Abstract

The oxidative folding of large polypeptides has been investigated in detail; however, comparatively little is known about the enzyme-assisted folding of small, disulfide-containing peptide substrates. To investigate the concerted effect of multiple enzymes on the folding of small disulfide-rich peptides, we sequenced and expressed protein-disulfide isomerase (PDI), peptidyl-prolyl cis-trans isomerase, and immunoglobulin-binding protein (BiP) from Conus venom glands. Conus PDI was shown to catalyze the oxidation and reduction of disulfide bonds in two conotoxins, α-GI and α-ImI. Oxidative folding rates were further increased in the presence of Conus PPI with the maximum effect observed in the presence of both enzymes. In contrast, Conus BiP was only observed to assist folding in the presence of microsomes, suggesting that additional co-factors were involved. The identification of a complex between BiP, PDI, and nascent conotoxins further suggests that the folding and assembly of conotoxins is a highly regulated multienzyme-assisted process. Unexpectedly, all three enzymes contributed to the folding of the ribbon isomer of α-ImI. Here, we identify this alternative disulfide-linked species in the venom of Conus imperialis, providing the first evidence for the existence of a "non-native" peptide isomer in the venom of cone snails. Thus, ER-resident enzymes act in concert to accelerate the oxidative folding of conotoxins and modulate their conformation and function by reconfiguring disulfide connectivities. This study has evaluated the role of a number of ER-resident enzymes in the folding of conotoxins, providing novel insights into the enzyme-guided assembly of these small, disulfide-rich peptides.

Highlights

  • Conotoxins can be utilized to investigate enzyme-assisted folding of disulfide-rich peptides

  • The in vitro interaction between another protein-disulfide isomerase (PDI) family member, PDIA3, and binding protein (BiP) was shown to improve folding rates of ribonuclease B and ␣-lactalbumin [12]. These findings indicate that members of the PDI family can recruit or are themselves recruited by other ER-resident enzymes and chaperones, such as BiP and/or peptidyl-prolyl cis-trans isomerase (PPI) B, to cooperatively act in the oxidative folding of at least some client substrates

  • Our findings indicate that the proper assembly of conotoxins relies upon the action of a number of ER-resident enzymes, including PDI, PPI B, and BiP

Read more

Summary

Background

Conotoxins can be utilized to investigate enzyme-assisted folding of disulfide-rich peptides. Oxidative folding rates of maurotoxin, a disulfide-rich peptide from scorpion venom, were highest in the presence of PDI and FKBP-12, a PPI isoform located in the cytosol [8] Another enzyme known to cooperate with PDI in the folding of disulfide-containing proteins is BiP, a member of the heat shock protein 70 (Hsp70) family. Our findings indicate that the proper assembly of conotoxins relies upon the action of a number of ER-resident enzymes, including PDI, PPI B, and BiP These enzymes appear to modulate the disulfide isomers of conotoxins, a process that generates additional conformational diversity and has the potential to enhance the functional diversity of these molecules

EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call