Role of hydroxyprolines in the in vitro oxidative folding and biological activity of conotoxins.

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.