Tài liệu Báo cáo khoa học: Physico-chemical characterization and synthesis of neuronally active a-conotoxins docx

MINIREVIEW Physico-chemical characterization and synthesis of neuronally active a-conotoxins Marion L. Loughnan and Paul F. Alewood Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia The high specificity of a-conotoxins for different neuronal nicotinic acetylcholine receptors makes them important probes for dissecting receptor subtype selectivity. New sequences continue to expand the diversity and utility of the pool of available a-conotoxins. Their identification andcharacterizationdependonasuiteoftechniques with increasing emphasis on mass spectrometry and micro- scale chromatography, which have benefited from recent advances in resolution and capability. Rigorous physico- chemical analysis together with synthetic peptide chemistry is a prerequisite for detailed conformational analysis and to provide sufficient quantities of a-conotoxins for activity assessment and structure–activity relationship studies. Keywords: a-conotoxins; Conus; peptide synthesis; post- translational modifications; sulfotyrosine. Classification, primary structure and biology of a-conotoxins Cone snails are a group of hunting gastropods that incapacitate their prey, which consists of worms, molluscs or fish, by envenomation. Conotoxins from the venom of cone snails are small disulfide-rich peptide toxins that act at many voltage-gated and ligand-gated ion channels. They can be grouped according to their molecular form into several superfamilies, each defined by characteristic arrange- ments of cysteine residues (not necessarily a single pattern), and characteristic highly conserved precursor signal sequence similarities. Individual conopeptide families within a superfamily are denoted by Greek letters and contain peptides that have a particular disulfide framework and target homologous sites on a particular receptor [1]. Each of the characterized conopeptides is named using a convention that indicates the activity (Greek letter), the source species from which the peptide was first isolated (Arabic letter(s)), the disulfide framework category (Roman numeral) and the order of discovery within that category (Arabic capital letter) [1]. For example a-AuIB belongs to the a-conotoxin family and was the second peptide, B, with that framework, I, isolated and reported from Conus aulicus [1,2]. The names of some conotoxins deviate from this nomenclature convention because their discovery preceded its formulation. Hence some a-conotoxin names do not conform to the alphabetical identifier system used to indicate order of discovery of peptides with a specified disulfide framework from the venom of any one species. The framework identifiers I and II are both used in reference to disulfide frameworks of the A superfamily without distinction. The A superfamily is so far comprised of the K + channel blocking jA familiy and the a and aA families, which together with the w family act at the nicotinic acetylcholine receptor (nAChR). No aAorw conopeptides have been reported to block neuronal nicotinic receptors with high affinity. Rather, they are generally muscle-specific nicotinic receptor antagonists [1]. The a-conotoxins fall into two categories depending on whether they act at muscle- type or neuronal-type receptors. The neuronally active a-conotoxins are the focus of this minireview. The known a-conotoxins consist of 12–19 amino acids. Most a-conopeptides have four cysteine residues and the general sequence GCCX m CX n C. The disulfide connectivity is between alternate cysteine residues (I-III, II-IV). The numbers of amino acid residues encompassed by the second and third cysteine residues (m) and the third and fourth cysteine residues (n) are the basis for a further division into several structural subfamilies (a3/5, a4/3, a4/6 and a4/7) [1,3,4]. For example a4/6-AuIB belongs to the 4/6 disulfide loop size subgroup of the a-conotoxin family. The neuronally active a-conotoxins are typically from the a4/7, a4/6 and a4/3 subfamilies (Table 1). Peptides from the most abundant a4/7subfamily are typically 16 residues in length and range from  1600 to  1900 Da in mass. However there have been recent additions to this subfamily in which Correspondence to P. F. Alewood, Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia. Fax: + 61 73346 2101, Tel.: + 61 73346 2982, E-mail: P.Alewood@imb.uq.edu.au Abbreviations: c-CRS, c-carboxylation recognition sequence; nAChRs, nicotinic acetylcholine receptors; RT, retention time; PTM, post-translational modification; TPST, tyrosyl-protein sulfotransferase; TCEP, tris(2-carboxyethyl)phosphine; M-biotin, maleimide-biotin; NEM, N-ethylmaleimide; IAM, iodoacetamide. (Received 22 January 2004, revised 16 March 2004, accepted 6 April 2004) Eur. J. Biochem. 271, 2294–2304 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04146.x there have been extensions at the N-terminus or C-terminus to a length of up to 19 residues and the mass range has been extended to almost 2200 Da [5]. For example, there are three additional residues at the N-terminus in the case of GID [5]. Peptides from the a4/3 and a4/6 subfamilies are typically 12 and 15 residues, respectively. Unusually, EI has the same disulfide framework as a4/7 conotoxins that target neuronal nAChRs but has been reported to antagonize the neuromuscular receptor as do the a3/5 and aA conotoxins [1,6]. There is a conserved proline between the second and third cysteine residues in almost all a-conotoxins except ImIIA and ImII [7,8]. However, the former has not been confirmed to be a neuronally active a-conotoxin, despite its sequence similarity with ImI and ImII. There is also a conserved serine residue between the second and third cysteine residues in many a-conotoxins. The residue N-terminal to the first cysteine residue of the sequence is in most cases glycine, although exceptions are the recently isolated peptides GID and PIA that have c-carboxyglutamic acid and proline, respectively, in that position (Table 1) [5,9]. More generally, the residues in the first loop tend to fit into defined categories, whereas the second loop seems to have greater heterogeneity of residues. There appears to be a relationship between selected sequence motifs and receptor subtype specificity and these sequence patterns may be a basis for further defining subclasses within the neuronally active members of the a-conotoxin family [9a]. There are many interesting features of the biology of Conus species and the functional applications of the a-conotoxins in their venom. It has been conjectured that of the estimated 500 Conus species, each appears to make at least one nAChR antagonist [1]. However, for some Conus species pharmacological screening of crude venom samples has not shown a-conotoxin activity (A. Nicke & M. Loughnan, unpublished results). Nonetheless it has become apparent that in any one species there may be multiple peptides that target nAChRs [1], and it seems likely that the complement of neuronally acting a-conotoxins in one species may cover a range of subtype specificities. There are examples of combinations of muscle-type and neuronal- type a-conotoxins in a single species, particularly in the case of the fish-eating Conus species. For example, C. geographus venom contains the muscle-acting a-conotoxins GI and GII together with the neuronally acting a-conotoxins GIC and GID [1,5,10] and C. magus venom contains the Table 1. Comparison of selected known a-conotoxins from a4/7, a4/6 and a4/3 families, their selectivity for mammalian nAChR subtypes, size, route of discovery, method of synthesis and reference for synthesis. Asterisks (*) indicate an amidated C-terminus. The letters O and Y denote hydroxyproline and sulfotyrosine, respectively. The letter Y ~~ denotes sulfotyrosine identified after original sequence was published. The symbol c denotes c-carboxyglutamic acid. Dashes indicate gaps in the sequence alignment. Mass (monoisotopic) in daltons, given for disulfide-bonded form. Conserved cysteine residues are shown in red and a highly conserved proline in the first loop is shown in green. The peptides Vc1.1, GIC, ImII and ImIIA were identified by prediction from the nucleic acid sequence. Other peptides were identified by isolation of the peptides from the venom ducts in response to activity assays or by physico-chemical characteristics. Im peptides are from C. imperialis,AufromC. aulicus,MfromC. magus,Ep from C. episcopatus,PnfromC. pennaceus,PfromC. purpurascens,GfromC. geographus and E from C. ermineus. Prey groups are denoted by p, m, v for piscivores, molluscivores and vermivores, respectively. Discovery and synthesis methods were as follows: a, Discovered by peptide activity at nAChRs in native tissues (e.g. bovine chromaffin cells, Aplysia neurons); b, Discovered by peptide activity at nAChRs heterologously expressed in Xenopus oocytes; c, Discovered by peptide physico-chemical characteristics, and confirmed by synthesis and assay; d, Discovered by gene sequencing with peptide sequence deduced from cDNA library obtained by RT-PCR of cone snail mRNA; e, Synthesised by Fmoc assembly, trifluoroacetic acid cleavage and directed disulfide formation (off-resin); f, Synthesised by Fmoc assembly, modified trifluoroacetic acid cleavage and air oxidation in ammonium bicarbonate for disulfide formation; g, Synthesised by tBoc assembly, HF cleavage and air oxidation in ammonium bicarbonate for disulfide formation; N/A, not available. Name Sequence Prey Selectivity Mass Methods Reference ImI GCCSDPRCAWR C* v a7 1350.5 c,a,e [20] ImIIA YCCHRGPCMVW C* v N/A 1451.6 d [7] ImII ACCSDRRCRWR C* v a7 1508.6 d,e [8] AuIB GCCSYPPCFATNPD-C* m a3/b4 1571.6 b,e [2] AuIA GCCSYPPCFATNSDYC* m (less active) 1724.6 b,e [2] AuIC GCCSYPPCFATNSGYC* m (less active) 1666.6 b,e [2] AnIA CCSHPACAANNQDYC* v a3/b2, a7 1673.6 c,f [21] AnIB GGCCSHPACAANNQDYC* v a3/b2, a7 1787.6 a,f [21] AnIC GGCCSHPACFASNPDYC* v a3/b2, a7 1805.6 a,f [21] MII GCCSNPVCHLEHSNLC* p a3b2; a6b2b3 1709.7 b,e [13] EpI GCCSDPRCNMNNPDYC* m a3b2/a3b4; a7 1866.6 c,a,f [24] Vc1.1 GCCSDPRCNYDHPEIC* m a3a7b4/a3a5b4 1805.7 d,a,g [22] PnIA GCCSLPPCAANNPDY ~~ C* m a3b2 a 1701.6 a,g,e [58,51] [A10L]PnIA GCCSLPPCALNNPDY ~~ C* a7 a 1663.7 g,e [60,51] PnIB GCCSLPPCALSNPDY ~~ C* m a7 a 1716.6 a,g,e [59,51] GIC GCCSHPACAGNNQHIC* p a3b2 1608.6 d,e [10] GID IRDcCCSNPACRVNNOHVC# p a3b2 2184.6 b,g [5] PIA RDPCCSNPVCTVHNPQIC* p a6b2b3 1980.8 d,e [9] EI RDOCCYHPTCNMSNPQIC* p (muscle-type) 2091.8 a [6] a Synthesis method and activity refer to the unsulfated peptides. Ó FEBS 2004 Characterization and synthesis of a-conotoxins (Eur. J. Biochem. 271) 2295 muscle-acting a-conotoxin MI and the neuronally acting a-conotoxin MII [11–13]. A biological interpretation of this emerging pattern of paired ligand types is that prey capture might rely on the combination of muscle-acting antagonists to cause paralysis and neuronally acting antagonists to inhibit the flight-or-fight response [10,14]. Although distinct peptide complements have been attributed to individual species [15], there are instances of a single a-conotoxin sequence occurring in more than one species. For example, GID from C. geographus has also been isolated from C. tulipa venom [5]. Conus venoms together provide an array of ligands with selectivity for various neuronal nAChR subtypes (Table 1, [9a]). Evolutionarily, this diversity of toxins has been generated by a hypermutation process that allows protection of conserved cysteine residues and high substi- tution rates for the intervening residues in the mature toxin peptides [3,16,17]. Each venom peptide is processed from a prepropeptide and the three defined regions of this precursor (signal sequence, proregion, mature toxin) have different rates of divergence [3,16]. Proposed diversifica- tion mechanisms include gene duplication and subsequent diversifying selection, or targeted gene mutation with some sophisticated molecular regulation, perhaps based on repair processes or recombination processes acting in discrete exon regions [16–19]. Prey-driven diversifying selection may be a factor for dominant expressed toxins, given the feeding specificity of cone snail species [17]. It has been suggested that nicotinic ligands from fish-hunters are more likely than those from snail and worm-hunters to target vertebrate nAChRs with high affinity [1]. Besides the piscivores (fish-hunters) C. magus, C. geographus and C. purpurascens, other species that have also yielded neuronally active a-conotoxins include the vermivores (worm-hunters) C. imperialis and C. anemone,andthe molluscivores (mollusc-hunters) C. aulicus, C. victoriae, C. pennaceus and C. episcopatus (Table 1) [2,5,7–10,13,20– 24]. Many more species are represented in a-conotoxin sequence information contained in patent documents, for example [25]. However these are not within the scope of this review because their activities have not been reported, although the patent applications reflect the commercial interest in this class of conopeptides as potential candi- dates for drug development. Many of the neuronally active a-conotoxins show a high conservation of the local backbone conformation although the surfaces are unique [3,25a]. The common structural scaffold suggests that the hypervariability of the sidechain groups confers peptide specificity for different neuronal nAChR subtypes [3]. Although the a-conotoxins are considered to have rigid structures, multiple inter- convertible isomers may exist in solution [1,3] (see also below). This potential heterogeneity is an important issue in the isolation, analysis and chemical synthesis of these peptides. Post-translational modifications of neuronally active a-conotoxins A feature of conotoxins in general is that they are relatively richly endowed with a wide spectrum of post-translational modifications (PTMs) and this aspect has been comprehen- sively reviewed elsewhere [14,15,26]. The classical published a-conotoxin sequences contained comparatively few mod- ifications apart from disulfide bridges and C-terminal amidation. However, more recently isolated peptides have expanded the list of modifications and they now seem comparable with the rest of the conotoxins in this respect (Table 2). A thorough exploration of the significance of these modifications for the function of a-conotoxins is yet to be completed and reported. Disulfide bridge formation is a basic feature of the a-conotoxins with their defined cysteine spacing and disulfide connectivity. Non-native disulfide-bonded cono- peptides are often considered to be inactive but this is not always the case. Intriguing results have been obtained in structure-function studies of a synthetic variant of a-AuIB with non-native disulfide bond connectivity, where an enhancement of biological activity was observed [27]. Hydroxylation of proline has been observed in several neuronally acting a-conotoxins but of these only GID has been described [5]. Hydroxyproline also occurs elsewhere in the A superfamily: in the muscle-specific a-EI, in aA-EIVA, EIVB and PIVA and in w-PIIIE [1]. The significance of this modification has not been determined. Many other cono- toxins that contain multiple hydroxyproline residues also have naturally occurring under-hydroxylated variants such as in the example of TVIIA [28] and perhaps the modifi- cation may not be critical. However there was no evidence of a variant of GID with proline in place of the single hydroxyproline residue [5]. Amidation of the C-terminus is a feature of most conotoxins and so far occurs in the majority of the a-conotoxins with the exceptions of GID and the muscle- Table 2. Post-translational modifications (PTMs) in neuronal a-conotoxins. PTMs in other conotoxins PTMS in a-conotoxins References Disulfide bridge formation Yes all Amidation of C-terminus Yes most (not in GID) [5] Sulfation of tyrosine Yes EpI, PnIA, PnIB, AnIA, AnIB, AnIC [24,31,21] Hydroxylation of proline Yes GID [5] Carboxylation of glutamic acid Yes GID [5] Cyclization of N-terminal Gln No O-Glycosylation No Bromination of tryptophan No Isomerization of tryptophan No Epimerization of other residues (L fi D) No 2296 M. L. Loughnan and P. F. Alewood (Eur. J. Biochem. 271) Ó FEBS 2004 acting SII [1,5]. The functional significance of the nature of the C-terminus in neuronal-type a-conotoxins has not been established. However a recent study of the effects of an amidated or free carboxyl C-terminus on activity of a synthetic a-conotoxin (AnIB) at nAChR subtypes expressed in oocytes showed subtype specific differences in activity [21]. Carboxylation of glutamic acid to c-carboxyglutamic acid has been reported in GID [5]. It has been estimated that 10% of conopeptides contain c-carboxyglutamic acid and the conantokins (NMDA receptor antagonists) have mul- tiple c-carboxyglutamic acid residues that are important for maintaining their three dimensional structure [15,29]. The existence of c-carboxylation recognition sequences (c-CRSs) in conopeptide precursors from some Conus species has been established and an enzyme responsible for glutamic acid carboxylation in conantokin G has been described [30]. The c-CRS regions in conantokin G and bromosleeper peptides [30] are highly dissimilar suggesting that thissequenceinformationcannotnecessarilybeextended to other conopeptide families. c-CRSs in a-conotoxin precursors have not been described. Sulfation of tyrosine has been observed in EpI, PnIA, PnIB, AnIA, AnIB and AnIC [21,24,31]. The mechanism of sulfation of tyrosine by an enzyme, tyrosyl-protein sulfo- transferase (TPST), has not been elucidated. It may involve a recognition sequence in the peptide precursor [15] or a consensus sequence in the mature peptide [32]. Alternat- ively, secondary structure may be the major determinant of sulfation and the TPST might broadly recognize any sufficiently exposed tyrosine residue [33,34]. The sulfotyro- sine-containing a-conotoxins have not previously been reported to have substantially different activity from the unmodified variants [15,24]. Recent comparisons of EpI with [Y15]EpI, and AnIB with [Y16]AnIB found about three-fold and ten-fold reduced activities, respectively, of the unsulfated forms relative to the sulfated peptides [21,35]. In the case of AnIB, tyrosine sulfation selectively influenced the binding to the mammalian a7 but not the a3b2 subtype [21]. The effects of substitution of phosphotyrosine for sulpho- tyrosine in a-conotoxins have not been investigated. Post-translational modifications such as O-glycosylation of serine or threonine residues, bromination of tryptophan, isomerization of tryptophan or L fi D epimerization of other residues have been observed in conotoxins from other families [1,15] but not reported for a-conotoxins. However, characterizations of a-conotoxins have not routinely inclu- ded tests for isomerization and epimerization modifications. Aspects of the biosynthesis of conotoxins in the cone snail and the mechanisms for incorporation of PTMs have garnered considerable interest [15]. Hypermutation of amino acid residues is a feature of the mature toxins but in contrast the prepropeptide precursor sequence, partic- ularly the signal sequence, seems to be highly conserved for each family of conotoxins [3,16]. Precursor sequences are available for conotoxins from most families but there have been relatively few precursors published in journals for the a-conotoxins (Table 3). Nevertheless it is interesting to examine the available prepropeptide sequence information for ImIIA and Vc1.1, ImII, GIC and PIA, each of which was identified by prediction from a genomic DNA clone [7–10,22]. Comparison of the peptide sequences GID, EI, ImIIA, GIC and PIA suggests that there are anomalies in Table 3. Precursor sequence or cleavage site information for selected a-conotoxins. The mature peptide is underlined. The putative cleavage site for generation of the mature toxin from the prepropeptide at one or more basic resisues is shown in bold. Name Species Reference Sequence GIC precursor C. geographus [10] SD GRNDAA KA FDLI-SSTV- KKGCCSHPAC AGNNQHICGR RR Vc1.1 precursor C. victoriae [22] MGMRMMFTVF LLVVLATTVV SSTSGRREFR GRNAAA KA SDLV-SLTDK KRGCCSDPRC NYDHPEICG ImIIA precursor C. imperialis [7] MGMRMMFTVF LLVVLATAVL PVTL-DRASD GRNAAANAKT PRLI-APFI- RDYCCHRGPC MVW CG ImII cleavage site C. imperialis [8] a RRACCSDRRC RWR CG ImI cleavage site C. imperialis [8] a RRGCCSDPRC AWR CG MII precursor C. magus [25] MFTVF LLVVLATTVV SFPS-DRASD GRNAAANDKA SDVITAL -KGCCSNPVC HLEHSNLCGR RR AuIB precursor C. aulicus [25] MFTVF LLVVLATTVV SFTS-DRASD GRKDAA SGLI-ALTM- -KGCCSYPPC FATNPD-CGR RR AuIA precursor C. aulicus [25] MFTVF LLVVLATTVV SFTS-DRASD GRKDAA SGLI-ALTI- -KGCCSYPPC FATNSDYCG EpI precursor C. episcopatus [25] MFTVF LLVVLATTVV SFTS-DRASD SRKDAA SGLI-ALTI- -KGCCSDPRC NMNNPDYCG PIA precursor C. purpurascens [9] SD GRDAAANDKA TDLI-ALTAR RDPCCSNPVC TVHNPQICGR R a EMBL/Genbank/DDBJ databases accession numbers P50983, Q816R5. Ó FEBS 2004 Characterization and synthesis of a-conotoxins (Eur. J. Biochem. 271) 2297 relation to GID, EI and PIA, which each have an N- terminal sequence motif RDX. It is tempting to speculate about the relationship of this motif to the potential dibasic cleavage sites for generation of the mature peptides from the prepropeptides, particularly in those cases where the residue at position X becomes post-translationally modified in the mature peptide. However there are also many other sequences reported in patent documents that have not been considered here and for many of those the deduced cleavage site does not have a dibasic motif [25]. There has been increasing utility of molecular biology techniques for identification of new a-conotoxin sequences. Reliance on the gene sequence of a conopeptide alone without verifica- tion from the peptide sequence might miss PTM sites, or misidentify the cleavage site for generation of the mature peptide. Much further work is necessary to elucidate the mechanisms of incorporation of PTMs in the biosynthesis of conopeptides. Appropriate methods of analysis need to be addressed to ensure recognition of these PTMs, if present, in the course of characterization of native a-conotoxins. Analysis of neuronally active a-conotoxins using HPLC and MS, including identification of post-translational modifications Isolation and identification Standard procedures for identification and isolation of a-conotoxins generally incorporate separations using reversed-phase HPLC, size exclusion or ion exchange chromatography in combination with mass-based screening and functional screening. Most of the a-conotoxins identi- fied so far are relatively hydrophilic and hence tractable. Mass-based screening entails searching by LC/MS or MS alone for components with a mass in the defined range for a-conotoxins and two disulfide bonds (identified by partial reduction and alkylation studies and MS/MS). It may also include diagnostic LC/MS for recognition of some post- translational modifications and possibly MS/MS for recog- nition of conserved sequence motifs. The small size range of a-conotoxins would seem ideal for MS-based sequence determination. However, complete de novo sequencing of conopeptides by MS/MS is still considered experimental, and primary sequence information is usually obtained by Edman degradation sequencing, interpreted in conjunction with MS data for the intact molecule. Efficient sequence analysis usually requires that the peptides are reduced and the cysteine residues alkylated in order to verify their identification. Although there are standard procedures for reduction and alkylation, their application to conopeptide analysis and characterization is by no means trivial, and often optimization on a case-by-case basis is required [14]. Sample amounts may be limiting even with the enhanced sensitivity of current automated sequence analysis instru- ments. Chromatography and structural heterogeneity Several a-conotoxins have a characteristic asymmetric peak under standard reversed-phase HPLC elution conditions [5,36]. The anomalous chromatographic behaviour is seen particularly with a-CnIA, a-MI, a-GI and a-GID for both native and synthetic forms and persists even after repeated refractionation [5,36,37]. The asymmetry may be more pronounced under isocratic elution conditions. It presum- ably reflects structural heterogeneity and can be interpreted as a slow interconversion between two conformers; this conclusion has been supported by the results of structure studies. NMR studies have shown the existence of two distinct interconvertible conformers for GI and multiple conformers of CnIA [36,37]. This heterogeneity may yet prove to be an important feature of some a-conotoxins in the understanding of structure-function relationships and the interaction of these ligands with the nAChR. Identification of PTMs Methods for the characterization of PTMs in conotoxins have been reviewed elsewhere, with particular emphasis on the utility of MS [14], but specific aspects relevant to a-conotoxins (identification of C-terminal amidation, sulfotyrosine, hydroxyproline and c-carboxyglutamic acid) are revisited here. The identification of the PTM is usually confirmed by synthesis of the modified a-conotoxin and comparison with the natural peptide. Identification of the nature of the C-terminus. The identification of C-terminal amidation or a free carboxyl terminus in an a-conotoxin is usually straightforward with the one mass unit difference between the two forms readily apparent from the monoisotopic mass determined by high resolution mass spectrometry. There may be difficulties in interpretation of MS data when there are ambiguities arising from, for example, asparagine to aspartic acid, or glutamine to glutamic acid changes [14]. The a-conotoxins EpI, PnIA, GIC, GID, AnIA and AnIB contain pairs of asparagine residues [5,10,21,23,24] (Table 1), and deamida- tion may confound MS data for these peptides. Determination of sulfotyrosine. Identification of sulfo- tyrosine in peptides is usually by mass spectrometry and the lability of the sulfogroup in mass spectrometry analysis allows recognition of the modification, and differentiation of sulfotyrosine and phosphotyrosine [38]. Characterization of the sulfotyrosine-containing a-cono- toxin EpI was undertaken by a combination of mass spectrometry and modified amino acid analysis [24]. The conotoxins a-PnIA and a-PnIB from C. pennaceus were initially identified and reported as unmodified sequences although an unidentified mass discrepancy had been recognized [23]. The verification of tyrosine sulfation in a-PnIA and a-PnIB and revision of those sequences were made in an investigation of labile sulfo- and phospho- peptides by electrospray MALDI and atmospheric pressure MALDI mass spectrometry [31]. The sulfation of three conotoxins from C. anemone was identified on the basis of LC/MS under different conditions together with the difference between the observed mass and that predicted from primary Edman sequence data [21]. The presence of either sulfation or phosphorylation may be indicated when liquid chromatography/electrospray ionization mass spectrometry shows doubly protonated species of the modified a-conotoxins with additional related 2298 M. L. Loughnan and P. F. Alewood (Eur. J. Biochem. 271) Ó FEBS 2004 ions at +40 m/z (+80 Da). Further examination by MALDI MS or ESI MS in both positive and negative ion modes, at high and low energy, or MALDI high-energy collision-induced dissociation, can be undertaken to confirm tyrosine sulfation by distinguishing features of ionization and fragmentation [31,38,39]. Determination of hydroxyproline and c-carboxyglutamic acid. The presence of these modified residues in a-conotoxins is generally determined by a combination of mass spectrometry, Edman N-terminal sequencing and amino acid analysis [14]. Hydroxyproline residues can be reliably identified in the course of N-terminal Edman sequencing of peptides [14]. Mass spectrometry has also been used for the analysis of peptides containing hydroxy- proline and bromotryptophan, by high-resolution, high- accuracy precursor ion scanning utilizing fragment ions with mass-deficient mass tags [40]. c-Carboxyglutamic acid residues are readily recognized in electrospray mass spectrometry under standard conditions because of the lability of the extra carboxyl group ()44 Da) ([5,41] and as shown in Fig. 1). This residue can not be reliably identified by standard N-terminal Edman sequencing procedures because inefficient extraction of the polar derivative generates a largely blank cycle [14,42], although there may be residual amounts of glutamic acid. Modified Edman sequencing procedures, modified amino acid analysis or colorimetric c-carboxyglutamic acid assays can also be used to confirm this residue. Disulfide linkage determination Identification of closely spaced cystine residues in peptides with complex disulfide linkage patterns is still considered a significant analytical challenge and determination of disul- fide linkages in even the relatively simple a-conotoxins can still pose difficulties. There have been several studies on characterization of closely spaced, complex disulfide bond patterns in peptides, usually based on stepwise reduction and differential alkylation of cysteine residues in a sequen- tial manner (Fig. 2), although there may be complications because of disulfide shuffling via the thiol-disulfide exchange reaction. These studies are of relevance to the characteriza- tion of neuronal a-conotoxins although many of the studies have been validated with other well-characterized peptides (some of them including a-conotoxin SI) and proteins. One of the original comprehensive studies of disulfide bond linkage relevant to conotoxins involved analysis of differ- entially alkylated products by Edman N-terminal sequen- cing and MS [43]. A subsequent study specific for conopeptide analysis described differential alkylation fol- lowed by tandem mass spectrometry to determine disulfide bond connectivity, and indicated that reduction and alky- lation under acidic conditions is preferred to avoid condi- tions that promote scrambling of disulfide bonds [44]. A further modification has been the use of iodination labelling of the peptide to obtain better separation of intermediates in partial reduction and alkylation studies [45]. Disulfide Fig. 2. Scheme of disulfide determination. Diagram of the general strategy for determination of disulfide linkage of conotoxins by dif- ferential reduction and alkylation of cystine residues and subsequent analysis by LC/ESI MS/MS or by Edman sequencing [43,44,46]. (A) Partial reduction of peptide containing disulfide bonds by incubation with a low concentration of tris(2-carboxyethyl)phosphine (TCEP) (0.1–0.5 m M )at65°C for 10–15 min, generating partially reduced peptides that were immediately alkylated by N-ethylmaleimide (NEM) (twotofive-foldmolarexcessovertheCysresiduesoftheanalyte) present in the TCEP solution. (B) Complete reduction with dithio- threitol and alkylation by iodoacetamide (IAM) to label the Cys residues of the peptide that were not reduced by TCEP. (C) Analysis of the resulting peptides differentially alkylated with NEM and IAM to identify the disulfide linkages. Fig. 1. LC/MS analysis of crude venom from C. geographus. Example of experiment approach using LC/ES MS of crude extract of C. geographus showing complexity of crude venom and showing the identification of a modified peptide, GID (2184.9 Da) [5]. Total ion chromatogram from positive ion analysis using LC/ES QqTOF mass spectrometry over a range m/z 500–2000. Chromatography was on a Zorbax 300SB C3, 2.1 · 150 mm, 5 lm column run at 300 llÆmin )1 with a gradient from 0 to 60% solvent B over 60 min. Solvent A was 0.1% formic acid in water and solvent B was 90% acetonitrile, 0.09% (v/v) formic acid in water. Location of the selected component has been indicated. Inset: Electrospray reconstructed mass spectrum of selected component from main figure revealing the presence of two components 44 Da apart, indicating the presence of a c-carboxyglut- amic acid residue. Doubly and triply charged ions were observed (data not shown). Ó FEBS 2004 Characterization and synthesis of a-conotoxins (Eur. J. Biochem. 271) 2299 linkage determination in peptides and proteins by LC/ electrospray ionization tandem mass spectrometry (LC/ESI MS/MS) in combination with partial reduction by tris (2-carboxyethyl)phosphine (TCEP) has been described recently [46]. The general procedure in these studies has been that peptides were treated with TCEP in the presence of an alkylation reagent such as maleimide- biotin (M-biotin) or N-ethylmaleimide (NEM), followed by complete reduction with dithiothreitol and alkylation by iodoacetamide (IAM). Subsequently, peptides that contained alkylated cysteine were analyzed by capillary LC/ESI MS/MS or other means to determine which cysteine residues were modified with M-biotin/NEM or IAM. The presence of the alkylating reagent (M-biotin or NEM) during TCEP reduction was found to minimize the occurrence of the thiol-disulfide exchange reaction [46]. In the determination of disulfide connectivity, it is advisable to undertake directed synthesis of mispaired as well as correctly disulfide-bonded conopeptides (as described below), and to compare their elution with the naturally occurring conopep- tide in chromatography studies. This is particularly applic- able for a-conotoxins because there are a manageable number of potential disulfide isomer variants. Authenticity is indicated by chromatography coelution of natural and synthetic peptides after coinjection. Ideally, disulfide con- nectivity would be further confirmed by assessing the structure of the naturally occurring peptide. However, the limitation of scarcity of most conopeptides usually precludes this. The possibility of activity of non-native disulfide- bonded isomers of a-conotoxins [27] precludes reliance on activity for identification of the correctly disulfide-bonded synthetic isomer. Peptide synthesis Chemical synthesis strategies The small size (10–25 amino acids) of a-conotoxins has made chemical synthesis the preferred route of synthetic access with both Boc and Fmoc chemistry widely employed. The incorporation of most post-translationally modified residues such as hydroxyproline, c-carboxyglut- amic acid or pyroglutamic acid into synthetic a-conotox- ins is relatively straightforward through incorporation of the suitably protected amino acid in the chain assembly step. By contrast, two areas of a-conopeptide synthesis can pose challenges and deserve particular attention: sulfotyrosine incorporation and disulfide bond formation together with selection of the desired disulfide bond isomer. Disulfide bond formation Synthetic strategies for the preparation of a-conotoxins vary between laboratories and often reflect different scientific Fig. 3. Schemes of directed disulfide bond formation. Schemes showing orthogonal strategies for selective disulfide bond formation in synthesis of a-conotoxins. (A) One-step directed disulfide formation [52]. (B) Two-step standard directed disulfide formation from Olivera and coworkers [2,8,10,13,20,50,51]. (C) Two-step directed disulfide formation, illustrated with ÔdiscreteÕ mispaired isomer with small disulfide loop closed first [54]. (D) Two-step directed disulfide forma- tion, in solution or resin-bound, with small disulfide loop closed first [55]. (E) Three possible disulfide isomers of a-conotoxins [4,53]. 2300 M. L. Loughnan and P. F. Alewood (Eur. J. Biochem. 271) Ó FEBS 2004 needs or preferences. The choice of simultaneous (non- selective) or selective oxidation (Fig. 3) for disulfide bond formation is a major point of difference. Information described in the following sections concerning detailed studies of selective disulfide formation with the muscle- specific a-conotoxin SI can be extended to the neuronal a-conotoxins. Nonselective disulfide bond formation. Several research groups have taken advantage of the fact that the native form (I-III, II-IV C-C connectivity) of the a-conotoxins is the predominant form accessible under simple oxidative condi- tions from the tetrathiol (i.e. fully reduced) peptide. Appropriate solvent conditions may influence the efficiency of the process and either standard Fmoc or Boc chemistry may be employed to generate the fully reduced conopeptide [47,48]. Disulfide bond formation is performed via a one- step procedure usually with 0.02–0.1 M aqueous ammonium bicarbonate, pH 6.7–10, or with minor variations such as the inclusion of 10–30% (v/v) of isopropanol, acetonitrile or dimethylsulfoxide where required. An exception to the generally used chain assembly and deprotection procedures employs a two-step deprotection and cleavage from methylbenzhydrylamine resin using trifluoroacetic acid and hydrogen fluoride, prior to nonselective disulfide formation and a shorter oxidation at higher pH (pH 10) [49]. Selective disulfide bond formation: off-resin approa- ches. Fmoc methodology for chain assembly together with trifluoroacetic acid-based deprotection and cleavage of peptide from resin is the most commonly used. Typical approaches have employed orthogonal cysteine protec- tion (Trityl, Acetamidomethyl) for a two-step disulfide bond formation, using reagents such as Tris buffer, 20 m M potassium ferricyanide with 0.1 M Tris, pH 7.5, or 10% dimethylsulfoxide in 0.02 M ammonium bicar- bonate, pH 6.7, for formation of the first disulfide bond, and iodine for the second disulfide bond (Fig. 3B) [2,8,10,13,20,50,51]. A novel approach by Cuthbertson & Indrevoll employed a one-pot regioselective formation of the two disulfide bonds of a-conotoxin SI [52] (Fig. 3A). By selecting temperature- sensitive orthogonal cysteine protecting groups, t-butyl and 4-methylbenzyl, the target molecule was efficiently obtained. Thus the first disulfide bridge was formed directly from the crude material by simultaneous cleavage and oxidation of the t-butyl groups in trifluoroacetic acid/dimethylsulfoxide/ anisole (97.9 : 2 : 0.1, v/v/v) at room temperature. The subsequent heating of this solution resulted in the cleavage of the 4-methylbenzyl groups with simultaneous oxidation yielding the desired bicyclic product [52]. Selective disulfide bond formation: on-resin approa- ches. Two detailed studies of selective disulfide formation in the muscle-specific a-conotoxin SI (Fig. 3) [49,53], employed both off and on-resin approaches. Syntheses of all three possible disulfide regioisomers, natural and disulfide-mispaired, with the sequence of a-conotoxin SI were described [53]. (Disulfide isomers: natural I-III, II-IV; mispaired ÔnestedÕ I-IV, II-III and mispaired ÔdiscreteÕ I-II, III-IV, Fig. 3E). It was possible to achieve the desired alignments with either order of loop formation (smaller loop before larger, or vice versa). The highest overall yields were obtained when both disulfides were formed in solution, while experiments where either the first or both bridges were formed while the peptide was on the solid support revealed lower overall yields and poorer selectivities towards the desired isomers. This and further studies with a-conotoxin SI illustrated novel protection schemes and oxidation strategies ([53–55] Fig. 3C,D). Synthesis of sulfated a-conotoxins Sulfopeptides can be prepared by chemical assembly with the incorporation of sulfated residues or less commonly by ÔglobalÕ modification of the completed peptide using enzymic or chemical methods of sulfation. The chemical synthesis of peptides containing O-sulfated hydroxy amino acids is still considered a difficult, delicate and laborious task for peptide chemists because of the intrinsic acid-lability of the sulfate moiety [50,56,57]. An efficient cleavage/deprotection procedure without loss of the sulfate remains to be elucidated for Fmoc-based solid-phase synthesis of sulfopeptides [24,50,57]. There have been a few reports of the solid phase synthesis of sulfotyrosine-containing a-conotoxins including EpI, PnIA, PnIB, AnIA, AnIB and AnIC and some ana- logues [21,24,56]. Most of the reported syntheses of PnIA, PnIB and the [A10L]PnIA analogue have been of the unsulfated forms [51,58–61]. A modified trifluoro- acetic acid-based protocol including low temperature steps and exclusion of thiol-containing scavengers, for example 95% (v/v) trifluoroacetic acid/triisopropylsilane or 90% (v/v) aqueous trifluoroacetic acid (0 °C, 8 h), is generally used for cleavage of sulfopeptides assembled with Fmoc chemistry [24,56]. Desulfation rate is strongly temperature-dependent whereas sidechain deprotections are less temperature-dependent and effective deprotection protocols can be developed accordingly. The use of tetrabutylammonium salts (rather than sodium or barium salts) of O-sulfated hydroxy amino acids minimized desulfation during Fmoc-based assembly, room tempera- ture trifluoroacetic acid cleavage and reversed-phase HPLC purification in applications for synthesis of cholecystokinin-12 and bradykinin containing tyrosine sulfate [57], and could perhaps be applicable to synthesis of a-conopeptides. Synthesis of variants of a-conotoxins: alanine scans and loop replacements In addition to replication of natural conotoxins, the standard synthesis procedures described above have been applied for strategies requiring synthesis of specific variants of conotoxins. The synthesis of alanine scan peptide variants containing systematic alanine replace- ments of the amino acids [62], and assessment in structure or function screening procedures, may reveal crucial amino acids or regions that are essential binding and structural determinants. A parallel of the alanine scan approach is the synthesis of a-conotoxin variants or ÔchimerasÕ in which whole loop regions have been swapped with those from other a-conotoxins with different attrib- Ó FEBS 2004 Characterization and synthesis of a-conotoxins (Eur. J. Biochem. 271) 2301 utes in an attempt to confer a different specificity or conformation [1,48,63]. Strategies for synthesis of multiple conotoxins in parallel Synthesis and evaluation of many different peptide mole- cules may be required for structure-function studies of a-conotoxins in the search for optimum synthetic variants. The rate-limiting step in these studies is often the time and effort required for peptide synthesis. Options for synthesis of multiple peptides in parallel have included synthesis on polyethylene pins, in Ôtea bagsÕ andonchipormembrane supports such as in the spot synthesis technique [64–66]. A study of [A10L]PnIA variants synthesized in parallel in a 96 well plate has been described [67]. However there have been few other references to the application of these techniques to the synthesis of a-conotoxins and their analogues, and quantities may be insufficient for conventional structure- function studies. There may be increasing scope for applications of parallel synthesis, particularly membrane- anchored synthetic peptide libraries, for structure-function analysis by in situ screening using binding assays or antibody recognition. Concluding remarks Comprehensive characterization of novel natural a-cono- toxin peptides has relied on a combination of analyses. These have included Edman N-terminal sequencing, MS and tandem MS for determination of the primary sequence, determination of disulfide linkages after differ- ential reduction and alkylation, and confirmation of the primary structure. Amino acid analysis in conjunction with Edman sequence analysis and diagnostic ion MS has been used to assess the possibility of other PTMs. Characterization would ideally include NMR analysis for proof of structure although this has often been precluded by scarcity of material. It may be the only means to assure certainty in determination of novel disulfide linkages. The primary structure of the peptide and the identified PTMs have usually been verified by subsequent synthetic chemistry and comparison with the natural peptide. The synthesized disulfide-bonded peptide, with minimum purity of 95–98% determined by reversed- phase HPLC, has been compared with the natural peptide, if available, by coinjection of the synthesized peptide and the naturally occurring peptide, and authen- ticity has been indicated by chromatography coelution. The correctness of the disulfide linkage has usually been confirmed by NMR spectroscopy. Newly identified pep- tides that clearly fit previously defined categories are often not subjected to such rigorous analysis. For variants such as residue replacement peptides and chimeras, where there is no corresponding natural peptide available for comparison, verification of authenticity, particularly correct disulfide linkage, has been reliant on NMR structure analysis. Many nonselective syntheses of a-conotoxins yield a single pre- dominant disulfide-bonded isomer, although in cases where multiple isomers are generated in substantial amounts, further characterization may be warranted. Thorough assessment of novel a-conotoxin folds may generate import- ant structure-function information. In conclusion, the physico-chemical characterization of the native peptides and chemical synthesis of neuronal a-conotoxins provide an important basis for the pharma- cology, structure and modelling studies that are the subject of further minireviews in this series. After this manuscript had been submitted for publication a newly published paper reported 16 conotoxin precursors of the A superfamily, from six Conus species, defining the A conotoxin gene superfamily [68]. Acknowledgements We thank Annette Nicke, David Craik, Gene Hopping, Alun Jones and Richard Lewis for their input. The LC/MS analysis shown in Fig. 1 was runbyAlunJones. References 1. 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