Eur. J. Biochem. 264, 271±275 (1999) q FEBS 1999

M I N I R E V I E W

Post-translationally modified neuropeptides from Conus venoms

A. Grey Craig1, Pradip Bandyopadhyay2 and Baldomero M. Olivera2

1The Clayton Foundation Laboratory for Peptide Biology, The Salk Institute, La Jolla, CA, USA; 2Department of Biology, University of Utah,

Salt Lake City, USA

Predatory cone snails (genus Conus) comprise what is arguably the largest living genus of marine animals (500

species). All Conus use complex venoms to capture prey and for other biological purposes. Most biologically

active components of these venoms are small disulfide-rich peptides, generally 7±35 amino acids in length. There

are probably of the order of 100 different peptides expressed in the venom of each of the 500 Conus species [1,2].

Peptide sequences diverge rapidly between Conus species, resulting in a distinct peptide complement for each

species. Thus, the genus as a whole has probably generated < 50 000 different peptides, which can be organized

into families and superfamilies with shared sequence elements [3]. In this minireview, we provide a brief

overview of the neuropharmacological, molecular and cell-biological aspects of the Conus peptides. However, the

major focus of the review will be the remarkable array of post-translational modifications found in these peptides.

Keywords: post-translational modification; Conus; neuropeptide toxins; g-carboxyglutamate; bromotryptophan.

O V E R V I E W O F N E U R O P H A R M A C O L O G Y :C O N U S P E P T I D E S A S L I G A N D S F O R I O NC H A N N E L S A N D R E C E P T O R S

Several Conus peptides are widely used research tools inneuroscience, and some are potential therapeutic agents. Theirutility is based on an unprecedented selectivity in targetingspecific molecular forms of either voltage-gated or ligand-gatedion channels (and in a few cases, G-protein linked receptors).

Over tens of millions of years, cone snails have evolved anelegant strategy for efficiently undermining diverse nervoussystems in prey, predators and competitors. A Conus peptidefamily generally targets a specific family of ion channels(i.e. a-conotoxins target nicotinic acetylcholine receptors,v-conotoxins target voltage-gated calcium channels). A listof Conus peptide families and the general ion channel familiesthey target is shown in Table 1. However, when differentmembers of a Conus peptide family (for example, differenta-conotoxins) are present in a single Conus venom, each isspecific for a different molecular form of the targeted ionchannel family. Several members of the a-conotoxin familyare known to target different members of the nicotinicreceptor family [4].

Peptides in the same family share both a common disulfidearrangement, as well as homologous target sites. Thus,v-conotoxins and d-conotoxins share the same arrange-ment of cysteines, but they do not have homologousphysiological target sites. Conversely, k-and kA-conotoxinsboth target K+ channels but have different disulfide patterns.

O V E R V I E W O F M O L E C U L A R A N DC E L L U L A R B I O L O G Y : C O N U S P E P T I D E SA S G E N E P R O D U C T S

Conus peptide precursors have been elucidated by cDNAcloning. The mRNA of each Conus peptide is initiallytranslated as a prepropeptide in which the biologically activepeptide is located at the C-terminus in single copy. Theprecursor has a typical signal sequence (the `pre-region') and avariable spacer (`pro-region'). A standard signal for proteolysis(XR) is generally found immediately before the mature peptidesequence. Members of a Conus peptide family not only exhibita similar arrangement of cysteine residues in the maturepeptide, but also show sequence similarity in both pre- and pro-regions. Particularly noteworthy is the extreme conservation ofsignal sequences within all members of a Conus peptidesuperfamily. The diversity of peptides in different Conusspecies is apparently generated by hypermutation of theC-terminal mature peptide region. The contrast between thestrikingly conserved signal sequences and the hypermutatedmature peptides is the most extreme example observed to datefor homologous gene products within a single genus oforganisms.

Presumably, after translation by ribosomes, the polypeptideprecursor enters the endoplasmic reticulum where furtherprocessing, including the specific proteolysis, must occur. Inaddition, several other post-translational modifications maytake place. Some are relatively common, and are found inpeptides from many other systems (such as C-terminalamidation and disulfide bond formation). However, manyConus peptides have unusual post-translational modificationsand some (such as post-translational bromination of Trp) werefirst discovered in Conus peptides. The post-translationalmodifications found in Conus peptides to date are listed inTable 2.

Post-translational modification of Conus peptides probablyrequires both specialized enzyme machinery as well as somerecognition signal sequence (or alternatively a characteristicstructural feature) in the peptide precursor to instruct the

Correspondence to B. M. Olivera, University of Utah, Department of

Biology, 201 Sth Building, Salt Lake City, Utah 84112-0840.

Fax: + 1 801 585 5010, Tel.: + 1 801 581 8370,

E-mail: oliveralab@bioscience.utah.edu

Abbreviations: Gla, g-carboxyglutamic acid; ACh, acetylcholine; 5HT3,

5-hydroxytryptamine type 3; i.c.v., intracerbral ventricular; NMDA,

N-methyl-d-aspartate.

(Received 17 February 1998, accepted 9 March 1999)

272 A. Grey Craig et al. (Eur. J. Biochem. 264) q FEBS 1999

enzymatic machinery as to which amino acid(s) to modify. Inalmost all cases, neither the enzymatic machinery nor therecognition signals have been elucidated.

g - C A R B O X Y L A T I O N O F G L U T A M A T ER E S I D U E S

The vitamin K-dependent g-carboxylation of glutamate resi-dues to g-carboxyglutamate (Gla) was originally characterizedin blood clotting factors such as prothrombin [5], and inproteins involved in mammalian bone metabolism [6].Recently, a novel mammalian g-carboxylated protein gas6,with a suggested role in the cell growth control [7] has beencharacterized [8].

The discovery of g-carboxylation in a Conus peptide,conantokin-G [9] was completely unexpected because it hadbeen thought that vitamin K-dependent g-carboxylation wasrestricted to higher vertebrates. A surprisingly large numberof Conus venom peptides contain Gla (< 10%) [10]. Inpreliminary experiments, many mechanistic features of vitaminK-dependent post-translational modification in the molluscansystem have exhibited striking homology to those of themammalian enzyme [11].

The role of Gla in Conus peptides has been best defined inthe conantokin family, which are N-methyl-d-aspartate(NMDA) receptor antagonists. It was originally suggestedthat Gla may play essentially the same role for conantokins asthat defined for blood clotting factors in mammals, i.e. that thepresence of Gla residues promoted formation of an a-helix[12]. This suggestion has been amply confirmed by NMRstudies (although there is some uncertainty about the proportionof a-helix and 3/10 helix) [13,14].

Vitamin K-dependent carboxylation is the only Conus post-translational modification for which a recognition signal hasbeen directly demonstrated [15]. The 21 to 220 region of theconantokin-G precursor has been shown to greatly increase theaffinity of a potential substrate for the g-carboxylation enzymefrom Conus venom ducts. These sequences can be regarded asg-carboxylation recognition signals (g-CRSs) for the Conusenzyme. Propeptide sequence 225 to 21 of another Conuspeptide, the bromosleeper, can also serve as a g-carboxylationrecognition signal for the Conus enzyme (B. Cook, B. M.

Olivera & P. Bandyopadhyay, unpublished results). g-CRSsequences of conantokin-G and bromosleeper are highlydissimilar, and in turn differ from g-CRS sequences of themammalian g-carboxylated peptides.

P O S T- T R A N S L A T I O N A L E P I M E R I Z A T I O NO F A N L T O A D A M I N O A C I D

An unusual modification in Conus peptides is the post-translational conversion of an l to a d amino acid which hasbeen found in one Conus peptide family, the contryphans.These are small peptides (7±9 amino acids) with a singledisulfide linkage. In most contryphans, one of two Trp residuesis epimerized from an l-Trp to a d-Trp [16]. The biologicalsignificance of this post-translational modification is notknown. Recently, an unusual contryphan, Leu-contryphan-Phas been shown to contain d-Leu instead of d-Trp.

Post-translational conversion of l to d amino acids has beendescribed in other biological systems, most notably in frog skinpeptides such as the deltorphins (opiate analogs). An enzymecatalyzing the post-translational epimerization to a d aminoacid in the spider toxin v-agatoxin IVB has been reported. Areview of post-translational epimerization has been recentlypublished [17].

S U L F A T I O N O F T Y R O S I N E R E S I D U E S

Sulfotyrosine was identified in the a-conotoxin EpI, isolatedfrom C. epicoscopatus venom [18] using amino acid analysisand mass spectrometric techniques. Several bioactive sulfatedpeptides are known in other systems such as caerulein [19],gastrin [20] and cholecystokinin [21,22]; where the hydroxylgroup of tyrosine is sulfated to sulfotyrosine by a tyrosylsulfotransferase. Prior to the identification of a-EpI, a sulfatedresidue had also been proposed as a possible explanation for ananalog of a-conotoxin PnIB; based on the analog's observedmass being 80 Da higher than that of PnIB [23].

An aspartate present in the 21 position of both EpI and PnIBand a proline residue at the 22 position are consistent withproposed consensus features for tyrosine sulfation [24,25]. It is,however, unusual to have a cysteine residue in the +1 positionas is the case for both EpI and PnIB [26]. The potency of both

Table 1. Conus peptide families with defined targets.

Conus peptide family Target Mode of action Cys pattern Reference

Peptide families targeted to ligand-gated ion channels

a-conotoxin Nicotine receptor Competitive antagonists CCÐCÐC [32]

aA-conotoxin Nicotine receptor Competitive antagonists CC±CÐCÐCÐC [33]

c-conotoxin Nicotine receptor Non-competitive antagonists CCÐC±C±CC [34]

s-conotoxin 5HT3 receptor Competitive antagonists CCÐCÐCÐCC [29]

Conantokins NMDA receptor ± Usually no disulfides [35,36]

Peptide families targeted to voltage-gated ion channels

m-conotoxin Na channels Channel blocker; Site I CCÐCÐCÐCC [37, 38]

mO-conotoxin Na channels Block conductance, not at Site I CCÐCÐCÐCC [39]

d-conotoxin Na channels Delay inactivation CCÐCÐCÐC [40,41]

kA-conotoxin K channels Inhibit conductance CCÐCÐCÐCÐC [30]

k-conotoxin K channels Channel blocker CCÐCÐCCÐCÐC [42]

v-conotoxin Ca channels Channel blocker CÐCÐCCÐCÐC [43]

Peptide families targeted to G-protein-linked receptors

Conopressin Vasopressin receptor Agonist C-C [44]

ContulakõÂn Neurotensin receptor Agonist No disulfides [45]

q FEBS 1999 Post-translationally modified neuropeptides from Conus venoms (Eur. J. Biochem. 264) 273

EpI and EpI(Tyr15) (a nonsulfated analog) were found to besimilar [18] as competitive nicotinic antagonists on bovineadrenal chromaffin cells (neuronal nicotinic ACh receptors).

B R O M I N A T I O N O F T R Y P T O P H A NR E S I D U E S

Post-translational bromination of tryptophan residues to 6-l-bromotryptophan has been demonstrated for four Conuspeptides: bromocontryphan [27], the bromosleeper peptide[28], the bromoheptapeptide [28] and s-conotoxin GVIIIA[29]. These peptides appear unrelated, and vary in size from 7to 41 amino acids with 1±5 disulfide bridges.

The presence of bromotryptophan was first suggested inConus peptides when it was observed that a number of peptidesgave no phenylthiohydantoin amino acid derivative duringchemical sequence analysis, and the observed mass wasapproximately 265 Da above that predicted. In addition, thesepeptides had intense absorption in the UV at 280 nm and anunusual intact molecule mass isotopomer distribution. Acombination of enzymatic hydrolysis, peptide synthesis andcoelution experiments were performed to identify the unknownresidue as 6-l-bromotryptophan.

No specific function has been attributed to the presence ofthe brominated tryptophan residue in any of the four knownConus peptides. However, s-conotoxin was isolated using itsactivity as a potent 5-hydroxytryptamine type 3 (5HT3)receptor antagonist as an assay [29]. The correspondingnonbrominated analog was not identified in the bioactivityscan and, based on the similarity between the structure ofserotonin and the bromotryptophan residue, it has beensuggested that the modified residue may be important foractivity.

G LY C O S Y L A T I O N O F S E R I N E A N DT H R E O N I N E R E S I D U E S

Two different families of O-linked glycopeptides have beenisolated from Conus venoms. The initial glyco-conotoxindescribed was the kA-conotoxin SIVA, which is 30 aminoacids long has a pentasaccharide O-linked to a serine residue[30]. The precise composition and stereochemistry of theglycan moiety has not been characterized.

The SIVA peptide elicits repetitive synaptic potentials in frog

nerve-muscle preparation and is presumed to act via inhibitionof voltage-gated K+ channels. The activity of the nonglycosy-lated SIVA(Ser7) analog was found to be significantly lowerthan the glycosylated SIVA peptide when administered in vivo.

ContulakõÂn-G was the second glycopeptide isolated fromConus venom to be described [31]. In contulakõÂn-G, theglycan has been characterized as the disaccharide Gal(b1! 3)Ga1NAc(a1!)Thr, using a combination of enzymaticdegradation (b-galactosidases and O-glycosidase) monitored bychromatography and mass spectrometry. The glycopeptidestructure was confirmed through chemical synthesis andcoelution of synthetic with native peptide.

Mice administered with contulakõÂn-G (intracerbral ventri-cular, i.c.v.) become very sluggish (the peptide's namecomes from the Filipino word for sluggish, `tulakõÂn'). TheC-terminus of this peptide bears significant identity toneurotensin. Both the synthetic glycosylated contulakõÂn-Gand nonglycosylated analog [contulakõÂn-G(Thr10)] have beenshown to be authentic neurotensin agonists. In vivo studiesindicate that the efficacy of the glycosylated contulakõÂn-G issignificantly greater than the nonglycosylated analog whenadministered i.c.v. into mice.

P E R S P E C T I V E S

The importance of post-translational modification for theactivity of a limited number of Conus neuropeptides has beendemonstrated. However, the detailed mechanistic role of thesemodifications in interactions of the peptide with its cognatereceptor target need to be elucidated. The rich array of post-translational modifications in Conus peptides provides animportant model for the more sophisticated design of highlysubtype-specific ligands.

A C K N O W L E D G E M E N T S

The work of the authors was supported primarily by Grant PO148677 from

the National Institute of General Medical Sciences. The work was

supported in part by the Primary Childrens Medical center Foundation,

University of Utah (P. B.), and the Foundation for Medical Research

(A. G. C.).

R E F E R E N C E S

1. Olivera, B.M., Rivier, J., Clark, C., Ramilo, C.A., Corpuz, G.P.,

Table 2. Post-translational modifications and the Conus peptides in which they were first identified. g , g-carboxyglutamate; Z, pyroglutamic acid; Y³,

tyrosine sulfate; W, d-tryptophan; W², 6-l-bromotryptophan; O, 4-trans hydroxyproline; S§, Hex3HexNAc2-Ser; * indicates amidated C-terminus.

Modification Peptide Sequence Enzyme Reference

Disulfide bridge formation GI ECCNPACGRHYSC* Disulfide isomerase [32]

Hydroxylation of proline GIIIA RDCCTOOKKCKDRQCKOQRCCA* Proline hydroxylase [37]

Amidation of C-terminus MI GRCCHPACGKNYSC* Protein amidating

monooxygenase

[46]

Carboxylation of glutamic acid Conantokin-G GEggLQVNQgLIRgKSN* g-Glutamate

carboxylase

[9]

Bromination of tryptophan Bromocontryphan GCOwEPW²C* Bromo peroxidase [27]

Isomerization of tryptophan Contryphan GCOwEPWC* Tryptophan epimerase [16]

Cyclization of N-terminal Gln Bromoheptapeptide ZCGQAW²C* Glutaminyl cyclase [28]

Sulfation of tyrosine EpI GCCSDPRCNMNNPY³C* Tyrosyl

sulfotransferase

[18]

O-glycosylation SIVA ZKSLVPS§VITTCCGYDOGTMCOOCRCTNSC* Polypeptide

HexNAc transferase

[30]

274 A. Grey Craig et al. (Eur. J. Biochem. 264) q FEBS 1999

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