Identification of Conus peptidylprolyl cis-trans isomerases (PPIases) and assessment of their role in the oxidative folding of conotoxins.
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.
Department of Biochemistry and Molecular Biology, The Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, 3010 Victoria, Australia.
Helena
H
Safavi-Hemami
Grzegorz
G
Bulaj
Baldomero M
BM
Olivera
Nicholas A
NA
Williamson
Anthony W
AW
Purcell
D000818:Animals; D001483:Base Sequence; D020916:Conotoxins / Q000096:biosynthesis* / Q000235:genetics; D052078:Conus Snail / Q000235:genetics / Q000378:metabolism*; D004330:Drosophila; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000502:physiology; D010084:Oxidation-Reduction; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D019696:Peptidylprolyl Isomerase / Q000235:genetics / Q000378:metabolism*; D011392:Proline / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding
D020916:Conotoxins; D007527:Isoenzymes; D010455:Peptides; D011392:Proline; D019696:Peptidylprolyl Isomerase