Bromocontryphan:  Post-Translational Bromination of Tryptophan

Ec, Jimenez; Ag, Craig; Watkins, Maren; Dr, Hillyard; Wr, Gray; Gulyás, József; Je, Rivier; Lj, Cruz; Olivera, B.M.

doi:10.1021/bi962840p

Cited by 117 publications

(79 citation statements)

References 17 publications

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“…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.…”

Section: Discussionmentioning

confidence: 99%

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

Tanaka

Yoshida

Miyamoto

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

181

217

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GPR7 and GPR8 are orphan G protein-coupled receptors that are highly similar to each other. These receptors are expressed predominantly in brain, suggesting roles in central nervous system function. We have purified an endogenous peptide ligand for GPR7 from bovine hypothalamus extracts. This peptide, termed neuropeptide B (NPB), has a C-6-brominated tryptophan residue at the N terminus. It binds and activates human GPR7 or GPR8 with median effective concentrations (EC50) of 0.23 nM and 15.8 nM, respectively. In situ hybridization shows distinct localizations of the prepro-NPB mRNA in mouse brain, i.e., in paraventricular hypothalamic nucleus, hippocampus, and several nuclei in midbrain and brainstem. Intracerebroventricular (i.c.v.) injection of NPB in mice induces hyperphagia during the first 2 h, followed by hypophagia. Intracerebroventricular injection of NPB produces analgesia to s.c. formalin injection in rats. Through EST database searches, we identified a putative paralogous peptide. This peptide, termed neuropeptide W (NPW), also has an N-terminal tryptophan residue. Synthetic human NPW binds and activates human GPR7 or GPR8 with EC 50 values of 0.56 nM and 0.51 nM, respectively. The expression of NPW mRNA in mouse brain is confined to specific nuclei in midbrain and brainstem. These findings suggest diverse physiological functions of NPB and NPW in the central nervous system, acting as endogenous ligands on GPR7 and͞or GPR8.T here are a large number of orphan G protein-coupled receptors (GPCR) whose cognate ligands have yet to be identified (1, 2). The search for endogenous ligands of orphan GPCRs is important because GPCRs are in general excellent drug targets (3). GPR7 and GPR8 are two orphan GPCRs (4). They were originally cloned from human genomic DNA by degenerative PCR using primers based on the sequences of the ␦-opioid receptor and somatostatin receptor. Indeed, GPR7 and GPR8 each share Ϸ40% overall amino acid identities with opioid and somatostatin receptors. Human GPR7 and GPR8 are 70% identical to each other at the nucleotide level and 64% identical at the amino acid level. Interestingly, no orthologue exists for the GPR8 gene in rodents, indicating that GPR8 may have originated as a replicate of GPR7 after divergence of the rodent from other species in mammalian evolution (5).The observation that GPR7 and GPR8 mRNAs are expressed in distinct areas in the central nervous system (CNS) (4, 5), together with the similarity of GPR7 and GPR8 with opioid and somatostatin receptors, strongly argue for the existence of endogenous peptide ligand(s) in CNS. In this study, we have purified a neuropeptide ligand for GPR7 from the bovine hypothalamus. This peptide, termed neuropeptide B (NPB), consists of 29 aa with a unique brominated N-terminal tryptophan. Through EST database searches, we also identified another isopeptide of the same family, termed neuropeptide W (NPW). While this manuscript was being prepared, three papers were published reporting two endogenous ligands for GPR7 and GPR8 (6-8). Sequ...

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Section: Discussionmentioning

confidence: 99%

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

Tanaka

Yoshida

Miyamoto

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

181

217

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“…The resulting product was transferred into competent DH5␣ cells as described (4). The nucleic acid sequences of the resulting clones were determined according to the standard protocol for Sequenase Version 2.0 DNA Sequencing kit as described (5).…”

Section: Methodsmentioning

confidence: 99%

α-Conotoxin BuIA, a Novel Peptide from Conus bullatus, Distinguishes among Neuronal Nicotinic Acetylcholine Receptors

Azam

Dowell

Watkins

et al. 2005

Journal of Biological Chemistry

Self Cite

105

117

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Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels. ␣ Subunits, together with ␤2 and/or ␤4 subunits, form ligand-binding sites at ␣/␤ subunit interfaces. Predatory marine snails of the genus Conus are a rich source of nAChR-targeted peptides. Using conserved features of the ␣-conotoxin signal sequence and 3 -untranslated sequence region, we have cloned a novel gene from the fisheating snail, Conus bullatus; the gene codes for a previously unreported ␣-conotoxin with unusual 4/4 spacing of amino acids in the two disulfide loops. Chemical synthesis of the predicted mature toxin was performed. The resulting peptide, ␣-conotoxin BuIA, was tested on cloned nAChRs expressed in Xenopus oocytes. The peptide potently blocks numerous rat nAChR subtypes, with highest potency for ␣3-and chimeric ␣6-containing nAChRs; BuIA blocks ␣6/␣3␤2 nAChRs with a 40,000-fold lower IC 50 than ␣4␤2 nAChRs. The kinetics of toxin unblock are dependent on the ␤ subunit. nAChRs with a ␤4 subunit have very slow off-times, compared with the corresponding ␤2 subunit-containing nAChR. In each instance, rat ␣x␤4 may be distinguished from rat ␣x␤2 by the large difference in time to recover from toxin block. Similar results are obtained when comparing mouse ␣3␤2 to mouse ␣3␤4, and human ␣3␤2 to human ␣3␤4, indicating that the ␤ subunit dependence extends across species. Thus, ␣-conotoxin BuIA also represents a novel probe for distinguishing between ␤2-and ␤4-containing nAChRs.

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“…Further confirmation of the presence of a sulfated amino acid being present in EpI was obtained using a high declustering potential (-220 V) and negative ion detection. The resultant mass spectrum revealed the presence of characteristic sulfate ions, SO 3 Ϫ and SO 4 Ϫ at m/z 80 and 96, respectively (data not shown). Comparison of the spectra of EpI to those of CCK-8 (a sulfated peptide) and MLP 111 (a phosphorylated peptide) confirmed that EpI was sulfated (data not shown).…”

Section: Isolation and Chemical Characterization Of Epimentioning

confidence: 99%

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

Loughnan

Bond

Atkins

et al. 1998

Journal of Biological Chemistry

109

100

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We have isolated and characterized ␣-conotoxin EpI, a novel sulfated peptide from the venom of the molluscivorous snail, Conus episcopatus. The peptide was classified as an ␣-conotoxin based on sequence, disulfide connectivity, and pharmacological target. EpI has homology to sequences of previously described ␣-conotoxins, particularly PnIA, PnIB, and ImI. However, EpI differs from previously reported conotoxins in that it has a sulfotyrosine residue, identified by amino acid analysis and mass spectrometry. Native EpI was shown to coelute with synthetic EpI. The peptide sequence is consistent with most, but not all, recognized criteria for predicting tyrosine sulfation sites in proteins and peptides.

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Bromocontryphan: Post-Translational Bromination of Tryptophan

Cited by 117 publications

References 17 publications

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

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

α-Conotoxin BuIA, a Novel Peptide from Conus bullatus, Distinguishes among Neuronal Nicotinic Acetylcholine Receptors

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

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