The Venom Repertoire of Conus Gloriamaris (Chemnitz, 1777), the Glory of the Sea

The Venom Repertoire of Conus gloriamaris (Chemnitz, 1777), the Glory of the Sea

Robinson, Samuel D; Li, Qing; Lu, Aiping; Bandyopadhyay, Pradip K; Yandell, Mark; Olivera, Baldomero M; Safavi-Hemami, Helena

Published in: Marine Drugs

DOI: 10.3390/md15050145

Publication date: 2017

Document version Publisher's PDF, also known as Version of record

Citation for published version (APA): Robinson, S. D., Li, Q., Lu, A., Bandyopadhyay, P. K., Yandell, M., Olivera, B. M., & Safavi-Hemami, H. (2017). The Venom Repertoire of Conus gloriamaris (Chemnitz, 1777), the Glory of the Sea. Marine Drugs, 15(5), [145]. https://doi.org/10.3390/md15050145

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Article The Venom Repertoire of Conus gloriamaris (Chemnitz, 1777), the Glory of the Sea

Samuel D. Robinson 1,*, Qing Li 2, Aiping Lu 3, Pradip K. Bandyopadhyay 1, Mark Yandell 2,4, Baldomero M. Olivera 1 and Helena Safavi-Hemami 1

1 Department of Biology, University of Utah, Salt Lake City, UT 84112, USA; [email protected] (P.K.B.); [email protected] (B.M.O.); [email protected] (H.S.-H.) 2 Eccles Institute of Human Genetics, University of Utah, Salt Lake City, UT 84112, USA; [email protected] (Q.L.); [email protected] (M.Y.) 3 School of Life Sciences and Technology, Institute of Protein Research, Tongji University, Shanghai 200092, China; [email protected] 4 USTAR Center for Genetic Discovery, University of Utah, Salt Lake City, UT 84112, USA * Correspondence: [email protected]; Tel.: +1-801-581-8370

Academic Editor: Peer B. Jacobson Received: 21 April 2017; Accepted: 17 May 2017; Published: 20 May 2017

Abstract: The marine cone snail Conus gloriamaris is an iconic species. For over two centuries, its shell was one of the most prized and valuable natural history objects in the world. Today, cone snails have attracted attention for their remarkable venom components. Many conotoxins are proving valuable as research tools, drug leads, and drugs. In this article, we present the venom gland transcriptome of C. gloriamaris, revealing this species’ conotoxin repertoire. More than 100 conotoxin sequences were identiﬁed, representing a valuable resource for future drug discovery efforts.

Keywords: venom; conotoxin; Conus; cone snail; Conus gloriamaris

1. Introduction For many years, the shell of the Glory of the Sea cone snail, Conus gloriamaris, was one of the most prized and valuable natural history objects in the world [1,2]. The shell is indeed remarkably beautiful (Figure1), but it is perhaps its rarity that contributed most to its fame—for over two centuries, C. gloriamaris was known from only half a dozen specimens. In 1837, Hugh Cuming, a British shell collector, found two live specimens on a reef on the island of Bohol, the Philippines [2]. Soon after, however, it would be reported that this reef, the only known location at which C. gloriamaris had been found alive, had been destroyed by an earthquake. Rumours followed that this already rare species had now been driven to extinction, and produced the obvious effect of making the few known specimens even more desirable. As recently as 1957, the number of known specimens was still only at two dozen. However, this would change as collectors entered into the waters of New Guinea and in 1969, a pair of SCUBA divers discovered over a hundred live specimens at Guadalcanal in the Solomon Islands [3]. Although still not considered to be abundant, C. gloriamaris is in fact reasonably widespread across the Indo-Paciﬁc. It has been found between 5 and 300 m, but is rarely encountered in shallow water (above 100 m) [4], explaining its historical rarity. Nevertheless, it remains probably the single most famous seashell, and because of its beauty and historic signiﬁcance, still attracts high prices from collectors.

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FigureFigure 1. The1. The shell shell of ofConus Conus gloriamaris gloriamaris.. AtAt oneone time, this this was was among among the the most most prized prized natural natural history history objectsobjects in in the the world. world.

Today, cone snails are attracting attention for an additional reason, the remarkable biomedical Today, cone snails are attracting attention for an additional reason, the remarkable biomedical potential of their venom components. Each one of the ~750 species of the genus Conus is venomous potential(number of of their extant venom species components. according Eachto WoRMS: one of thehttp://www.marinespecies.org/). ~750 species of the genus Conus Theyis use venomous their (numbervenom offor extant prey capture species and according defense to [5]. WoRMS: Some species http://www.marinespecies.org/ prey on fish, whilst others prey). They on marine use their venomworms for or prey molluscs. capture C. and gloriamaris defense is [5 ].believed Some species to be a preymollusc on fish,‐hunter whilst [4]. othersTheir venoms prey on are marine complex worms or molluscs.mixtures typicallyC. gloriamaris containingis believed more than to be 100 a mollusc-hunterbioactive peptides [4 (known]. Their as venoms conotoxins). are complex Furthermore, mixtures typicallyeach species containing of Conus more is thanthought 100 to bioactive harbour peptides an almost (known unique as repertoire conotoxins). of conotoxins. Furthermore, Many each speciesconotoxins of Conus haveis unmatched thought to potency harbour and an selectivity almost unique profiles repertoire at their respective of conotoxins. molecular Many targets conotoxins [6]. haveAdditionally, unmatched while potency only and a minute selectivity fraction profiles of the at their estimated respective total molecularnumber of targets conotoxins [6]. Additionally, has been whileinvestigated only a minute to date, fraction several of have the estimated already proven total number valuable of as conotoxins research tools, has beendrug investigatedleads, and drugs. to date, severalω‐MVIIA, have alreadya conotoxin proven from valuable the venom as researchof Conus magus tools,, drug is used leads, for the and treatment drugs. ω of-MVIIA, chronic a pain conotoxin [7], fromwhile the several venom others of Conus are magus under, isdevelopment used for the for treatment the treatment of chronic of various pain pathologies [7], while several [8], including others are underepilepsy, development neuropathic for thepain, treatment and diabetes. of various pathologies [8], including epilepsy, neuropathic pain, and diabetes.Conotoxins are produced, by the cone snail, in a specialized venom gland. Conotoxin transcripts are ribosomally‐translated into precursor peptides that undergo folding and Conotoxins are produced, by the cone snail, in a specialized venom gland. Conotoxin transcripts post‐translational processing before being secreted into the lumen of the venom gland [9]. In are ribosomally-translated into precursor peptides that undergo folding and post-translational general, conotoxin precursor peptides are characterized by an N‐terminal signal peptide followed by processing before being secreted into the lumen of the venom gland [9]. In general, conotoxin precursor a propeptide region, and, encoded at the C‐terminus, a single copy of the bioactive mature toxin. N peptidesRecent are characterized advances in high by an throughput-terminal ‘next signal‐generation’ peptide sequencing followed by have a propeptide made it possible region, to and, encodedsequence at the the Centire-terminus, transcriptome a single of copy a tissue of the in bioactivea rapid and mature cost‐effective toxin. manner. The application of thisRecent technology advances to the in venom high glands throughput of Conus ‘next-generation’ offers an avenue sequencingto acquiring a have comprehensive made it possible picture to sequenceof a species’ the entire conotoxin transcriptome repertoire of[10–13]. a tissue in a rapid and cost-effective manner. The application of this technologyThe venom to the of venomC. gloriamaris glands ofhasConus yet tooffers be ancomprehensively avenue to acquiring studied. a comprehensiveOnly 10 conotoxin picture of asequences species’ conotoxinhave so far repertoire been reported [10–13 from]. this species [14] and only two conotoxins (Gm9a and GmVIA)The venom have ofbeenC. gloriamarisfunctionallyhas characterised yet to be comprehensively [15,16]. In this article, studied. we Only present 10 conotoxin the venom sequences gland havetranscriptome so far been of reported C. gloriamaris from, this revealing species the [ 14venom] and repertoire only two of conotoxins this iconic (Gm9a species. and GmVIA) have been functionally characterised [15,16]. In this article, we present the venom gland transcriptome of C. gloriamaris2. Results , revealing the venom repertoire of this iconic species. Sequencing generated from C. gloriamaris whole venom gland RNA yielded a total of 42,602,912 2. Resultsdemultiplexed raw reads (40,363,512 following adapter‐trimming and quality‐trimming/filtering). UsingSequencing Trinity [17], generated 16,353 fromtranscriptsC. gloriamaris were assembledwhole venom with an gland E90 of RNA 238 (the yielded number a total of transcripts of 42,602,912 demultiplexed raw reads (40,363,512 following adapter-trimming and quality-trimming/filtering). Mar. Drugs 2017, 15, 145 3 of 20

Mar. Drugs 2017, 15, 145 3 of 21 Using Trinity [17], 16,353 transcripts were assembled with an E90 of 238 (the number of transcripts that arethat supported are supported by 90% by of 90% the expressionof the expression data). Transcriptsdata). Transcripts annotated annotated as conotoxin as conotoxin precursors precursors made up ~70%made of theup total~70% expression of the total data, expression as well asdata, the bulkas well of the as mostthe bulk highly of expressedthe most highly transcripts, expressed i.e., all of thetranscripts, top 60 annotated i.e., all of transcripts the top 60 annotated were conotoxin transcripts precursors. were conotoxin precursors. ConotoxinConotoxin precursors precursors can can be be grouped grouped into into gene gene families families (or (or superfamilies), superfamilies), based based on on their their signal peptidesignal peptide sequence sequence identity identity [18]. [18]. A total A total of 31of 31 distinct distinct conotoxin conotoxin gene families families were were identified identified in in C.C. gloriamaris gloriamaris.. Not Not all all toxintoxin genegene families were were expressed expressed at at equal equal levels. levels. In Infact, fact, 95% 95% of conotoxin of conotoxin expressionexpression was was derived derived from from just nine gene gene families families (O2, (O2, T, T, O1, O1, J, H, J, H, P, P,U, U, A, A,and and M; M;from from highest highest to to lowest)lowest) (Figure (Figure2). 2). From the 31 toxin gene families, a total of 108 individual conotoxin transcripts were identified. From the 31 toxin gene families, a total of 108 individual conotoxin transcripts were identified. Again, there are substantial differences in the number of individual toxins per gene family. Almost Again, there are substantial differences in the number of individual toxins per gene family. Almost all of all of the toxin gene families identified were represented by three or fewer individual toxins. Only the toxin gene families identified were represented by three or fewer individual toxins. Only five toxin five toxin gene families were represented by more than four individual conotoxin transcripts (O2, T, geneO1, familiesM, and MSRLF) were represented (Figure 2). by more than four individual conotoxin transcripts (O2, T, O1, M, and MSRLF) (Figure2).

Figure 2. Distribution of toxin gene families in C. gloriamaris. Conotoxin expression is dominated by onlyFigure a few 2. conotoxinDistribution gene of toxin families. gene Valuesfamilies represent in C. gloriamaris transcripts. Conotoxin per million expression transcripts is dominated (TPM) countsby obtainedonly a few for conotoxin all sequences gene belonging families. Values to the represent gene family. transcripts The number per million of individual transcripts sequences (TPM) counts per toxin geneobtained family for is providedall sequences in parenthesis. belonging to the gene family. The number of individual sequences per toxin gene family is provided in parenthesis. 2.1. O2-Superfamily 2.1. O2‐Superfamily InInC. C. gloriamaris gloriamaris,, thethe O2-superfamilyO2‐superfamily is is the the single single most most-highly‐highly expressed expressed toxin toxin gene gene family, family, accountingaccounting for for closeclose to half half of of the the total total conotoxin conotoxin expression. expression. It is Italso is a also diverse a diverse superfamily: superfamily: 17 17individual individual O2 O2-superfamily‐superfamily conotoxins conotoxins were were detected detected (Figure (Figure 3). Of3 these,). Of these,16 have 16 the have typical the type typical typeVI/VII VI/VII cysteine cysteine framework framework (C‐C‐ (C-C-CC-C-C)CC‐C‐C) and one and belongs one belongs to a subclass to a subclass known known as contryphans. as contryphans. Of Ofthe the two two O2 O2-superfamily‐superfamily conotoxins conotoxins characterized characterized so far so (TxVIIA far (TxVIIA and PnVIIA), and PnVIIA), both appeared both appeared to be to bemollusc mollusc-specific‐specific in in activity, activity, producing producing strong strong paralytic paralytic effects effects in molluscs, in molluscs, but butwith with no paralytic no paralytic effectseffects on on arthropods arthropods or vertebrates or vertebrates [19,20 ].[19,20]. Notably, Notably, for a number for a ofnumber the C. gloriamarisof the C.O2-superfamily gloriamaris conotoxinsO2‐superfamily identified conotoxins here, anidentified identical here, or an nearly identical identical or nearly sequence identical was sequence previously was previously described in Conusdescribed victoriae in [Conus13]. C. victoriae victoriae [13].is a closely-relatedC. victoriae is a species closely belonging‐related species to the belonging same subgenus to the ( Cylindersame ), endemicsubgenus to ( theCylinder coast), endemic of North-Western to the coast Australia. of North‐ AsWestern is discussed Australia. below, As is a discussed striking similaritybelow, a to C.striking victoriae similarityis observed to C. for victoriae almost is all observed of the toxin for almost families all of identified. the toxin families identified.

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FigureFigure 3. Sequence 3. Sequence alignment alignment of C. gloriamarisof C. gloriamarisO2-superfamily O2‐superfamily precursor precursor sequences. sequences. Precursor Precursor sequences of O2_Vc6.22,sequences of O2_Vc6.18, O2_Vc6.22, O2_Vc6.21, O2_Vc6.18, O2_Vc6.26, O2_Vc6.21, O2_Vc6.24,O2_Vc6.26, O2_contryphan_Vc2O2_Vc6.24, O2_contryphan_Vc2 [13], PnVIIA [13], [20 ], Figure 3. Sequence alignment of C. gloriamaris O2‐superfamily precursor sequences. Precursor andPnVIIA TxVIIA [20], [19] and are shownTxVIIA for [19] comparison are shown infor grey comparison and marked in grey with and *; Cys, marked yellow; with Signal *; Cys, peptides yellow; are sequences of O2_Vc6.22, O2_Vc6.18, O2_Vc6.21, O2_Vc6.26, O2_Vc6.24, O2_contryphan_Vc2 [13], underlinedSignal peptides in purple are and underlined predicted maturein purple peptides and predicted are underlined mature in peptides black/grey. are This underlined color scheme in black/grey.PnVIIA [20], This and color TxVIIA scheme [19] is are used shown in all for subsequent comparison figures. in grey and marked with *; Cys, yellow; is usedSignal in allpeptides subsequent are underlined figures. in purple and predicted mature peptides are underlined in 2.2. Tblack/grey.‐Superfamily This color scheme is used in all subsequent figures. 2.2. T-Superfamily 2.2. TIn‐Superfamily C. gloriamaris , the T‐superfamily is the second most highly expressed toxin gene family, makingIn C. up gloriamaris 22% of the, the total T-superfamily conotoxin expression. is the second It is also most the most highly diverse: expressed 21 individual toxin genesequences family, In C. gloriamaris, the T‐superfamily is the second most highly expressed toxin gene family, makingwere upidentified 22% of (Figure the total 4), conotoxin including expression. the previously It is reported also the mostGm5.1 diverse: and Gm5.2 21 individual [21]. Nineteen sequences of making up 22% of the total conotoxin expression. It is also the most diverse: 21 individual sequences werethese identified had a type (Figure V cysteine4), including framework the previously (CC‐CC), one reported a type Gm5.1XIII framework, and Gm5.2 and [ one21]. a Nineteen type I/X of were identified (Figure 4), including the previously reported Gm5.1 and Gm5.2 [21]. Nineteen of thesecysteine had a framework type V cysteine (CC‐C‐ frameworkC). Several molecular (CC-CC), targets, one a type including XIII framework, presynaptic andcalcium one channels a type I/X these had a type V cysteine framework (CC‐CC), one a type XIII framework, and one a type I/X cysteine[22], TTX framework‐sensitive (CC-C-C). sodium channels Several molecular[23], and somatostatin targets, including receptors presynaptic [24], have calcium been reported channels for [22 ], cysteine framework (CC‐C‐C). Several molecular targets, including presynaptic calcium channels TTX-sensitiveconotoxins sodiumof the channelsformer subclass, [23], and while somatostatin the toxins receptors of the [ 24latter], have subclass been reported are norepinephrine for conotoxins [22], TTX‐sensitive sodium channels [23], and somatostatin receptors [24], have been reported for of thetransporter former inhibitors subclass, [25]. while For several the toxins of the of C. the gloriamaris latter subclassT‐superfamily are norepinephrineconotoxins identified transporter here, conotoxins of the former subclass, while the toxins of the latter subclass are norepinephrine inhibitorsan identical [25]. or For nearly several identical of the sequenceC. gloriamaris was previouslyT-superfamily described conotoxins in C. victoriae identified. here, an identical transporter inhibitors [25]. For several of the C. gloriamaris T‐superfamily conotoxins identified here, or nearly identical sequence was previously described in C. victoriae. an identical or nearly identical sequence was previously described in C. victoriae.

Figure 4. C. gloriamaris T‐superfamily precursor sequences. *, precursor sequences of T_Vc5.7,

T_Vc5.20, T_Vc5.23, T_Vc5.16, T_Vc5.8, T_Vc13.1, T_Vc5.19, T_Vc5.22, T_Vc10.1 [13], and MrIA [26] Figure 4. C. gloriamaris T‐superfamily precursor sequences. *, precursor sequences of T_Vc5.7, Figureare shown 4. C. gloriamaris for comparison.T-superfamily precursor sequences. *, precursor sequences of T_Vc5.7, T_Vc5.20, T_Vc5.20, T_Vc5.23, T_Vc5.16, T_Vc5.8, T_Vc13.1, T_Vc5.19, T_Vc5.22, T_Vc10.1 [13], and MrIA [26] T_Vc5.23, T_Vc5.16, T_Vc5.8, T_Vc13.1, T_Vc5.19, T_Vc5.22, T_Vc10.1 [13], and MrIA [26] are shown are shown for comparison. for comparison.

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2.3.2.3. O1-Superfamily O1‐Superfamily AA total total of of 12 12 O1-superfamily O1‐superfamily conotoxinconotoxin precursors were identified identified in C. gloriamaris gloriamaris (Figure(Figure 55A),A), includingincluding fourfour sequences sequences previously previously reported reported from from this this species species (Gm6.1, (Gm6.1, Gm6.2, Gm6.2, Gm6.5 Gm6.5 [27], and [27], andGmVIA GmVIA [28]). [28 GmVIA]). GmVIA is a is δ‐ a conotoxin,δ-conotoxin, which which delays delays the the inactivation inactivation of of voltage voltage-gated‐gated sodium channelschannels and and produced produced convulsions convulsions in molluscs in molluscs [28]. All[28]. O1-superfamily All O1‐superfamily precursors precursors from C. gloriamaris from C. encodegloriamaris mature encode peptides mature with peptides the typicalwith the type typical VI/VII type cysteineVI/VII cysteine framework, framework, and all,and except all, except one low-expressedone low‐expressed transcript, transcript, fit into fit into the the same sameδ/ δµ/functionalμ functional subclass subclass as as GmVIA. GmVIA. Again,Again, for several several ofof thethe C.C. gloriamarisgloriamaris O1-superfamilyO1‐superfamily conotoxinsconotoxins identifiedidentified here, an identical or close match was was previouslypreviously described described in inC. C. victoriae victoriae..

Figure 5. (A) C. gloriamaris O1-superfamily; (B) J-superfamily; (C) H-superfamily precursor sequences. Figure 5. (A) C. gloriamaris O1‐superfamily; (B) J‐superfamily; (C) H‐superfamily precursor *, precursor sequences of O1_Vc6.36, O1_Vc6.41, O1_Vc6.35, O1_Vc6.30, O1_Vc6.28, O1_Vc6.37, sequences. *, precursor sequences of O1_Vc6.36, O1_Vc6.41, O1_Vc6.35, O1_Vc6.30, O1_Vc6.28, J_Vc14.1, J_Vc14.2, J_Vc14.4, H_Vc7.2, and H_Vc1 [13] are shown for comparison. O1_Vc6.37, J_Vc14.1, J_Vc14.2, J_Vc14.4, H_Vc7.2, and H_Vc1 [13] are shown for comparison.

2.4. J-Superfamily 2.4. J‐Superfamily C. gloriamaris FourFour J-superfamily J‐superfamily conotoxins conotoxins were were identified identified in the in venom the venom gland transcriptome gland transcriptome of of C. (Figuregloriamaris5B). They(Figure are 5B). similar They to are the similar sequence to the previously sequence identified previously in identifiedC. victoriae in [C.13 victoriae], but differ [13], from but thediffer previously from the characterised previously characterised J-superfamily J‐superfamily conotoxin pl14a, conotoxin which pl14a, produces which excitatory produces symptomsexcitatory (shaking,symptoms barrel-rolling, (shaking, barrel and‐ seizures)rolling, and in mice seizures) on intracranial in mice on injection intracranial and wasinjection shown and to was be an shown inhibitor to of the voltage-gated potassium channel subtype K 1.6 [29]. be an inhibitor of the voltage‐gated potassium channelv subtype Kv1.6 [29]. 2.5. H-Superfamily 2.5. H‐Superfamily Both cysteine-rich (one), and cysteine-poor (two) H-superfamily conotoxins were identified Both cysteine‐rich (one), and cysteine‐poor (two) H‐superfamily conotoxins were identified (Figure5C). The cysteine-poor conotoxins are both closely related to H-Vc1, previously identified in (Figure 5C). The cysteine‐poor conotoxins are both closely related to H‐Vc1, previously identified in C. victoriae, while the predicted mature peptide of the cysteine-rich H-Gm7.1, differs only by a single C. victoriae, while the predicted mature peptide of the cysteine‐rich H‐Gm7.1, differs only by a single residue from that of Vc7.2 from C. victoriae. The biological function is yet to be reported for any residue from that of Vc7.2 from C. victoriae. The biological function is yet to be reported for any H-superfamily conotoxin. H‐superfamily conotoxin. 2.6. P-Superfamily 2.6. P‐Superfamily Three P-superfamily transcripts were identified in the venom gland transcriptome of C. gloriamaris Three P‐superfamily transcripts were identified in the venom gland transcriptome of C. (Figure6A). One of these is Gm9a (previously identified from C. gloriamaris [15]) and another appears to gloriamaris (Figure 6A). One of these is Gm9a (previously identified from C. gloriamaris [15]) and be an allelic variant of this sequence, differing by only a single synonymous substitution. Gm9a elicited another appears to be an allelic variant of this sequence, differing by only a single synonymous hyperactivity and spasticity in mice on intracranial injection. The third precursor, P-Gm14.1, encodes substitution. Gm9a elicited hyperactivity and spasticity in mice on intracranial injection. The third precursor, P‐Gm14.1, encodes a 30 residue mature peptide cysteine framework XIV (C‐C‐C‐C). It is

Mar. Drugs 2017, 15, 145 6 of 20 Mar. Drugs 2017, 15, 145 6 of 21 aclosely 30 residue related mature to Vc14.5 peptide from cysteine C. victoriae framework, differing XIV in (C-C-C-C). its predicted It is mature closely peptide related toby Vc14.5 only three from C.residues. victoriae , differing in its predicted mature peptide by only three residues.

Figure 6. (A) C. gloriamaris P-superfamily; (B) U-superfamily; (C) A-superfamily precursor sequences. Figure 6. (A) C. gloriamaris P‐superfamily; (B) U‐superfamily; (C) A‐superfamily precursor *, precursor sequences of P_Vc9.2, P_Vc14.5, U_Vc7.3, Vc22.1 [13], Tx1 [30], Vc1.1 [31], and the textile sequences. *, precursor sequences of P_Vc9.2, P_Vc14.5, U_Vc7.3, Vc22.1 [13], Tx1 [30], Vc1.1 [31], convulsant mature peptide [32], are shown for comparison. and the textile convulsant mature peptide [32], are shown for comparison.

2.7.2.7. U-Superfamily U‐Superfamily AA singlesingle precursorprecursor sequencesequence belongingbelonging toto thethe U-superfamilyU‐superfamily was identified identiﬁed in C. gloriamaris (Figure(Figure6 B).6B). The The predicted predicted mature mature peptide peptide is identical is identical to the to previously the previously described described Vc7.3 from Vc7.3C. from victoriae C. andvictoriae differs and by differs one residue by one from residue the “Textilefrom the convulsant “Textile convulsant peptide” frompeptide”C. textile from, C. which textile produces, which convulsionsproduces convulsions when injected when intracranially injected intracranially in mice [32 in]. mice [32].

2.8. A-Superfamily 2.8. A‐Superfamily ThreeThree A-superfamilyA‐superfamily conotoxinconotoxin precursorsprecursors were identiﬁedidentified in the C. gloriamaris venom gland gland transcriptometranscriptome (Figure 66C).C). The most most highly highly expressed expressed A A-superfamily‐superfamily sequence sequence in C. in gloriamarisC. gloriamaris was α wasGm1.1. Gm1.1. The Thepredicted predicted mature mature peptide peptide is similar is similar to several to several previously previously described described α‐conotoxins-conotoxins and is andlikely is to likely also tobe also an inhibitor be an inhibitor of neuronal of neuronal subtypes subtypes of nicotinic of nicotinicacetylcholine acetylcholine receptors. receptors. Gm22.1 Gm22.1belongs belongs to an unusual to an unusual subclass subclass that was that previously was previously described described in C. in victoriaeC. victoriae [13].[ 13A]. comparison A comparison of ofGm22.1 Gm22.1 with with Vc22.1 Vc22.1 is isshown shown in in Figure Figure 6C.6C. While While there there are are minor differences in the signalsignal andand propeptide,propeptide, the the predicted predicted maturemature peptidespeptides areare identicalidentical betweenbetween species. The third precursor shares shares thethe A-superfamily A‐superfamily signalsignal sequence,sequence, butbut appears to encode an unusually large (87 residues) mature mature peptidepeptide with with 12 12 cysteines, cysteines, and and is is unrelated unrelated to to anyany previouslypreviously describeddescribed sequence.sequence. 2.9. M-Superfamily 2.9. M‐Superfamily C. gloriamaris InIn C. gloriamaris, seven, seven M-superfamily M‐superfamily conotoxins conotoxins were detectedwere detected (Figure 7(FigureA), including 7A), including the previously the reportedpreviously Gm3-WP04 reported (GenBank:Gm3‐WP04 JF510902.1).(GenBank: JF510902.1). All, bar one, All, belong bar toone, the belong “mini-M” to the subclass “mini‐ [M”33], whichsubclass produce [33], which excitatory produce symptoms excitatory when symptoms injected intracraniallywhen injected in intracranially mice, but for in which mice, a but general for molecularwhich a targetgeneral is yetmolecular to be defined. target The is remainingyet to be sequence defined. encodes The remaining a cysteine-free sequence conomarphin. encodes a cysteine‐free conomarphin.

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Figure 7. (A) C. gloriamaris M‐superfamily; (B) I2‐superfamily; (C) B‐superfamily precursor sequences. *, precursor sequences of M_Vc3.1, M_Vc3.6, M_Vc3.9, M_conomarphin_Vc1, I2_Vc11.8, FigureI2_Vc11.7, 7. (A) C.conantokin_Vc1 gloriamaris M-superfamily; [13], TxIIIA [34], (B) I2-superfamily;and the mature (peptideC) B-superfamily of MrIIIA [35] precursor are shown sequences. for Figure 7. (A) C. gloriamaris M‐superfamily; (B) I2‐superfamily; (C) B‐superfamily precursor *, precursorcomparison. sequences of M_Vc3.1, M_Vc3.6, M_Vc3.9, M_conomarphin_Vc1, I2_Vc11.8, I2_Vc11.7, sequences. *, precursor sequences of M_Vc3.1, M_Vc3.6, M_Vc3.9, M_conomarphin_Vc1, I2_Vc11.8, conantokin_Vc1 [13], TxIIIA [34], and the mature peptide of MrIIIA [35] are shown for comparison. 2.10.I2_Vc11.7, I2‐Superfamily conantokin_Vc1 [13], TxIIIA [34], and the mature peptide of MrIIIA [35] are shown for comparison. 2.10. I2-SuperfamilyThree I2‐superfamily conotoxins were identified in C. gloriamaris (Figure 7B). There are similarities to previously identified sequences, in particular those of C. victoriae, but there is little 2.10.Three I2‐Superfamily I2-superfamily conotoxins were identiﬁed in C. gloriamaris (Figure7B). There are similarities similarity to any of the I2‐superfamily toxins with a known function (as potassium channel blockers to previouslyThree identiﬁedI2‐superfamily sequences, conotoxins in particular were identified those of C. in victoriae C. gloriamaris, but there (Figure is little 7B). similarity There toare any [36–38]). of thesimilarities I2-superfamily to previously toxins identified with a known sequences, function in particular (as potassium those channel of C. victoriae blockers, but [36 there–38]). is little similarity2.11. B2‐Superfamily to any of the I2‐superfamily toxins with a known function (as potassium channel blockers 2.11.[36–38]). B2-Superfamily A single B2‐supefamily sequence was detected in C. gloriamaris (Figure 8A). The A single B2-supefamily sequence was detected in C. gloriamaris (Figure8A). The B2-“superfamily” 2.11.B2‐“superfamily” B2‐Superfamily refers to an unusual class of sequence that is found at a high frequency in the refersvenom to an glands unusual of all class species of sequence of Conus thatexamined is found [13]. at The a high functional frequency role inof this the venomclass of glandssequences of all A single B2‐supefamily sequence was detected in C. gloriamaris (Figure 8A). The speciesremains of Conus unclear.examined [13]. The functional role of this class of sequences remains unclear. B2‐“superfamily” refers to an unusual class of sequence that is found at a high frequency in the venom glands of all species of Conus examined [13]. The functional role of this class of sequences remains unclear.

Figure 8. (A) C. gloriamaris B2-superfamil; (B) Con-insulin; (C) prohormone-4; (D) conoCAP precursor sequences.Figure 8. *, ( precursorA) C. gloriamaris sequences B2‐superfamil; of B2_Vc1 [(B13) ],Con Con-Ins‐insulin; Tx1 (C [)39 prohormone], Con-Ins‐ Vc1,4; (D PH4-Vc1) conoCAP [40 ], precursor sequences. *, precursor sequences of B2_Vc1 [13], Con‐Ins Tx1 [39], Con‐Ins Vc1, PH4‐Vc1 and conoCAP [41] are shown for comparison. [40], and conoCAP [41] are shown for comparison. Figure 8. (A) C. gloriamaris B2‐superfamil; (B) Con‐insulin; (C) prohormone‐4; (D) conoCAP 2.12. B-Superfamily (Conantokins) precursor sequences. *, precursor sequences of B2_Vc1 [13], Con‐Ins Tx1 [39], Con‐Ins Vc1, PH4‐Vc1 [40], and conoCAP [41] are shown for comparison. One conantokin was identified in the C. gloriamaris venom gland transcriptome (Figure7C) .

Conantokins are cysteine-free peptides, some of which are antagonists of vertebrate

N-methyl-D-aspartate receptor (NMDA) receptors [42]. Conantokin-Gm1 is very similar to

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2.12. B‐Superfamily (Conantokins) Mar. DrugsOne2017 conantokin, 15, 145 was identified in the C. gloriamaris venom gland transcriptome (Figure 7C).8 of 20 Conantokins are cysteine‐free peptides, some of which are antagonists of vertebrate N‐methyl‐D‐aspartate receptor (NMDA) receptors [42]. Conantokin‐Gm1 is very similar to the single the single conantokin sequence identified in C. victoriae. Minor differences are seen in the signal and conantokin sequence identified in C. victoriae. Minor differences are seen in the signal and propeptide, while the encoded mature peptides are identical between the two species. propeptide, while the encoded mature peptides are identical between the two species. 2.13. Con-Insulin 2.13. Con‐Insulin A single venom insulin transcript was identified in C. gloriamaris (Figure8B). Specialised insulins A single venom insulin transcript was identified in C. gloriamaris (Figure 8B). Specialised are a venom component of many species of Conus [39]. One of these, Con-Ins G1 from C. geographus, insulins are a venom component of many species of Conus [39]. One of these, Con‐Ins G1 from C. hasgeographus been characterised,, has been characterised, and binds to and the vertebratebinds to the insulin vertebrate receptor, insulin inducing receptor, “insulin inducing shock” “insulin in the fishshock” prey. in the fish prey. TheThe venom venom insulin insulin fromfrom C.C. gloriamaris gloriamaris belongsbelongs to the to thesame same gene gene family family as Con as‐Ins Con-Ins G1 [43]. G1 The [ 43]. Thepredicted predicted signal signal sequences sequences of ofboth both venom venom insulins insulins are are highly highly similar, similar, a characteristic a characteristic of secreted of secreted venomvenom components components that that belong belong toto thethe samesame gene family. However, However, the the predicted predicted mature mature insulins insulins differdiffer significantly. significantly. The The insulin insulin from fromC. gloriamaris C. gloriamarisis longer is longer and hasand an has additional an additional interchain interchain disulfide bond.disulfide These bond. differences These differences can readily can be readily rationalized be rationalized by comparing by comparing the two the venom two venom insulins insulins with the endogenouswith the endogenous insulins of the insulins zebrafish of the and zebrafish the snail andLymnaea the snail stagnalis Lymnaea(Figure stagnalis9). The (FigureC. gloriamaris 9). Theinsulin C. hasgloriamaris the same organizationinsulin has the and same a greater organization sequence and similarity a greater to thesequenceLymnaea similarityinsulin, to while the Con-InsLymnaea G1, theinsulin, insulin while from C.Con geographus‐Ins G1, ,the shows insulin a much from greater C. geographus similarity, shows to the a much zebrafish greater hormone. similarityC. geographus to the is azebrafish fish-hunting hormone. species, C. geographus while C. gloriamaris is a fish‐huntingis a snail species, hunter. while Thus, C. in gloriamaris each case, is the a snail structure hunter. of the venomThus, insulin in each appears case, the to structure reflect aof strong the venom selection insulin for appears efficacy to in reflect the distinct a strong prey selection of each for species. efficacy in the distinct prey of each species.

FigureFigure 9. 9.Similarity Similarity in in cysteine cysteine arrangement,arrangement, chain chain lengths, lengths, and and amino amino acids acids between between the the A and A and B B chainschains of of Con-Ins Con‐Ins Gm Gm and and an an endogenous endogenous molluscan molluscan insulin, insulin, and and Con-Ins Con‐Ins G1 G1 and and an an endogenous endogenous fish insulin.fish insulin. Comparative Comparative alignments alignments of Con-Ins of Con Gm‐Ins andGm theand giant the giant pond pond snail snailLymnaea Lymnaea stagnalis stagnalisinsulin 1 [Uniprot:insulin 1 [Uniprot: P07223], P07223], and Con-Ins and Con G1‐Ins from G1 the from venom the venom of C. of geographus C. geographusand and the the zebrafish zebrafishDanio Danio rerio insulinrerio [Uniprot:insulin [Uniprot: O73727]. O73727]. Chain lengthsChain lengths and the and cysteine the cysteine number number are shown are shown following following the sequence. the Cysteinessequence. are Cysteines highlighted are highlighted in yellow. Post-translational in yellow. Post‐translational modifications modifications are omitted. are omitted.

2.14.2.14. Prohormone-4 Prohormone‐4 TwoTwo prohormone-4 prohormone‐4 precursors precursors were identified identified in in C.C. gloriamaris gloriamaris (Figure(Figure 8C).8C). Prohormone Prohormone-4‐4 is a is a neuropeptideneuropeptide that was initially identified identified in in the the brain brain of of the the honeybee, honeybee, ApisApis melifera melifera, but, but was was more more recentlyrecently identified identified as as a a venom venom component component inin somesome species of of ConusConus [40].[40]. A second class of prohormone‐4‐like precursors that do not appear to encode any mature A second class of prohormone-4-like precursors that do not appear to encode any mature peptide peptide have been reported in all Conus examined to-date [40]. A transcript belonging to this class have been reported in all Conus examined to-date [40]. A transcript belonging to this class was also was also identified in C. gloriamaris. identified in C. gloriamaris.

2.15. I1-Superfamily Two I1-superfamily conotoxins were identiﬁed in C. gloriamaris (Figure 10A). Closely-related sequences were previously reported in C. victoriae [13]. The predicted mature peptide of Gm11.2 differs Mar. Drugs 2017, 15, 145 9 of 21

2.15. I1‐Superfamily Mar. Drugs 2017, 15, 145 9 of 20 Two I1‐superfamily conotoxins were identified in C. gloriamaris (Figure 10A). Closely‐related sequences were previously reported in C. victoriae [13]. The predicted mature peptide of Gm11.2 by adiffers single by amino a single acid amino to that acid of to I1-Vc11.5. that of I1‐ TheVc11.5. I1-superfamily The I1‐superfamily conotoxins conotoxins characterized characterized so far produceso far excitatoryproduce symptoms excitatory insymptoms mice, and in one, mice, RXIA, and producesone, RXIA, these produces through these the through agonism the of voltage-gatedagonism of sodiumvoltage channels‐gated sodium [44]. channels [44].

Figure 10. (A) C. gloriamaris I1-superfamily; (B) I4-superfamily; (C) cono-NPY; (D) N-superfamily Figure 10. (A) C. gloriamaris I1‐superfamily; (B) I4‐superfamily; (C) cono‐NPY; (D) N‐superfamily precursor sequences. *, precursor sequences of I1_Vc11.5, I1_Vc11.1, I4_Vc12.1 [13], cono-NPY [45], precursor sequences. *, precursor sequences of I1_Vc11.5, I1_Vc11.1, I4_Vc12.1 [13], cono‐NPY [45], and Mr15.1 [10] are shown for comparison. and Mr15.1 [10] are shown for comparison.

2.16.2.16. I4-Superfamily I4‐Superfamily FourFour I4-superfamily I4‐superfamily precursors precursors were were identiﬁed identified in C. in gloriamaris C. gloriamaris(Figure (Figure 10B). 10B). The predictedThe predicted mature peptidemature of I4-Gm12.1peptide of differsI4‐Gm12.1 from differs that of from the previouslythat of the previously described I4_Vc12.1described [I4_Vc12.113], by only [13], two by residues. only Notwo function residues. has No yet function been ascribed has yet to been any ascribed conotoxin to any belonging conotoxin to thebelonging I4-superfamily. to the I4‐superfamily.

2.17.2.17. ConoCAP ConoCAP A conoCAPA conoCAP precursor precursor was was identiﬁed identified in in the theC. C. gloriamaris gloriamarisvenom venom gland gland transcriptome transcriptome (Figure (Figure8D). ConoCAPs8D). ConoCAPs are short are short single single disulphide-containing disulphide‐containing peptides peptides identiﬁed identified in the the venom venom of ofsome some worm-huntingworm‐hunting cone cone snails, snails, whichwhich reduce the the heart heart rate rate and and blood blood pressure pressure (in vertebrates) (in vertebrates) [41]. In [41 ]. Incontrast to to other other ConusConus venomvenom peptides, peptides, multiple multiple conoCAPs conoCAPs are are encoded encoded on ona single a single long long neuropeptide-likeneuropeptide‐like precursor. precursor. The The C.C. gloriamaris gloriamaris conoCAPconoCAP precursor precursor encodes encodes three individual three individual mature maturepeptides. peptides.

2.18.2.18. Cono-NPY Cono‐NPY Cono-NPYCono‐NPY was was the the name name given given toto twotwo peptidespeptides previously previously isolated isolated from from the the venom venom of ofthe the worm‐hunter Conus betulinus because of their similarity to neuropeptide‐Y [45]. Here, we report, in worm-hunter Conus betulinus because of their similarity to neuropeptide-Y [45]. Here, we report, in the the venom gland transcriptome of C. gloriamaris, a precursor that encodes a mature peptide with a venom gland transcriptome of C. gloriamaris, a precursor that encodes a mature peptide with a sequence sequence similarity to the previously reported cono‐NPY peptides (Figure 10C). High expression in similarity to the previously reported cono-NPY peptides (Figure 10C). High expression in the venom the venom gland and a secretory signal peptide are consistent with the role of this peptide as a toxin glandin andthe avenom secretory of signalC. gloriamaris peptide. areFurthermore, consistent withpreliminary the role ofinvestigations this peptide asindicate a toxin that in the other venom of C.mollusc gloriamaris‐hunting. Furthermore, cone snails preliminary share closely investigations related precursors indicate in that their other venoms mollusc-hunting (unpublished cone snailsobservation). share closely related precursors in their venoms (unpublished observation).

2.19.2.19. N-Superfamily N‐Superfamily A singleA single N-superfamily N‐superfamily precursor, precursor, Gm15.1, Gm15.1, was found in in the the venom venom gland gland transcriptome transcriptome of C. of C. gloriamarisgloriamaris(Figure (Figure 10D). 10D). It shows It shows a high a sequencehigh sequence similarity similarity to Mr15.1, to Mr15.1, one of theone sequences of the sequences previously identiﬁed in C. marmoreus [10]. The biological function is yet to be reported for any conotoxin of the N-superfamily. Mar. Drugs 2017, 15, 145 10 of 21 Mar. Drugs 2017, 15, 145 10 of 20 previously identified in C. marmoreus [10]. The biological function is yet to be reported for any conotoxin of the N‐superfamily. 2.20. E-Superfamily 2.20.A singleE‐Superfamily E-superfamily sequence was identiﬁed in C. gloriamaris (Figure 11A). The alignment of this sequenceA single E‐ withsuperfamily that of sequenceC. victoriae wasreveals identified seven in C. differences gloriamaris (Figure across the11A). 90 The residue alignment precursor. of Nothis biological sequence function with that has of been C. victoriae reported reveals for any seven conotoxin differences of this across “superfamily”. the 90 residue precursor. No biological function has been reported for any conotoxin of this “superfamily”.

FigureFigure 11. 11.(A ()AC.) C. gloriamaris gloriamarisE-superfamily; E‐superfamily; ( B)) con con-ikot-ikot;‐ikot‐ikot; (C (C) )conorfamide; conorfamide; (D ()D S)‐superfamily S-superfamily precursorprecursor sequences. sequences. *, precursor*, precursor sequences sequences of of E_Vc1, E_Vc1, S_Vc8.1 S_Vc8.1 [13 [13],], con-ikot-ikot con‐ikot‐ikot [46 [46],], and and CNF_Vc1 CNF_Vc1 [ 47] are[47] shown are shown for comparison. for comparison.

2.21.2.21. Con-ikot-ikot Con‐ikot‐ikot TwoTwo con-ikot-ikot con‐ikot‐ikot precursor precursor sequences sequences were identifiedwere identified in C. gloriamaris in C. gloriamaris(Figure 11 B).(Figure Con-ikot-ikots 11B). areCon a class‐ikot‐ ofikots conotoxin are a class predominantly of conotoxin found predominantly in the venoms found of in fish-hunters. the venoms Con-ikot-ikot of fish‐hunters. from ConusCon striatus‐ikot‐ikotinhibits from Conus AMPA striatus receptor inhibits channel AMPA desensitization receptor channel [46] and desensitization was recently used[46] and as a was tool to recently used as a tool to co‐crystalize and reveal the activation mechanism of this receptor [48]. co-crystalize and reveal the activation mechanism of this receptor [48]. However, the C. gloriamaris However, the C. gloriamaris sequences differ substantially, such that a similar function would not be sequences differ substantially, such that a similar function would not be expected. expected. 2.22. Conorfamide 2.22. Conorfamide A single conorfamide precursor sequence was identified in C. gloriamaris (Figure 11C). It bares A single conorfamide precursor sequence was identified in C. gloriamaris (Figure 11C). It bares a a striking similarity to CNF-Vc1, the conorfamide identified in C. victoriae [47], with no differences in striking similarity to CNF‐Vc1, the conorfamide identified in C. victoriae [47], with no differences in thethe signal signal peptide, peptide, one one in thein Cthe-terminal C‐terminal propeptide, propeptide, and and two two in the in maturethe mature peptide. peptide. CNF-Vc1 CNF‐ causedVc1 excitatorycaused symptomsexcitatory symptoms in mice on in intracranial mice on injection,intracranial as injection, well as the as depolarization well as the depolarization of sensory neurons. of sensory neurons. 2.23. S-Superfamily 2.23.A singleS‐Superfamily S-superfamily precursor sequence was identified in the venom gland transcriptome of C. gloriamarisA single(Figure S‐superfamily 11D). Two precursor conotoxins sequence from was the identified S-superfamily in the venom have been gland characterized transcriptome so of far; oneC. is gloriamaris an inhibitor (Figure of serotonin 11D). Two receptors conotoxins [49 from], while the S the‐superfamily other is a have nicotinic been acetylcholinecharacterized so receptor far; inhibitorone is an [50 ].inhibitor of serotonin receptors [49], while the other is a nicotinic acetylcholine receptor inhibitor [50]. 2.24. Conodipine 2.24. Conodipine The precursor sequence of a single conodipine was detected in the C. gloriamaris venom gland transcriptomeThe precursor (Figure sequence 12A). Conodipines of a single conodipine are a class ofwas secretory detected phospholipase-A in the C. gloriamaris2 enzyme venom foundgland in Conustranscriptomevenoms. (Figure 12A). Conodipines are a class of secretory phospholipase‐A2 enzyme found in Conus venoms. 2.25. O3-Superfamily A single precursor sequence belonging to the O3-superfamily was identified in C. gloriamaris (Figure 12B). In contrast to most previously reported O3-superfamily sequences, it lacks cysteines and appears to encode a cysteine-free mature peptide. A single cysteine-free O3-superfamily sequence has been previously identified in C. victoriae, to which the C. gloriamaris sequence is clearly closely related, differing by only three residues across the entire 73 residue precursor.

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Figure 12. (A) C. gloriamaris conodipine; (B) O3-superfamily; (C) F-superfamily; (D) conopressin Figure 12. (A) C. gloriamaris conodipine; (B) O3‐superfamily; (C) F‐superfamily; (D) conopressin precursor sequences. *, precursor sequences of conodipine, O3_Vc1, F_Vc1 [13], and conopressin-G [11] precursor sequences. *, precursor sequences of conodipine, O3_Vc1, F_Vc1 [13], and conopressin‐G are shown for comparison. [11] are shown for comparison.

2.26.2.25. F-Superfamily O3‐Superfamily AA single single F-superfamily precursor sequence sequence belonging was identified to the O3‐ insuperfamilyC. gloriamaris was (Figureidentified 12 inC). C. Similar gloriamaris to the E-superfamily,(Figure 12B). thisIn contrast “superfamily” to most ispreviously defined by reported one precursor O3‐superfamily sequence, sequences, each from it C.lacks marmoreus cysteines[ 10] andandC. victoriaeappears [13to ],encode and no a biological cysteine‐free function mature has peptide. been reported. A single The cysteine alignment‐free ofO3 the‐superfamilyC. gloriamaris sequencesequence with has that been of previouslyC. victoriae identifiedreveals complete in C. victoriae identity, to which across the the C. entiregloriamaris peptide sequence precursor. is clearly closely related, differing by only three residues across the entire 73 residue precursor. 2.27. Conopressin 2.26. F‐Superfamily A single conopressin transcript was identified (at low expression levels) in the venom gland transcriptomeA single of F‐C.superfamily gloriamaris sequence(Figure 12wasD). identified Conopressins, in C. gloriamaris analogues (Figure of the 12C). mammalian Similar hormoneto the vasopressin,E‐superfamily, have this been “superfamily” described in is the defined venoms by one of Conusprecursor[51 ].sequence, Gonopressin-Gm each from C. is closelymarmoreus related [10] to conopressin-Gand C. victoriae from [13],C. and geographus no biological, and function the predicted has been mature reported. conopressin The alignment peptide of isthe identical. C. gloriamaris sequence with that of C. victoriae reveals complete identity across the entire peptide precursor. 2.28. Putative Conotoxins (MKAVA,MSRLF, MMLFM, MLSML) 2.27. Conopressin Four additional putative conotoxin gene families were identified in the C. gloriamaris A single conopressin transcript was identified (at low expression levels) in the venom gland transcriptome: The class of transcript we temporarily refer to as the “MKAVA-superfamily” transcriptome of C. gloriamaris (Figure 12D). Conopressins, analogues of the mammalian hormone (Figure 13A) was previously reported in a C. geographus venom gland transcriptome [52], but was vasopressin, have been described in the venoms of Conus [51]. Gonopressin‐Gm is closely related to C. gloriamaris mis-annotatedconopressin‐G as from the C. I1-superfamily. geographus, and In the predicted mature, a single conopressin transcript peptide sharing is identical. the same signal peptide and a closely related predicted mature peptide sequence, was identified at a very high expression2.28. Putative level. Conotoxins A preliminary (MKAVA, examination MSRLF, MMLFM, of other MLSML) species has revealed that related transcripts are very widespread in Conus (unpublished observation). Four additional putative conotoxin gene families were identified in the C. gloriamaris Several transcripts sharing the same signal peptide as “new superfamily 1”, also previously transcriptome: The class of transcript we temporarily refer to as the “MKAVA‐superfamily” (Figure reported in C. geographus [52], were identified in C. gloriamaris (Figure 13B), some at very high 13A) was previously reported in a C. geographus venom gland transcriptome [52], but was expressionmis‐annotated levels. as Tothe avoid I1‐superfamily. ambiguity, In weC. gloriamaris temporarily, a single refer transcript to this group sharing of the sequences same signal as the “MSRLF-superfamily”.peptide and a closely Similarrelated topredicted what was mature observed peptide in C.sequence, geographus was, theidentifiedC. gloramaris at a verysequences high appearexpression to encode level. cysteine-freeA preliminary matureexamination peptides. of other A species preliminary has revealed examination that related of other transcripts species are has revealedvery widespread that this superfamily in Conus (unpublished is very widespread observation). and diverse in Conus (unpublished observation). TheSeveral “MMLFM-superfamily” transcripts sharing wasthe same initially signal represented peptide byas “new a single superfamily sequence from1”, also the previously worm-hunter Conusreported caracteristicus in C. geographus(GenBank: [52], were B0L0Y6.1), identified with in C. other gloriamaris sequences (Figure recently 13B), some reported at very in high several worm-huntingexpression levels. species To [ 53avoid]. A singleambiguity, transcript, we temporarily sharing the refer same to generalthis group precursor of sequences structure, as the signal peptide“MSRLF sequence,‐superfamily”. and cysteine Similar framework to what was of thisobserved class wasin C. identified, geographus at, the a reasonably C. gloramaris high sequences expression, in C.appear gloriamaris to encode(Figure cysteine 13C),‐free indicating mature thatpeptides. this classA preliminary of transcripts examination is not limited of other to worm-hunters.species has A preliminaryrevealed that examination this superfamily of other is very species widespread has revealed and diverse that in related Conus (unpublished transcripts are observation). widespread in Conus (unpublishedThe “MMLFM observation).‐superfamily” was initially represented by a single sequence from the wormTwo‐hunter transcripts, Conus which caracteristicus we will temporarily(GenBank: B0L0Y6.1), refer to as with the “MLSML-superfamily”,other sequences recently were reported identified in several worm‐hunting species [53]. A single transcript, sharing the same general precursor structure, (Figure 13D). These shared a secretory signal peptide sequence and reasonably high expression levels. signal peptide sequence, and cysteine framework of this class was identified, at a reasonably high The predicted mature peptides are large and each contained 12 cysteine residues. These appear to be expression, in C. gloriamaris (Figure 13C), indicating that this class of transcripts is not limited to closely related to a transcript recently reported in the venom gland transcriptome of Conus lenavati

Mar. Drugs 2017, 15, 145 12 of 21

worm‐hunters. A preliminary examination of other species has revealed that related transcripts are widespread in Conus (unpublished observation). Two transcripts, which we will temporarily refer to as the “MLSML‐superfamily”, were Mar.identified Drugs 2017 ,(Figure15, 145 13D). These shared a secretory signal peptide sequence and reasonably high12 of 20 expression levels. The predicted mature peptides are large and each contained 12 cysteine residues. These appear to be closely related to a transcript recently reported in the venom gland transcriptome (Cln_SF6_1)of Conus lenavati [54], and (Cln_SF6_1) again, a preliminary [54], and again, examination a preliminary of other examination species has of revealed other species that related has transcriptsrevealed are that widespread related transcripts in Conus are(unpublished widespread in observation). Conus (unpublished observation).

Figure 13. (A) C. gloriamaris MKAVA-superfamily; (B) MSRLF-superfamily; (C) MMLFM-superfamily; Figure 13. (A) C. gloriamaris MKAVA‐superfamily; (B) MSRLF‐superfamily; (C) (D) MLSML-superfamily precursor sequences. *, precursor sequences of G114, G118, G120 [52], MMLFM‐superfamily; (D) MLSML‐superfamily precursor sequences. *, precursor sequences of C. caracteristicus ‘putative conotoxin’ (GenBank: B0L0Y6.1), and Cln_SF6_1 [54] are shown for comparison. G114, G118, G120 [52], C. caracteristicus ‘putative conotoxin’ (GenBank: B0L0Y6.1), and Cln_SF6_1 [54] are shown for comparison. 3. Discussion 3. Discussion The original characterization of the crude venom of C. gloriamaris [28] revealed that this species’ venom isThe particularly original characterization enriched in a single of the component, crude venomδ -conotoxinof C. gloriamaris GVIA. [28] Consistent revealed that with this this, species’ GVIA is onevenom of the mostis particularly highly expressed enriched individual in a single toxins component, in our transcriptomic δ‐conotoxin GVIA. dataset. Consistent In fact, in allwith members this, of theGVIA subgenus is one ofCylinder the most thathighly have expressed been examined individual so toxins far, inδ-conotoxins our transcriptomic are highly dataset. expressed In fact, in [13 ]. A comparisonall members of of a selectionthe subgenus of the Cylinderδ-conotoxins that have from beenC. gloriamaris examined toso some far, δ‐ fromconotoxins two other are specieshighly in thisexpressed subgenus, [13].C. textileA comparisonand C. victoriae of a selection, is shown of the in δ‐ Tableconotoxins1 δ-conotoxin from C. gloriamaris GmVIA and to some other from molluscan two δ-conotoxinsother species cause in this hyperactivity subgenus, C. in textile the envenomated and C. victoriae snail, is shown prey [ 28in, 55Table]; it 1 has δ‐conotoxin been suggested GmVIA that and the other molluscan δ‐conotoxins cause hyperactivity in the envenomated snail prey [28,55]; it has been function of the extreme excitatory effects of this venom component is to prevent prey from escaping suggested that the function of the extreme excitatory effects of this venom component is to prevent (since the envenomated snail is observed to ﬂail in an uncontrolled, seizure-like manner). After this, prey from escaping (since the envenomated snail is observed to flail in an uncontrolled, seizure‐like the prey snail is often extended out of its shell, and therefore remains easily accessible to the predator manner). After this, the prey snail is often extended out of its shell, and therefore remains easily coneaccessible snail (instead to the predator of the natural cone snail tendency (instead of of snails the natural to withdraw tendency deep of snails into to their withdraw shells deep at the into ﬁrst signtheir of danger). shells at the first sign of danger). In allIn all three three species, species, a venoma venom insulin insulin is is also also expressedexpressed (Table (Table 11).). The presence presence of of venom venom insulins insulins suggestssuggests that that after after the the initial initial uncontrolled uncontrolled excitatoryexcitatory motor response, response, the the snail snail would would become become hypoglycemichypoglycemic and and therefore therefore transformedtransformed into into a a more more quiescent quiescent state state (while (while remaining remaining outside outside its its shell).shell). Other components of of the the venom, venom, including including the the α‐αtoxins-toxins that that block block nicotinic nicotinic acetylcholine acetylcholine receptors,receptors, would would then then presumably presumably cause cause a a generalized generalized flaccid paralysis. paralysis. Thus, Thus, the the results results suggest suggest at at leastleast two two phases phases of of prey prey capture: capture: a a state state of of hyper-excitability hyper‐excitability that that guarantees guarantees that that the the envenomated envenomated preyprey is unable is unable to withdraw to withdraw into itsinto shell, its shell, and a and transition a transition to a hypoglycemic to a hypoglycemic state and state flaccid and paralysis.flaccid Whenparalysis. the prey When is quiescent the prey andis quiescent paralyzed and outside paralyzed its shell,outsideC. its gloriamaris shell, C. gloriamariscan begin can to engulfbegin to and digestengulf the and now digest helpless, the now easily helpless, accessible easily prey. accessible Together, prey. our Together, data suggest our data a shared suggest prey-capture a shared strategyprey‐capture for C. gloriamaris strategy forand C. othergloriamaris species and in otherCylinder speciesfor in capturing Cylinder theirfor capturing snail prey, their which snail involves prey, thewhich use, among involves other the use, toxins, among of bothother excitatorytoxins, of bothδ-conotoxins excitatory δ‐ andconotoxins venom insulins.and venom It insulins. seems likely It seems likely that other intriguing insights into the chemical strategies used by these snails may that other intriguing insights into the chemical strategies used by these snails may emerge as emerge as these venoms are studied further.The venoms of mollusc‐hunting species of Conus have these venoms are studied further.The venoms of mollusc-hunting species of Conus have so far not so far not been heavily studied. The venom gland transcriptomes of three other species, C. marmoreus been heavily studied. The venom gland transcriptomes of three other species, C. marmoreus [10], C. victoriae [13], and C. episcopatus [56] have been reported. Of these, C. victoriae is of the same subgenus, Cylinder, as C. gloriamaris (Figures 14 and 15A). As indicated above, these species appear to share a similar venom repertoire. Indeed, clear similarities emerge on a comparison of the toxin repertoire of C. gloriamaris to that of C. victoriae [13]. Of the 23 conotoxin gene families detected in C. victoriae [13,40,47], all are present in C. gloriamaris. Only two additional conotoxin gene families were detected here in C. gloriamaris (N and conopressin) that were looked, for but not found, in C. victoriae. Mar. Drugs 2017, 15, 145 13 of 20

Both were present here as single transcripts at relatively low expression levels (626.1 and 22.5 TPM, respectively). The increased sensitivity provided by larger raw read numbers (40,363,512 reads (Illumina) in this study versus 701,536 reads (454) in the prior C. victoriae study) may have facilitated their detection here. Both species also shared the absence of certain conotoxin gene families (C, D, G, I3, K, L, V, and Y). Thus, the two species share what appears to be a near identical toxin gene family repertoire.

Table 1. δ-conotoxins and venom insulins of C. gloriamaris and other species of the subgenus Cylinder.

δ-Conotoxins Primary Structure GmVIA VKPCRKEGQLCDPIFQNCCRGWNCVLFCV Vc6.36 (C. victoriae)[13] GKPCHKEGQLCDPFLQNCCLGWNCVFVCI Tx6.1 (C. textile) QVKPCRKEHQLCDLIFQNCCRGWYCVVLSCT Gm6.1 CRLGAESCDVISQNCCQGTCVFFCLP Vc6.41 (C. victoriae)[13] CRLGAESCDVISQNCCQGTCVFFCLP TxVIA (C. textile)[55] WCKQSGEMCNLLDQNCCDGYCIVLVCT TxVIB (C. textile)[19] WCKQSGEMCNVLDQNCCDGYCIVFVCT Venom Insulins Primary Structure A-chain GIVCECCKNVCTKEEFTEYCPPLTESS * Con-Ins Gm B-chain SEYSCSYNDPDHPEGKCGPQLSEHVEEKCEEEEAEQGGTNNARSNT * Con-Ins Vc1 A-chain GIVCECCKHHCTKEELTEYCH (C. victoriae)[40] B-chain HVCWLGDPNHPKGICGPQLSDIVEIRCEEKEAEQGGANNARAYT * Con-Ins Tx1 A-chain GIVCECCKHHCTKEEFTEYCH (C. textile)[39] B-chain GEHVCWLGDPNHPQGICGPQVADIVEIRCEEKEAEQGGANNARANT * Predicted or known mature peptides are shown. *, C-terminal amidation (other post-translational modiﬁcations are not predicted).

The two species also share a similar total number of conotoxin transcripts. A total of 97 conotoxin transcripts (from the 23 conotoxin superfamilies reported in both species) were identified here in C. gloriamaris, compared with 119 reported in C. victoriae [13,40,47]. It should be noted that the venom glands of multiple individuals of C. victoriae were used, while in this study, the venom gland of a single individual of C. gloriamaris was used. Thus, the population level genetic polymorphism, as reported in other Conus species [57], potentially contributes to the slightly greater total number of transcripts reported for C. victoriae. There are also clear similarities in the distribution of transcripts between toxin gene families (Figure 15B). Those, in C. gloriamaris, with a high diversity of transcripts (T, O2, O1, and M) also exhibit a high diversity in C. victoriae [13]. Likewise, those of a low diversity, where only one or two transcripts exist, are shared between the two species. A comparison of the absolute number of individual conotoxins in each gene family is given in Figure 15B (comparisons based on the expression level were not appropriate since the C. victoriae data were generated from a cDNA library that was normalized). Thus, on multiple levels, it is clear that the venom repertoire of C. gloriamaris is remarkably similar to that of the closely related C. victoriae. However, does this similarity extend to the individual toxin level? Of the 97 C. gloriamaris conotoxin transcripts (from the 23 conotoxin gene families reported in both species), 14 encode mature peptides with identical matches in C. victoriae, and numerous others encode mature peptides with near-identical matches (i.e., one to a few residues difference). To our knowledge, this is the highest rate of identical toxins between any species of Conus so far examined, and it indicates that certain species can share substantial overlap in venom composition. While the similarities are striking, it is important to not lose sight of the fact that more than 85% of the venom components of C. gloriamaris do not have an identical match in C. victoriae. The relatively subtle differences between the venom repertoires of the two closely related species are largely seen at the mature peptide level and may be a reflection of the differences in prey specialization. For example, C. gloriamaris is a deep-water offshore species, while C. victoriae occupies shallower marine habitats, and while both are mollusk-hunters, differences in their specific prey might be expected. Mar. Drugs 2017, 15, 145 14 of 21

families reported in both species), 14 encode mature peptides with identical matches in C. victoriae, and numerous others encode mature peptides with near‐identical matches (i.e. one to a few residues difference). To our knowledge, this is the highest rate of identical toxins between any species of Conus so far examined, and it indicates that certain species can share substantial overlap in venom composition. While the similarities are striking, it is important to not lose sight of the fact that more than 85% of the venom components of C. gloriamaris do not have an identical match in C. victoriae. The relatively subtle differences between the venom repertoires of the two closely related species are largely seen at the mature peptide level and may be a reflection of the differences in prey Mar. Drugs 2017, 15, 145specialization. For example, C. gloriamaris is a deep‐water offshore species, while C. victoriae occupies 14 of 20 shallower marine habitats, and while both are mollusk‐hunters, differences in their specific prey might be expected.

Mar. Drugs 2017, 15, 145 15 of 21

predicted for only a handful. Even for these, differences in the primary structure may impact subtype selectivity. For example, A‐Gm1.1, which shows a sequence similarity to known nAChR blockers, would be predicted to have a similar function, but perhaps a novel subtype selectivity profile. Others belong to classes which are demonstrably bioactive (i.e. Gm9a, contryphan‐Gm, CNF‐Gm, U‐Gm7.2, Gm10.1, and each the conotoxins of the M‐superfamily), but for which a specific molecular target or mechanism of action is yet to be defined. However, the vast majority of the Figure 14. C. gloriamaris and some other species in the subgenus Cylinder. Left, C. gloriamaris; Second Figureconotoxins 14. C. gloriamarispresentedcolumn, C. victoriaestilland remain (Top), some C. completely textile other (Bottom); species uncharacterized. Third incolumn, the Conus subgenus legatusGiven; Fourth theCylinder successfulcolumn,. Conus Left, history C. gloriamaris of the ; Secondsmall column, fractionC.retifer of victoriaeconotoxins (Top), Conus(Top), aureus characterized (Bottom).C. textile so(Bottom); far, it seems Third probable column, that Conusa further legatus exploration; Fourth of this column, C. gloriamaris conotoxin library has the potential to yield new research tools, if not drug leads or Conus retifer (Top),TheConus venoms aureus of mollusc(Bottom).‐hunting species of the cone snail have proven their potential as a source therapeutics.of therapeutically ‐relevant peptides [8] (e.g. χ‐MrIA and Vc1.1). Moreover, to the drug discoverer, the comparison between the venoms of C. gloriamaris and C. victoriae highlights two important points: i) That the venom of each species of Conus represents a near‐unique library of natural products; and ii) the venom repertoire of certain species can mirror that of closely related species, essentially representing a library of naturally‐occurring analogues. This type of knowledge can be valuable in guiding targeted drug discovery efforts from Conus venoms. The toxin repertoire of C. gloriamaris (summarized in Table 2) should offer rich grounds for the discovery of new functions. Of the 108 conotoxins identified, a molecular target can be confidently

Figure 15. Conus gloriamaris shares a near‐identical toxin gene family repertoire to the closely‐related Figure 15.speciesConus Conus gloriamaris victoriae. (sharesA) Phylogenetic a near-identical tree of various toxin Conus gene species family highlighting repertoire close to relatedness the closely-related species Conusof C. gloriamaris victoriae.( andA) C. Phylogenetic victoriae (subgenus tree ofCylinder various). NeighborConus‐speciesjoining tree highlighting of concatenated close 12S, relatedness 16S of C. gloriamarisand COIand sequencesC. victoriae was(subgenus generated (JukesCylinder‐Cantor). Neighbor-joining model) using Geneious tree version of concatenated 8.1.3. Sequences 12S, 16S and COI sequenceswere obtained was generated from GenBank. (Jukes-Cantor Conus imperialis model) was using used as Geneious an outgroup. version Branch 8.1.3. labels Sequences show were consensus support (%); (B) A comparison of the expressed toxin gene family repertoire of C. obtained from GenBank. Conus imperialis was used as an outgroup. Branch labels show consensus gloriamaris and C. victoriae. Heat map is indicative of individual conotoxin number in each gene support (%);family. (B ) A comparison of the expressed toxin gene family repertoire of C. gloriamaris and C. victoriae. Heat map is indicative of individual conotoxin number in each gene family.

Mar. Drugs 2017, 15, 145 15 of 20

The venoms of mollusc-hunting species of the cone snail have proven their potential as a source of therapeutically-relevant peptides [8] (e.g., χ-MrIA and Vc1.1). Moreover, to the drug discoverer, the comparison between the venoms of C. gloriamaris and C. victoriae highlights two important points: (i) That the venom of each species of Conus represents a near-unique library of natural products; and (ii) the venom repertoire of certain species can mirror that of closely related species, essentially representing a library of naturally-occurring analogues. This type of knowledge can be valuable in guiding targeted drug discovery efforts from Conus venoms. The toxin repertoire of C. gloriamaris (summarized in Table2) should offer rich grounds for the discovery of new functions. Of the 108 conotoxins identified, a molecular target can be confidently predicted for only a handful. Even for these, differences in the primary structure may impact subtype selectivity. For example, A-Gm1.1, which shows a sequence similarity to known nAChR blockers, would be predicted to have a similar function, but perhaps a novel subtype selectivity profile. Others belong to classes which are demonstrably bioactive (i.e., Gm9a, contryphan-Gm, CNF-Gm, U-Gm7.2, Gm10.1, and each the conotoxins of the M-superfamily), but for which a specific molecular target or mechanism of action is yet to be defined. However, the vast majority of the conotoxins presented still remain completely uncharacterized. Given the successful history of the small fraction of conotoxins characterized so far, it seems probable that a further exploration of this C. gloriamaris conotoxin library has the potential to yield new research tools, if not drug leads or therapeutics.

Table 2. Functional diversity encoded in the venom gland transcriptome of Conus gloriamaris. Each conotoxin superfamily is divided into groups according to the cysteine framework, with the number identiﬁed in C. gloriamaris and a summary of biological activity associated with each group indicated.

Cysteine # Identified in Superfamily Associated Activities Reference Framework C. gloriamaris nAChRs inhibitors, GABA receptor agonists, A I 1 B [25,58,59] α1-adrenoceptor inhibitor - XXII 1 N.D. - - XXXVI 1 N.D. - conantokin (B) Cysteine-free 1 NMDA receptor inhibitors [42] Con-insulin - 1 Insulin receptor agonists [39] conodipine - 1 Phospholipase-A2 [60] cono-NPY Cysteine-free 1 [45] Convulsions in mice (IC), conorfamide Cysteine-free 1 [47] sensory neuron depolarization conopressin Single disulfide 1 Vasopressin receptor agonists [51] I1 XI 2 NaV agonists [44] I2 XI 3 K+ channel modulators [37,38] J XIV 4 nAChR inhibitor and KV inhibitor [29] M III 7 Excitatory symptoms in mice (IC) [35,61,62] conomarphin (M) Cysteine-free 1 N.D. - (M) Single disulfide 1 N.D. - Na agonists, K blockers, Na blockers O1 VI/VII 12 V V V [63–66] or CaV blockers O2 VI/VII 17 Neuronal pacemaker modulators [19] contryphan (O2) Single disulfide 1 Ca2+ channel modulators [67–69] O3 Cysteine-free 1 N.D. - P IX 2 Hyperactivity and spasticity in mice (IC) [15] - XIV 1 N.D. - S VIII 1 5-HT3 receptor inhibitor, nAChR inhibitor [49,50] Na inhibitor, presynaptic Ca2+ channel inhibitor T V 19 V [22–24] (or GPCR modulator), sst3 GPCR antagonist - XIII 1 N.D. - - X 1 Noradrenaline transporter inhibitors [25] U VI/VII 1 Convulsions in mice (IC) [70]

CaV, voltage-gated calcium channel; GABA, γ-aminobutyric acid; GPCR, G protein-coupled receptor; IC, intracranial injection; KV, voltage-gated potassium channel; nAChR, nicotinic acetylcholine receptor; NaV, voltage-gated sodium channel; N.D., not determined; NMDA, N-Methyl-D-aspartate; sst, somatostatin. Other gene families detected for which there is no reported associated activity (B2, E, F, H, I4, N, con-ikot-ikot, MKAVA, MSRLF, MMLFM, MLSML) are omitted from this table. Mar. Drugs 2017, 15, 145 16 of 20

4. Materials and Methods A single live adult specimen of C. gloriamaris was collected from Balicasag Island in the Philippines using gill nets—a fine mesh net was laid out on the sea bottom, at a depth of ~120 m, for approximately three months, after which time the net was raised and the colonized molluscs were collected. The venom gland was dissected (~7 cm in length) and stored in RNA-later, before being transferred for storage at −80 ◦C. Total RNA extraction was performed using the Direct-zol RNA extraction kit (Zymo Research, Irvine, CA, USA), with on-column DNase treatment, according to the manufacturer’s instructions. cDNA library preparation and sequencing was performed by the University of Utah High Throughput Genomics Core Facility: Total RNA quality and quantity were first validated on an Agilent 2200 TapeStation (Agilent Technologies, Santa Clara, CA, USA). A dual-indexed library was constructed with the Illumina TruSeq Stranded mRNA Sample Prep Kit with oligo (dT) selection and an average insert size of approximately 150 bp. The library was validated on an Agilent 2200 TapeStation and using a qPCR assay (Kapa Biosystems Library Quantification Kit for Illumina, Boston, MA, USA), and was pooled in a batch of 13 samples. 125 cycle paired-end sequencing was performed on an Illumina HiSeq2000 instrument (San Diego, CA, USA) at an 80% standard cluster density. Adapter trimming of de-multiplexed raw reads was performed using fqtrim [71], followed by quality trimming and filtering using prinseq-lite [72]. Error correction was performed using the BBnorm ecc tool, part of the BBtools package. Trimmed and error-corrected reads were assembled using Trinity (version 2.2.1) [17] with a k-mer length of 31 and a minimum k-mer coverage of 10. Assembled transcripts were annotated using a blastx [73] search (E-value setting of 1e-3) against a combined database derived from UniProt (downloaded April 2015), Conoserver [14], and an in-house conotoxin library. Transcripts per million transcripts (TPM) counts were generated using the Trinity RSEM [74] plugin (align_and_estimate_abundance) and expression data were analysed using the trinity utilities: abundance_estimates_to_matrix and contig_ExN50_statistic. An in-house script was used to extract conotoxin transcripts, trim to open-reading frame, and discard redundant and partial sequences. The final list of assembled conotoxin transcripts was then manually examined using the Map-to-Reference tool of Geneious, version 8.1.7 [75]. Conotoxin precursor sequences from this Transcriptome Shotgun Assembly project have been deposited at DDBJ/EMBL/GenBank [accession: GFNK00000000]. The version described in this paper is the first version, GFNK01000000. Raw sequencing data has been deposited in the NCBI sequence read archive [SRA accession: SRR5499408].

Acknowledgments: We would like to thank Noel Saguil, Iris Bea Ramiro, and Alexander Fedosov for their help with sample collection and dissection, and the University of Utah High Throughput Genomics Core Facility for cDNA library preparation and sequencing. This work was supported in part by National Institutes of Health Grants GM 48677 (to Baldomero M. Olivera) and GM 099939 (to Mark Yandell and Pradip K. Bandyopadhyay), an International Outgoing Fellowship Grant from the European Commission (CONBIOS 330486, to Helena Safavi-Hemami), the National Natural Science Foundation of China (Grant No. 31500626, to Aiping Lu) and a pilot grant from the University of Utah Diabetes and Metabolism Center to Helena Safavi-Hemami. Author Contributions: Samuel D. Robinson, Aiping Lu, Baldomero M. Olivera, and Helena Safavi-Hemami conceived and designed the experiments; Samuel D. Robinson, Qing Li, Aiping Lu, and Helena Safavi-Hemami performed the experiments; Samuel D. Robinson analyzed the data; Pradip K. Bandyopadhyay, Mark Yandell, Baldomero M. Olivera, and Helena Safavi-Hemami contributed reagents/materials/analysis tools; Samuel D. Robinson wrote the paper with input from all authors. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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