A Novel Post-translational Modification Involving Bromination of Tryptophan

Abstract

We report a novel post-translational modification involving halogenation of tryptophan in peptides recovered from the venom of carnivorous marine cone snails (Conus). The residue, L-6-bromotryptophan, was identified in the sequence of a heptapeptide, isolated from Conus imperialis, a worm-hunting cone. This peptide does not elicit gross behavioral symptoms when injected centrally or peripherally in mice. L-6-Bromotryptophan was also identified in a 33-amino acid peptide from Conus radiatus; this peptide has been shown to induce a sleep-like state in mice of all ages and is referred to as bromosleeper peptide. The sequences of the two peptides Pca-Cys-Gly-Gln-Ala-Trp*-Cys-NH 2 were determined using a combination of mass spectrometry, amino acid, and chemical sequence analyses, where Pca ‫؍‬ pyroglutamic acid, Hyp ‫؍‬ hydroxyproline, Gla ‫؍‬ ␥-carboxyglutamate, and Trp* ‫؍‬ L-6-bromotryptophan. The precise structure and stereochemistry of the modified residue were determined as L-6-bromotryptophan by synthesis, co-elution, and enzymatic hydrolysis experiments. To our knowledge this is the first documentation of tryptophan residues in peptides/proteins being modified in a eukaryotic system and the first report of halogenation of tryptophan in vivo. SEQUENCE 1 andPolypeptides encoded by genes are primarily made up of the 20 common amino acids that are directly translated using the genetic code. However, many of these amino acids can be further modified post-translationally to yield a set of additional amino acids that contribute to the function of the mature protein. Together the 20 primary amino acids and these "secondary" amino acids which are found in proteins comprise the set of proteinogenous amino acids.These modified amino acids include amino acid conjugates where the side chain is linked to a glycosyl, phosphate, or sulfate group and amino acids such as 5-hydroxylysine, 4-hydroxyproline, and ␥-carboxyglutamate. One notable set of modified amino acids are halogenated derivatives of tyrosine and histidine. Naturally occurring halogenated tyrosine and histidine residues have previously been identified from proteins (1-4). A particularly well documented example of in vivo posttranslational halogenation of an amino acid residue in a protein is afforded by thyroglobulin (5). After iodination of several tyrosine residues, selective cleavages release the iodinated thyroid hormones thyroxine (3,5,3Ј,5Ј-tetraiodothyronine or T 4 ) 1 ; 3,5,3Ј-triiodothyronine (T 3 ), and 3,3Ј-di-iodothyronine. The presence of free T 3 and T 4 has also been shown in protochordates, suggesting a primitive thyroid function exists in tunicates (2).Of the 20 common amino acids, alanine, glycine, isoleucine, leucine, and valine, which lack side chain functional groups, have not been implicated in post-translational modifications (6). In 1907 it was proposed that hydroxytryptophan was a proteinogenous amino acid (7), but this proposal has not been verified (1), and chemical oxidation of tryptophan is likely to be responsible for the observed ...

α-Conotoxin EpI, a Novel Sulfated Peptide from Conus episcopatusThat Selectively Targets Neuronal Nicotinic Acetylcholine Receptors

“…The crystal structure of [Tyr 15 ]EpI and a detailed comparison with PnIA and PnIB are reported elsewhere. 2 The activities of synthetic EpI and its unsulfated analogue [Tyr 15 ]EpI were similar. Both caused competitive inhibition of nicotine-induced catecholamine release in bovine adrenal chromaffin cells (predominantly neuronal ␣3␤4 nAChRs (46)) but neither had an effect on the rat phrenic nerve-hemidiaphragm (muscle nAChRs).…”

“…The recent finding of Kulak et al (14) shows that ␣-conotoxins with appropriate nAChR-subtype specificity have potential as novel therapeutics. Given that its structure is now well defined, 2 EpI provides a useful template for the design of selective small molecule inhibitors of neuronal nAChRs.…”

“…15 ]EpI-Solid phase chemical synthesis of EpI was undertaken to confirm the proposed structure of EpI and to provide sufficient material for further biological (present study) and structural characterization. 2 The cleavage conditions selected to remove reduced EpI from resin were a compromise between achieving adequate removal of the side chain protecting groups and preventing undesirable desulfation. Problems were encountered with the incomplete removal of the tBu and Pmc protecting groups.…”

“…Following a 3-day incubation at 37°C in an atmosphere of 10% carbon dioxide, the culture was allowed to equilibrate to room temperature for 5 min. To remove the incubation medium, the culture was washed with modified Locke's buffer of the following composition in mM: NaCl, 154; KCl, 2.6; CaCl 2 10; and bovine serum albumin, 0.5%. The cells were preincubated in the presence or absence of synthetic conotoxin (0 -10 M) for 5 min at room temperature and then incubated for 5 min with 4 M nicotine Ϯ conotoxin.…”

Characterization of a family of endogenous neuropeptide ligands for the G protein-coupled receptors GPR7 and GPR8

“…Other than this example, to our knowledge, no posttranslationally brominated polypeptides have been reported in terrestrial animals. Some peptides from the venom of carnivorous marine cone snails, such as -conotoxin, are shown to have brominated tryptophan(s) (22)(23)(24)(25). Like NPB, brominations on these peptides of conus venom were found at the C-6-position of the indole moiety.…”