides (Safavi-Hemami et al, 2010). In plants, these proteins are involved in flowering (Wang et al, 2010) and in controlling cell proliferation, since PPIase expression increases in the presence of cytokinin (Vittorioso et al, 1998).…”

Modulation of Conotoxin Structure and Function Is Achieved through a Multienzyme Complex in the Venom Glands of Cone Snails

“…Primary reverse transcription PCR (RT-PCR) was performed in volumes of 50 l containing 4 l of cDNA (200 ng), 0.5 l of Velocity Taq DNA polymerase (Bioline), 1ϫ PCR buffer (Bioline), 200 M each deoxynucleoside triphosphate (dNTPs; Invitrogen), and 0.2 M PDI sense (CGA CCA TAT GGA TGA TAT CAA ACA GGA GGA A) and antisense oligonucleotide primers (CGC TCG AGC AGT TCA TCT CTT GGC AGA TC). PCR amplicons were analyzed by gel electrophoresis, cloned, and sequenced as described previously (17). The novel Conus PDI sequence was deposited in GenBank TM (National Center for Biotechnology Information, United States National Library of Medicine, Bethesda, MD).…”

“…Cloning, Expression, and Purification of Recombinant Enzymes-Conus PPI B was expressed and purified as described previously (17). Conus PDI and BiP lacking the N-terminal signal sequences were cloned into the pET22bϩ and pET30cϩ expression vector, respectively (Novagen).…”

“…Although PDI and PPI have been identified in the venom gland of Conus (16) and are known to play a role in the in vitro folding of conotoxins (17,18), their combined effect has not been investigated. We recently sequenced Conus BiP from the venom gland of Conus novaehollandiae and demonstrated it to be highly expressed in the venom glandular cells (16).…”

Embryonic Toxin Expression in the Cone Snail Conus victoriae

“…To rule out false amplification of genomic DNA, a negative control was performed using a reverse transcription reaction from which the enzyme reverse transcriptase was excluded. Nested PCRs were performed as described above except 2 l of the 1:5 diluted primary PCR was used as DNA template, and oligonucleotides were replaced with 0.2 M of nested oligonucleotides (supplemental Table 1), and the annealing temperature was reduced to 43°C for 30 s. All PCR amplicons were analyzed by gel electrophoresis, cloned into pGEM-T plasmid vectors (Promega), and subsequently sequenced as described previously (27). All sequences analyzed in this study were deposited in GenBank TM (National Center for Biotechnology Information, National Library of Medicine, Bethesda).…”