Role of Hydroxyprolines in the in Vitro Oxidative Folding and Biological Activity of Conotoxins

Abstract

Hydroxylation of proline residue occurs in specific peptides and proteins derived from plants and animals, but the functional role of this modification has been characterized primarily in collagen. Marine cone snails produce disulfide-rich peptides that have undergone a plethora of posttranslational modifications, including proline hydroxylation. Although Conus snails extensively utilize proline hydroxylation, the consequences of this modification remain largely unexplored. In this work, we investigated the function of 4-hydroxyproline (Hyp) in conotoxins from three distinct gene families: mu-, omega-, and alpha-conotoxins. Analogues of mu-GIIIA, omega-MVIIC, alpha-GI, and alpha-ImI were synthesized with either Pro or Hyp, and their in vitro oxidative folding and biological activity were characterized. For GIIIA, which naturally contains three Hyp residues, the modifications improved the ability to block NaV1.4 sodium channels but did not affect folding. In contrast, the presence of Hyp in MVIIC had a significant impact on the oxidative folding but not on the biological activity. The folding yields for the MVIIC[Pro7Hyp] analogue were approximately 2-fold higher than for MVIIC under a variety of optimized oxidation conditions. For alpha-conotoxins ImI and GI, the hydroxylation of the conserved Pro residue improved their folding but impaired their activities against target receptors. Since prolyl-4-hydroxylase and protein disulfide isomerase coexist as a heterotetramer in the ER, we discuss the effects of Hyp on the folding of conotoxins in the context of cis-trans isomerization of Pro and Hyp. Taken together, our data suggest that proline hydroxylation is important for both in vitro oxidative folding and the bioactivity of conotoxins.

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

“…For the -conotoxin GIIIA, the modification enhances bioactivity but does not affect folding. In contrast, Pro hydroxylation impairs activity but improves folding yields in the two ␣-conotoxins ImI and GI, which naturally contain a conserved Pro residue (18). These findings emphasize the importance of Pro hydroxylation in the structure and bioactivity of conotoxins.…”

“…Folding reactions were analyzed by reversed-phase HPLC on a C18 column (Vydac, 5 m particle size, 4.6 ϫ 250 mm; Grace) under the conditions described above. Native peptides were distinguished from linear peptides and other forms on the basis of characteristic HPLC elution profiles (18,47), mass spectrometric analyses of manually collected reversed-phase fractions (MALDI-TOF mass spectrometer, Ultraflex II in positive reflector mode, Bruker Daltonics, Bremen, Germany), and for the GIIIA experiments coelution with chemically synthesized biologically active GIIIA (18) (supplemental Figs. 1 and 2).…”

“…yields (18). Hydroxylation of Pro leads to a 2-fold increase in folding yields, whereas the neurotoxic activity of the toxin is maintained.…”

“…All three peptides had been studied previously in detail with respect to their oxidative folding properties (18,53). GIIIA is 22 amino acids in length with a C-terminal amidation and six cysteines forming three disulfide bonds.…”

“…The -conotoxin MVIIC is 26 amino acids in length, C-terminally amidated, and contains six cysteine residues forming three disulfide bonds (Table 2). MVIIC, which contains one proline residue in position 7, is likely to adopt both cis and trans conformations (18). However, as the peptide sequence was originally deduced from cDNA sequence information, hydroxylation of this proline residue may occur in vivo.…”