Olga Buczek

Conotoxins and the posttranslational modification of secreted gene products

Abstract.The venoms of predatory cone snails (genus Conus) have yielded a complex library of about 50–100,000 bioactive peptides, each believed to have a specific physiological target (although peptides from different species may overlap in their target specificity). Conus has evolved the equivalent of a drug development strategy that combines the accelerated evolution of toxin sequences with an unprecedented degree of posttranslational modification. Some Conus venom peptide families are the most highly posttranslationally modified classes of gene products known. We review the variety and complexity of posttranslational modifications documented in Conus peptides so far, and explore the potential of Conus venom peptides as a model system for a more general understanding of which secreted gene products may have modified amino acids. Although the database of modified conotoxins is growing rapidly, there are far more questions raised than answers provided about possible mechanisms and functions of posttranslational modifications in Conus.

Characterization of D‐amino‐acid‐containing excitatory conotoxins and redefinition of the I‐conotoxin superfamily

Post‐translational isomerization of l‐amino acids to d‐amino acids is a subtle modification, not detectable by standard techniques such as Edman sequencing or MS. Accurate predictions require more sequences of modified polypeptides. A 46‐amino‐acid‐long conotoxin, r11a, belonging to the I‐superfamily was previously shown to have a d‐Phe residue at position 44. In this report, we characterize two related peptides, r11b and r11c, with d‐Phe and d‐Leu, respectively, at the homologous position. Electrophysiological tests show that all three peptides induce repetitive activity in frog motor nerve, and epimerization of the single amino acid at the third position from the C‐terminus attenuates the potency of r11a and r11b, but not that of r11c. Furthermore, r11c (but neither r11a nor r11b) also acts on skeletal muscle. We identified more cDNA clones encoding conopeptide precursors with Cys patterns similar to r11a/b/c. Although the predicted mature toxins have the same cysteine patterns, they belong to two different gene superfamilies. A potential correlation between the identity of the gene superfamily to which the I‐conotoxin belongs and the presence or absence of a d‐amino acid in the primary sequence is discussed. The great diversity of I‐conopeptide sequences provides a rare opportunity for defining parameters that may be important for this most stealthy of all post‐translational modifications. Our results indicate that neither the chemical nature of the side chain nor the precise vicinal sequence around the modified residue seem to be critical, but there may be favored loci for isomerization to a d‐amino acid.

Oxidative folding of conotoxins sharing an identical disulfide bridging framework

Conotoxins are short, disulfide‐rich peptide neurotoxins produced in the venom of predatory marine cone snails. It is generally accepted that an estimated 100 000 unique conotoxins fall into only a handful of structural groups, based on their disulfide bridging frameworks. This unique molecular diversity poses a protein folding problem of relationships between hypervariability of amino acid sequences and mechanism(s) of oxidative folding. In this study, we present a comparative analysis of the folding properties of four conotoxins sharing an identical pattern of cysteine residues forming three disulfide bridges, but otherwise differing significantly in their primary amino acid sequence. Oxidative folding properties of M‐superfamily conotoxins GIIIA, PIIIA, SmIIIA and RIIIK varied with respect to kinetics and thermodynamics. Based on rates for establishing the steady‐state distribution of the folding species, two distinct folding mechanisms could be distinguished: first, rapid‐collapse folding characterized by very fast, but low‐yield accumulation of the correctly folded form; and second, slow‐rearrangement folding resulting in higher accumulation of the properly folded form via the reshuffling of disulfide bonds within folding intermediates. Effects of changing the folding conditions indicated that the rapid‐collapse and the slow‐rearrangement mechanisms were mainly determined by either repulsive electrostatic or productive noncovalent interactions, respectively. The differences in folding kinetics for these two mechanisms were minimized in the presence of protein disulfide isomerase. Taken together, folding properties of conotoxins from the M‐superfamily presented in this work and from the O‐superfamily published previously suggest that conotoxin sequence diversity is also reflected in their folding properties, and that sequence information rather than a cysteine pattern determines the in vitro folding mechanisms of conotoxins.

Erratum: Characterization of D-amino-acid-containing excitatory conotoxins and redefinition of the I-conotoxin superfamily (FEBS Journal (2005) 272 (4178-4188))

Table 3. legend should read: Amino acid sequences of I-superfamily peptides determined from cDNA clones. Peptides with the following precursor consensus sequence: MKLCVTFLLVLMILPSVTG ⁄EKSSERTLSGALLRGVKRR; defined as the I1 superfamily (Group A and B). Peptides with the following precursor consensus sequence: MMFRVTSVGCFLLVIVFLNLVVLTDA; defined as the I2 superfamily (Group C). O, 4-trans-hydroxyproline; F, d-phenylalanine; L, d-leucine; ˆ , C-terminal free acid; c, c-carboxyglutamate; *, C-terminal amidation. The underlined parts of sequences are presumed to be post-translationally cleaved by carboxypeptidase. Underlined l-amino acids are presumed to be post-translationally modified to the corresponding d-isomers, as in the case of r11a, r11b and r11c (see Table 1) encoded by cDNA clones R11.6, R11.14 and R11.4, respectively. It is likely that the C-terminal Arg residue in Fi11.1, M11.1 and S11.2 will be hydrolyzed by carboxypeptidase.