snake venome review plus esi spectrum of pla2

8/11/2019 Snake Venome REVIEW Plus ESI Spectrum of PLA2

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Next-generation snake

venomics: protein-locusresolution through venomproteome decomplexationExpert Rev. Proteomics Early online, 115 (2014)

Juan J Calvete

Instituto de Biomedicina de Valencia,

Consejo Superior de Investigaciones

Cientficas, Jaime Roig 11, 46010

Valencia, Spain

Tel.: +34 963 391 778

Fax: +34 963 690 800

[email protected]

Venom research has been continuously enhanced by technological advances. High-throughputtechnologies are changing the classical paradigm of hypothesis-driven research totechnology-driven approaches. However, the thesis advocated in this paper is that full proteomecoverage at locus-specific resolution requires integrating the best of both worlds into a protocolthat includes decomplexation of the venom proteome prior to liquid chromatographytandemmass spectrometry matching against a species-specific transcriptome. This approach offers thepossibility of proof-checking the species-specific contig database using proteomics data.Immunoaffinity chromatography constitutes the basis of an antivenomics workflow designed toquantify the extent of cross-reactivity of antivenoms against homologous and heterologousvenom toxins. In the authors view, snake venomics and antivenomics form part of abiology-driven conceptual framework to unveil the genesis and natural history of venoms, andtheir within- and between-species toxicological and immunological divergences and similarities.Understanding evolutionary trends across venoms represents the Rosetta Stone for generatingbroad-ranging polyspecific antivenoms.

KEYWORDS:affinity chromatography antivenom antivenomics mass spectrometry snakebite envenoming

snake venom venomics

The evolutionary link in venom research

Venoms represent an adaptive trait and anexample of both divergent and convergent evo-lution [1]. The ecological advantages conferredby the possession of a venom system are evi-dent from the extraordinarily diverse range ofanimals that have evolved venoms for hunting,defense efficiency or competitor dissuasion.Every ecosystem on Earth supporting life con-tains venomous organisms, and the extant suite

of venomous animals includes over 170,000species throughout all major phyla of the evo-lutionary tree of the animal kingdom fromancient cnidarians through annelids, nemer-tines, echinoderms, mollusks, arthropods andchordates.

Snakes are represented on earth today by some3150 species, which represent a single massivediversification event that occurred after the K-Tboundary at the time of the extinction of thedinosaurs[2]. Extant snakes are found throughout

most of the world (including the oceans), exceptfor a few islands, frozen environments and highaltitudes. Venomous snakes, represented by600 or so species within Viperidae, Elapidaeand

Atractaspidinae, have developed muscularizedvenom glands and tubular front fangs [3]. Inaddition, venom in an increasing number ofrear-fanged snakes (Serpentes, superfamily Colu-broidea) has also been documented and studiedat proteomic and transcriptomic levels ( [49]andreferences therein). Furthermore, the demonstra-

tion that snakes and all lizard lineages possessingtoxin-secreting oral glands (Helodermatidae,

Anguidae, Varanidae and Iguana) form aclade [10] for which the previously suggestedname Toxicofera [11](Greek for those who beartoxins) was adopted, provided overwhelmingsupport for a single, early origin of the venomsystem in lizards and snakes [10]. The proposedcommon ancestor of venomous squamate reptilespecies may have originated at the base of thecolubroid radiation, approximately 6080 million

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years ago [2,912]. Though the role of venom of lizards and colu-brids in their feeding behavior, adaptive ecology or defence frompredators remains a matter of study and debate, the venom systemsof these taxa represent an untapped source of novel-led com-pounds potentially useful in drug design and discovery. In addi-tion, despite the venoms of front-fanged snakes provide modelsystems for investigating predatorprey interactions, the molecularbases for evolutionary and ecological adaptations, the generation ofchemical and pharmacological novelty [13] and strategies for theknowledge-based design of antivenoms to reduce the burden ofthe neglected pathology of snakebite envenoming that kills ormaims thousands of healthy individuals every year [14,15], onlyrecently has the research on venoms began to flourish [13,16]. Dur-ing the last decade or so, advances in instrumentation and high-throughput methodologies have fueled an expansion of the scopeof biological studies and strategies for assessing the toxin composi-tion of snake venoms (snake venomics), directly (throughproteomics-centered approaches) or indirectly (via venom gland

transcriptomics and bioinformatic analysis) in a relatively rapidand cost-effective manner. For an overview of snake lineages forwhich proteomics or transcriptomics venom composition datahave become available, please consult [6]and Table 1 of[16]. Snakevenomics on a few more taxa have been reported since the publica-tion of this review[16]and can be consulted addressing the NCBIsPubMed database [17]. More recently, the genomes of the worldslongest venomous King cobra (Ophiophagus hannah) and the non-venomous Burmese python (Python molurus bivittatus) have beensequenced [18,19], and other snakes have been targeted for genomesequencing [20]. These studies provide an insight into the biologyof the venom in snakes and allow the understanding of the evolu-tion of venom genes at the genome structural level. The Burmese

python and King cobra studies represent a significant addition tosystems genomics and the foundation of the field of comparativesnake genomics. Thus, comparison between these genomes hasrevealed dynamic evolution and adaptation in the snake venomsystem, which seemingly occurs in response to an evolutionaryarms race between venomous snakes and their prey. These adapta-tions include the massive and rapid expansion of gene families thatproduce venom toxins that correlate directly with their functionalimportance in prey capture.

Refined during eons of biological evolution, snakes produce astaggering number of compounds in their venoms with therapeuticand pharmacological potential, a fact that is attracting increasinginterest in academic, industrial and medical arenas [21]. Their highmolecular specificity and potency have long made venom a promis-ing source of novel compounds for use in drug design and develop-ment. More than 30 years ago, the US FDA approved the firstvenom-derived drug, a therapy for hypertension, called Captopryl,developed from bradykinin-potentiating peptides isolated fromvenom of the South American pit viper Bothrops jararaca [21]. Ven-oms exhibiting anticoagulant properties are extensively studied forpossible medical applications, and a handful of venom-deriveddrugs, such as Tirofiban, Eptifibatide, Batroxobin, has alreadybeen approved for cardiovascular diseases [21]. The pipeline fromfang to pharmacy is expanding with the discovery of peptides

isolated from black mamba venom that block neuronal acid-sensingion channels that play a key role in the pain pathway[22].

The basic and applied aspects of venom research represent twosides of the same coin: understanding the principles governing theevolution of venomous systems is not only of medical relevance, forunderstanding the molecular basis for adaptive variations in snakevenom phenotypes is of outmost importance for improving currentantivenoms, and also for learning how to use deadly toxins as thera-peutic agents. Developing the full potential of venom researchrequires the integration of data across the biological system withinthe frame of an evolutionary hypothesis. Thus, although initiallyconceived as a technological platform for the proteomics characteri-zation of the chemical space [23], natural history and immunoreactiv-ity of snake venoms with homologous and heterologous antivenoms[24,25], the inclusion of the evolutionary hypothesis for clustering ven-oms based on within- and between-species shared traits and trends,has catalyzed the revival of snake venomicsas a conceptual frame-work for the comprehensive analysis of venoms [16]. This review

focuses on the concepts and technological alliances that guided thistransition from the mere inventory of toxins to biology and discussesforeseeable future developments in the field.

Decomplexing the venom proteomes: an opportunity

to quantify the relative abundances of the venom

components

Snake venom toxins originated by duplication of ordinary genesand the accidental expression of the duplicated copies in thevenom gland, followed by amplification of the co-opted genes tomultigene families via a birth and deathmode of evolution [26],and the rapid diversification of these families by extensive neo-functionalization of some copies and the transformation into

pseudogenes of nonfunctional forms [1,2,2729]. As a result of this,after approximately 100 million years of evolution, venoms ofextant snakes comprise moderate complex mix of


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of and immunoreactivity trends across venoms represents theapproach of choice.

The development of sample preparation protocols (nano-uPLC,multidimensional-high performance liquid chromatography(HPLC), 2D electrophoresis [2DE]) coupled to soft ionization(matrix-assisted laser-desorption ionization and electrospray ioniza-tion) mass spectrometry has been pivotal to get an accurate pictureof the startling complexity of venoms [44]. Different approacheshave been applied to unlock the peptide and protein compositionof a number of snake venoms including shotgun proteomics (alsoknown as discovery proteomics) and hyphenated separation tech-nologies [36,37]. In a shotgun data-dependent acquisition (DDA)method, the first scan is a survey scan (full MS scan) where theprecursor ions are isolated and subsequently activated. Theobtained fragments are analyzed in a second stage (data-dependentscan) of mass spectrometry. In many sample types, including ven-oms, the complexity and dynamic range of compounds can bevery large. This poses challenges for the traditional data-dependent

workflows, requiring very high-speed tandem mass spectrometry(MS/MS) acquisition to deeply interrogate the sample. Data-independent acquisition (DIA) strategies have been used toincrease the reproducibility and comprehensiveness of data collec-tion. In DIA mode, an expanded mass isolation window is steppedacross a mass range covering the mass-to-charge distribution ofpeptides, and all ions present at a given time are activated and dis-sociated without selection. In this case, instead of the serialcollision-induced dissociation (CID) of peptide ions, parallel CIDof ion mixtures take place in. A large mass range can be interro-gated in an liquid chromatography (LC)MS time frame becauseof the larger mass steps. To assign subsequent fragments to correctprecursors, data are continuously acquired by alternating between

high (precursor filtering) and low (dissociation and fragment filter-ing) voltage potentials [45]. This strategy is also called LCMSE

[46]. Another variant of the emerging class of DIA workflows isMS/MSALL with Sequential Windowed data-independent Acquisi-tion of the Total High-resolution Mass Spectra (SWATH

TM

-MS)[47]. In SWATH-MS mode, a 25 amu window is transmittedthrough the MS1 analyzer into the collision cell. MS2 data areacquired by repeatedly cycling through 32 consecutive 25 Daprecursor isolation windows (swaths), which cover the full4001200 m/z range and monitoring all fragment ions. The highresolution of MS2 spectra (10 p.p.m.) ensures the specificity ofpeptide identifications. For a comparative overview of the MSinstrumental principles of conventional shotgun proteomics andSWATH MS analysis, refer [48].

Tryptic digestion of the whole venom followed by LCCIDMS/MS, no matter whether they are acquired by DDA or DIAmode, allows the identification of the toxin classes present in thevenom but does not inform about the quaternary structure of indi-vidual toxins. In addition, a limitation inherent to this methodol-ogy is its inability to distinguish among protein family membersexhibiting high levels of sequence redundancy. Further, quantifica-tion by label-free spectral counting of the relative abundances ofvenom components based on the information provided by theshotgun MS data set on the types, number and ion intensities of

the different peptide ion spectra associated with each protein byMS/MS, requires correction for differential MS detectability of thecontributing peptides (i.e., due to sequence properties that affectpeptide ionization) and protein size (larger proteins contributemore peptides) or the addition of proteotypic peptides.

2D electrophoretic analysis provides a more realistic view ofvenom complexity. However, small peptides are essentially lost,and colorimetric quantification of individual toxins or toxin fami-lies is not straightforward. Thus, despite the existence of sophisti-cated software tools, 2DE gel image analysis still remains a seriousbottleneck, which traditionally involves a preprocessing step to sup-press noise, correct background and remove artifacts; spot bound-aries delineation; and expression quantification. The latter step isdone by estimating the relative spot volume: Vspot_i/

PVspotsin the

2DE gel = mass [in SI units, g]% of spoti. On the other hand, frac-tionation of venom components by chromatographic methods(usually reversed-phase HPLC) prior to MS analysis (FIGURE 1; step1) is a powerful approach for proteome decomplexation, and in

conjunction with apparent molecular mass determination by SDSPAGE under reducing and nonreducing conditions (FIGURE1;step 2)provides a true appreciation of both the proteolytic processing andquaternary structure of the venom proteins [23]. Reversed-phaseHPLC represents the method of choice for the fine separation andquantitative recovery in a single run of peptides (0.47 kDa) andproteins (7150 kDa) commonly present in snake venoms. Inaddition, the fact that the chromatographic fractions are obtainedin a solvent compatible with downstream mass spectrometricanalysis, allows the (on-line or off-line) determination of theaccurate molecular mass of the native venom components (FIG-URE 1; step 3) and their number of reduced (sulphydryl groups)and oxidized (disulfide linkages) cysteine residues [23]. The

binomial molecular mass and number of disulphide bonds isa toxin-specific parameter that discriminates between mosttoxin classes found in snake venoms [23].

An important challenge in the characterization of venoms isthe inability of any single method to analyze all unique pro-teins in the venom proteome. However, the need to decomplexthe venom proteome represents also an opportunity to quanti-tate the relative abundances of the different venom compo-nents. Thus, by monitoring the reversed-phase column eluateat the absorbance wavelength of the peptide bond, 215220 nm, peak area integration allows an estimation of the rela-tive abundances (expressed as percentage of the total venomproteins) of the different chromatographic fractions (FIGURE 1;step 4). According to the LambertBeer Law, A = ecl; (wheree = molar extinction coefficient [M-1cm-1], c = concentration[M], and l = path length [cm]). Expressed in this form, theextinction coefficient allows for estimation of the molar concen-tration of a solution from its measured absorbance: A/e = molarconcentration. The calculated figures (%A of component i) cor-respond thus to the mol% of peptide bonds in component i. Therelative contributions of different proteins eluting in the same chro-matographic fraction can be estimated by densitometric scanningafter SDSPAGE gel analysis (FIGURE1; step 2). These figures shouldclosely match transcriptomic-based measurements of the contents

Next-generation venomics Review

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m/z,amu

m/z,amu

1500155016001650

A

B

Review Calvete

doi: 10.1586/14789450.2014.900447 Expert Rev. Proteomics


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of venom toxin transcripts, if the number of reads mapping to aparticular transcript are used as a measure of its abundance [49].However, to estimate the relative contribution of each toxin ortoxin family as protein molecules/100 molecules of total venomproteins, the molar percentages of peptide bonds of each familyshould be normalized for the number of peptide bonds (aminoacids) in the full-length sequence of a representative member of theprotein family. This way of expressing the relative concentrations oftoxins in the venom allows the direct comparison of proteomic andtranscriptomic data if the relative expression of a given toxin pro-tein (family) (mol%) is calculated as the number of reads assignedto this protein (family) (Ri) normalized by the length (in nucleoti-des [nt]) of the reference transcript sequence (ntREF) and expressedas the percentage of total reads in the transcriptome (Sreads): mol% toxin (family) i= %[(Ri/ntREF)/Sreads) [32].

It must be stressed that quantitative venomic approaches onlyyield relative, not absolute, toxin quantification. The bestapproach currently available for absolute quantitation of a particu-

lar venom component in venom involves the generation of stan-dard curves for synthetic stable isotope-labeled proteopeptidesadded as internal standards to the sample [5052]. In a typicalAbsolute Quantification strategy, the sample is spiked withdefined amounts of isotope-labeled analog(s) of specific proteo-lytic peptide(s), digested and analyzed by shotgun LCMS.Quantification can be performed in the MS mode by comparingthe extracted ion signal (peak height or peak area) of the isotopelabeled and the native forms of a given proteotypic peptide. In thetandem mode (MS/MS), a comparison of ion fragment signalsfrom standard and native peptides can also be performed forquantification [41,42]. Absolute quantification of full-length pro-teins would require a Protein Standard Absolute Quantification

strategy. Although an isotope-labeled equivalent of the full-lengthtarget protein appears to constitute the standard of choice, a cur-rent limitation for applying the Protein Standard Absolute Quan-tification method in a quantitative venomics protocol is thedifficulty to produce natural (chemically labeled) or recombinant(isotope-labeled) venom protein standards.

Species-specific transcriptomics: the Rosetta Stone for

achieving locus resolution in venomics analysis

The presence in the same venom of a diversity of proteins of thesame family but differing from each other in their amino acidsequences and pharmacological activities reflects the accelerated

adaptive molecular evolution of snake venom toxins via positiveselection [13,53,54]. The occurrence of multiple isoforms within eachmajor toxin family evidences the emergence of paralogous groupsof multigene families across taxonomic lineages where gene dupli-cation events occurred prior to their divergence and suggests animportant role for balancing selection in maintaining high levels offunctional variation in venom proteins within populations [55].Omics analysis of venoms across and between different generaoffers the possibility of understanding the molecular evolution oftoxins, the ecological forces and mechanisms driving venom vari-ability, and the relationship of the components in the venom andthe pathological outcomes of snake envenomings, thereby pavingthe way for developing a new generation of broad-range polyspe-cific antivenoms that are clinically more effective [14,15].

Biologically motivated questions require genome/transcriptomeannotation. However, in these 10 years of first-generation snakevenomics [16,24,56], high-throughput proteomic studies on snakevenoms have been somewhat hampered by the lack of species-

specific venom sequence databases to match the MS/MS data. Not-withstanding this drawback,de novo interpretation of high-qualitypeptide ion fragmentation spectra (FIGURE 1; step 5), in conjunctionwith Basic Local Alignment Search Tool analysis of the MS/MS-derived amino acid sequences (FIGURE 1; step 6), usually allows theunambiguous identification of an homolog protein in the currentdatabases[23,24]. The relative abundance (%) of each protein familyrepresented in the venom can be computed from the relation of thesum of the areas of the reversed-phase chromatographic peaks con-taining proteins from the same family to the total area of venomprotein peaks in the reversed-phase chromatogram (FIGURE1;step 7).

Incomplete sequence coverage resulting from the low-throughput venomics approach described above and schematized

in FIGURE 1, steps 17 yields toxin family resolution, being usuallyunable to distinguish between different isoforms or proteoformsof toxin family members due to extensive sequence similarity. Toa certain degree, this is being addressed by the production oftranscriptome databases from snake venom glands. However, theavailability of snake venom gland transcriptomes has increasedslowly since the pioneer work of Ho and co-workers in 1995 [57].For a list of snake species for which transcriptomic, proteomicand joint transcriptomic and proteomic analyses have beenreported, please refer Table 1 in reference [16].

The majority of the snake venom gland transcriptomic studiesreported to date involve the low-throughput sequencing of clones

Figure 1. Scheme of the second-generation snake venomics platform. Venom proteins are separated by reversed-phase HPLC (1)and analyzed by SDSPAGE under nonreduced and reduced conditions (2A), and ESIMS (3). The relative abundance of the proteinseluting in the reversed-phase separation are quantitated by combination of densitometry (2B) and LambertBeer law (4). Electrophoreticbands are in-gel digested with trypsin and the peptide ions in the digests de novo sequenced by data-dependent CID-MS/MS (5) followedby protein family identification via BLAST search (6) or data-independent MSE (8) and product ion spectra matching to a species-specificvenom gland transcriptomic database (9). Combining qualitative (protein family or locus identifications) (7) and quantitative (relativeabundances derived from absorption data by the LambertBeer Law, c = A/eL) (4) data allow an accurate overview of the toxincomposition of the venom. Comparison of experimental ESIMS toxin masses (3) and those calculated for the species-specifictranscriptome-predicted full-length proteins (10) is a simple and accurate method for discriminating between protein isoforms whoseamino acid sequences share a high degree of identity.BLAST: Basic Local Alignment Search Tool; CID: Collision-induced dissociation; LCMS: Liquid chromatographymass spectrometry;MS/MS: Tandem mass spectrometry; RP-HPLC: Reverse-phase high performance liquid chromatography.




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randomly picked from a cDNA library constructed by reverse tran-scription of the RNA molecules expressed in the venom gland [58].These partial expressed sequence tags cluster into groups of contig-uous sequences (contigs), which only occasionally cover the entireextension of the original RNA molecule [58]. Recently, a significanteffort has been directed at generating comprehensive high-throughput venom gland transcriptomic databases comprising full-length toxin-coding transcript sequences through next-generationsequencing platforms [47,56,59]. Next-generation sequencing tech-nologies are revolutionizing the field of transcriptomics by rapidlyreducing the time and cost per base sequenced.

The most complete characterizations to date of the genesexpressed in active venom glands have been achieved with Illuminatechnology [33,49,59]: using 95,643,958 pairs of quality filtered,100 bp Illumina reads, Rokyta and co-workers [49] identified123 unique, full-length toxin-coding sequences in the venom glandtranscriptome of an eastern diamondback rattlesnake (Crotalus ada-manteus). Subsequent nanospray LC/MSE analysis of a whole

trypsin-digested venom sample yielded peptide evidence for 52 ofthe 78 unique toxin transcript clusters although only 36 toxinswere unambiguously identified based on unique peptide evi-dence [33]. Clearly, the availability of species-specific full-lengthvenom gland transcript sequences as a reference database has greatlyenhanced the efforts of MS-based venom proteomics, circumvent-ing the need for de novo MS sequencing. However, maximizingproteome coverage at locus-specific resolution requires the integra-tion into a single second-generation venomicsprotocol of the ben-efits of coupling venom proteome decomplexation (FIGURE 1; steps1 & 2) and matching the output of DDA or DIA shotgun MS/MSmass spectrometry against the species-specific transcriptome (FIGURE1;steps 8 & 9). For example, through the combination of RP-HPLC

separation, SDSPAGE analysis and MS/MS characterization ofthe venom components, Wagstaff and colleagues reported the pres-ence of a multidomain PIV metalloproteinase in Echis ocellatus[60].In a shotgun approach, the snake venom metalloprotease (SVMP),disintegrin-like, cysteine-rich and C-type lectin-like (CTL)domains, would have been detected separately, and the informationon the quaternary structure of this metalloproteinase, which consti-tutes about 20% of the venom toxins, is lost. Furthermore, sincethe covalent linkage of CTL domains to a PIII-SVMP is a post-translational modification[6163], the quaternary structure of a PIV-SVMP is not reflected in the transcriptome. The key to identifyingthis class of SVMPs is as simple as an SDSPAGE analysis, undernonreducing and reducing conditions, of the RP-HPLC-isolatedvenom fractions, followed by MS/MS identification of domain-specific peptides in the nonreduced protein band [60].

The identification of highly homologous proteins eluting in dif-ferent chromatographic fractions or migrating differentially onSDSPAGE may indicate the coexistence of protein isoforms inthe venom sample. Accurate determination of the molecularmass (FIGURE 1; step 10) of protein isoforms whose amino acidsequences share a high degree of identity may be key to identifythe mRNAs that encode them in a species-specific transcriptome[51,6466]. Linking transcriptome and proteome by mass profilingare particularly useful in the case of toxin families such as

disintegrin, phospholipase A2 (PLA2), three-finger toxin, snakevenom metalloproteinase of class PI (PI-SVMP), CTL, cysteine-rich secretory protein, Kunitz-type inhibitor, cystatin, ohanin, b-defensin-like myotoxin (refer Table 1 in [16]), in which the onlydocumented post-translational modification is the formation ofdisulfide bonds. In a recent paper, Conlon and colleagues [66]iden-tified transcripts encoding cytotoxic PLA2 molecules in species-specific transcriptomic databases using a combination of electro-spray ionizationMS measurement of the masses of the nativeHPLC-isolated proteins and LCMSE analysis of tryptic digests.Venoms from a number of terrestrial and marine elapids are rela-tively simple, comprising distinct repertoires of toxins typicallybelonging to two major protein families, PLA2and short and longthree-finger toxins [4,34,6773]. LC separation of venom componentscoupled to mass spectrometry fingerprinting is a very simple andaccurate method for quantifying and bridging the gap betweenproteome and transcriptome. Determination of similarities anddifferences in the composition of individual molecules across the

geographical range of a species through mass profiling may alsorepresent a useful tool in chemotaxonomy[67]and for inferring[74]or complementing [75] phylogeographical patterns generated bygenetic (mtDNA) approaches [76].

Proteotranscriptomics: proof checking the quality &

annotation accuracy of a species-specific contig

database using proteomics data

An interesting derivation of the next-generation snake venomicsprotocol described above is the possibility of using the proteo-mics data to check back the accuracy of the original transcrip-tome assembly and translation (FIGURE 2). Thus, when the set of(tryptic) peptides derived by proteolysis of an SDSPAGE-

separated protein band followed by data-independent MS/MSacquisition (FIGURE 2; step 2) do not match a single species-specificcontig sequence (FIGURE2;step 3), three distinct scenarios are possi-ble: (i) some peptides are shared between different proteins eachof which also possess unique peptides (FIGURE 3A); (ii) the set oftryptic fragments comprises different subsets of peptides thatmap onto nonoverlapping regions of different contigs (FIGURE 3B);and (iii) different set of tryptic peptides match sequences coveredby different reading frames of the same contig(FIGURE3C). The firstpossibility clearly points out to the presence of a mixture ofhomologous proteins in the same electrophoretic band. Possibil-ity iiwould indicate the erroneous assembly of contigs B1 andB2 (i.e., due to false overlap of short reads), whereas scenario iiiis seemingly due to a read sequencing error leading to a readingframe shift. All these assembly errors can easily be detected andcorrected by carefully annotating the transcriptomic data basedon proteomic findings (FIGURE 3; steps 5 & 6). Although lowthroughput and time-consuming, the resolving power of check-ing back the contig database using proteomics data, critically relieson implementing strategies to decomplex the venom proteome,ideally achieving the level of individual toxins, prior to LCMS/MS analysis. Digesting the venom proteins without any samplefractionation step greatly suppresses any downstream opportunityof bridging the gap between proteome and transcriptome.

Review Calvete



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Proteotranscriptomics.

Proposedschemeforusingtheproteomicsinformationgatheredthroughasecond

-generationsnakevenomicsworkflow

displayedinFIGURE1

toreassesstheassemblyqualityandannotationofaspecies-specifictranscriptomicsdatabase.

MatchingtheMS/MS-d

erivedpeptidesequences(steps1&2)againstaspecies-

specifictranscriptomicdatabaseallowsthedetectionofmixtureofisotoxins

(4A),misassembledcontigs(4B)andtrans

lationframeshifts(4C).Transcriptomesequenceerrorscan

thenbecorrectedbymatchingth

eproteomicsdatatoachievetranscriptom

eproteomeconcordance(FIGURE3;steps5&6).

MS/MS:Tandem

massspectrometry.




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The term proteogenomics has been coined [7780] to describethe mapping of MS/MS-identified peptides to the specificgenome locus coding for these amino acid sequences. By anal-ogy, the use of proteomics data to reassess the quality of the six

possible translation frames of a species-specific transcriptomicsdatabase, and to match MS/MS-derived peptide sequences totranscriptomics loci, thereby contributing to accurate assemblyand locus annotation, should be called proteotranscriptomics.

Antivenomics: a translational venomics platform

Snakebite envenoming is largely a neglected threat to publichealth in tropical and subtropical regions of Africa, Asia, Latin

America and Oceania, affecting some of the worlds poorest ruralcommunities. An estimated 5.5 million people are bitten bysnakes each year, resulting in about 400,000 amputations, and

between 20,000 and 125,000 deaths; however, the true scale ofthis disease of povertymay be much greater than these hospital-based statistics [14,8184]. Despite these figures, which affect mainlypeople involved in subsistence farming activities in vast regions of

the world, the burden of human suffering caused by snakebiteshas only been added to the WHOs list of neglected condition in

April 2009 [85]and represents the most neglected of the neglectedtropical diseases [14,15]. The timely parenteral administration of anappropriate antivenom remains for more than a century after thedevelopment of the first serum antivenimeuxby Calmette [8689],and Phisalix and Bertrand [9092], the only currently effective treat-ment for snakebite envenomings [93]. Poor access to health servicesin these settings and, in some instances, a scarcity of antivenomoften leads to poor outcomes and considerable morbidity andmortality[83,84].

2.0

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Cerastes cerastes (Morocco)

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Figure 3. Immunocapture efficacy of the F(ab)2 antivenom CcMo_AV toward Moroccan Cerastes cerastes venom. Immunore-

activity of the Moroccan monospecific (C. cerastes) CcMo_AV toward proteins from the venom of C. cerastes from Morocco assessed bysecond-generation antivenomics.(AC)Show, respectively, reversed-phase separations of 0.25 mg of the whole venom; the nonimmuno-captured venom components; and the proteins retained and recovered from the F(ab)2 antivenom affinity matrix. Column eluates weremonitored at 215 nm. Proteins within the immunocaptured and the flow-through fractions were identified by the venomics approachdescribed in the text and schematized in F IGURE1, and quantified by comparing the areas of homologous peaks in the two fractions.CTL: C-type lectin-like; DD: Dimeric disintegrin; LAO: L-amino acid oxidase; PIII-SVMP: Snake venom Zn2+-metalloproteinase of class III;PLA2: Phospholipase A2.Adapted from FIGURE4 of [104]. Picture ofCerastes cerastes: author, H Krisp, Ulm, Germany; reproduced from [120] under Creative CommonsAttribution 3.0 Unported (CC BY 3.0).

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Because each snake possesses its ownvenom with a distinctive mix of chemicals,an antivenom developed for one snake maynot work against the venom of another spe-cies. To complicate matters, the evolutionof venomous species and their venoms donot always follow the same course, and theidentification of structural and functionalconvergences and divergences among ven-oms is often unpredictable by a phyloge-netic hypothesis. Thus, a key issue in themanufacture of antivenoms is the selectionof the venoms that are used in the immu-nizing mixtures [14,94]. Owing to the largeintra- and interspecific variability in venomcomposition [95], it is necessary to ensurethat the venoms used for immunizationgenerate antibodies effective in the neutrali-

zation of the most medically relevant toxi-nological activities in a geographical settingof deployment. The study of the preclinicalefficacy of antivenoms has experienced asignificant evolution in the last decades. Ini-tially, the only test performed to ensuresuch efficacy was the neutralization oflethality, using animal models, most fre-quently mice [95]. Although this testremains as the gold standard for antivenomin neutralizing efficacy[9698], the complex-ity of the pathophysiology of snakebiteenvenomings demands a more meticulous

analysis of efficacy, based on the testing of neutralization of othertoxic effects in addition to lethality. Hence, simple experimentalprotocols have been implemented for the analysis of the neutraliza-tion of hemorrhagic, myotoxic, coagulant, edema forming anddefibrinogenating activities, as well as for the neutralization ofenzymatic activities such as proteinase, PLA2 and hyaluronidase(reviewed in [96]). On the other hand, the design of venom mix-tures should be based on a rigorous analysis of epidemiological,clinical, proteomic, immunological and toxicological information.In this sense, knowledge of the ontogenetic, individual and geo-graphical intraspecific venom variability has applications for thequality control of the reference venom pool for generating newand more effective broad-ranging antidotes [94]. On the otherhand, assessing the cross-reactivity of a polyvalent antivenomagainst venoms not included in the immunization mixture mayaid in expanding their range of clinical application [14,94]. The com-bination of antivenomics and neutralization assays of toxic activi-ties provides a powerful methodological platform to analyze, indepth, the preclinical efficacy of antivenoms.

Confronting the problem of snakebite envenoming requires thefull potential of combining venomic tools and preclinical testing ofantivenom efficacy using functional neutralization assays [96]. Inthis respect, our laboratory collaborates with the Global SnakebiteInitiative [14,15,94,99,100], an international collaborative project

aimed, among other goals, at developing new regional polyvalentantivenoms for Asia and Africa. We have developed a proteomics-centered tool, termed antivenomics, for aiding in the design ofoptimized immunization venom mixtures and assessing the immu-nological cross-reactivity of antivenoms toward homologous andheterologous venoms [25,101103]. The most recent antivenomicsworkflow, dubbed second-generation antivenomics, consists of astep of immunoaffinity chromatography step on Sepharose-coupled IgG, F(ab)2, Fab or Fab molecules, followed by theproteomic analysis of the nonimmunocaptured (flow through frac-tion) and the immunocaptured fractions (FIGURE4) [25]. The percent-age of a particular venom toxin retained on the immunoaffinitycolumn represents a measure of the preclinical potential of the

immobilized antivenom against that venom toxin present in thesampled venom. This figure can be experimentally derived as 100-([NRi/(Ri+NRi)] x 100), where NRi and Ri are the chro-matographic peak areas of toxin i in the chromatogram of thenonretained and the immunocaptured and eluted affinity columnfractions recovered in steps 2 and 3 of FIGURE 4A. Experiments inwhich venoms are incubated with mock matrix and with matrix-coupled preimmune antibodies, run in parallel to the immunoaf-finity antivenomics analysis, serve as matrix and immunospecificitycontrols, respectively (FIGURE 4B). Its quantitative character andmolecular resolution suggest the possibility for antivenomics of

Step 1 Step 2 Step 3

Antivenomimmobilization

Incubation ofvenom and

antivenom

Elution

Non-retainedvenom components

Immunocapturedvenom

components

Venom

Antivenom

NHS-sepharose

Venom

Non-specific IgG

NHS-sepharose

Controls

Matrix control Specificity control

Analysis of retained and non-retainedvenom components

A B

Figure 4. Second-generation antivenomics.Scheme of the immunoaffinity capturingantivenomics protocol developed by Pla et al. in 2012 [25]. (A) Whole venom is appliedto an immunoaffinity column (step 2) packed with antivenom antibodies immobilizedonto Sepharose beads (step 1). After eluting the nonretained venom components, thecolumn is thoroughly washed and the immunocaptured proteins eluted (step 3). (B)Cartoon of the specificity controls of the immunoaffinity-based antivenomics protocolschematized in(A). Mock Sepharose 4 Fast Flow matrix, (matrix control) or Sepharosebeads coated with preimmune IgG molecules (immunospecificity control), was incubatedwith venom and developed in parallel to the immunoaffinity column. Qualitative andquantitative analysis of the column eluates are illustrated in F IGURE3.NHS: N-hydroxysuccinimide.




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supplanting the use of immunoassays and western blots, the mostpopular techniques for assessing the immunoreactivity of antibod-ies. The capability of this simple knowledge-based analyticalmethod to formulate hypotheses as to how improved venom mix-tures might be designed or redesigned for the manufacture ofimproved therapeutic antivenoms has been documented in a num-ber of investigations in recent years (FIGURE 3) (reviewedin[16,103,104]). The following examples illustrate this point.

The ability of EchiTAb-Plus-ICP antivenom to immunode-plete and neutralize the venoms of African spitting cobras was alsoassessed by antivenomics and neutralization tests [25,71,96]. Theantivenom neutralized the dermonecrotic and PLA2 activities ofall African Naja venoms tested, whereas lethality was eliminatedin the venoms ofNaja nigricollis,Naja mossambicaandNaja pal-lida, but not in those ofNaja nubiaeand Naja katiensis. Antive-nomics analysis indicated that the impaired binding capability ofthe antivenom to a type-1 a-neurotoxin, high abundant in N.nubiae(12.6% of the total venom proteome), N. katiensis(4.4%)

and N. pallida (2.8%) venoms but absent in the N. nigricollisvenom used in the immunization mixture, appears to be responsi-ble for the inability of EchiTAb-Plus-ICP antivenom to neutralizelethality of ofN. nubiaeandN. katiensisvenoms. The antivenom-ics results suggest that N. nubiaevenom should be included aspart of a improved immunization mixture, either in addition ofor substitutingN. nigricollisvenom.

Hypotheses are proposed explanations for observable phenom-ena. The scientific method requires that one can test them, andvenomics- and antivenomics-based hypotheses are no exceptions.In this regard, recent genus-wide venom proteomics analyses acrossLachesis revealed a high conservation of the overall composition ofCentral and South American bushmaster venoms [105]. This find-

ing suggested that a monospecific antivenom generated against thevenom of any Lachesis species may exhibit paraspecific protectionagainst the toxic activities of all other venoms of congeneric spe-cies [105]. This venomics-guided hypothesis was validated in a sub-sequent study in which the paraspecificity of two antivenoms,produced at Instituto Vital Brazil (Brazil) and Instituto ClodomiroPicado (Costa Rica) using, respectively, Lachesis muta rhombeataandL. stenophrysin the immunization mixtures, was assessed usinggenus-wide comparative antivenomics [106]. Similarly, a combinedvenomics and antivenomics investigation on the venoms of theTaiwanese snakes, Protobothrops mucrosquamatusand Viridoviperastejnegeri, showed that a bivalent antivenom generated against thesevenoms was unable to neutralize the lethality induced by P.mucrosquamatus[107]. This deficiency was associated by antivenom-ics analysis with the antivenoms inability to recognize the crotoxinB-like neurotoxic PLA2, trimucrotoxin, which accounts for 5.2%of the total venom proteins ofP. mucrosquamatus[107]. This find-ing suggested that neutralization of the neurotoxic activity of tri-mucrotoxin should neutralize the lethal activity of P.mucrosquamatusvenom. This hypothesis was verified by showingthat the anti-Crotalus durissus terrificus antivenom produced atInstituto Butantan (Sao Paulo, Brazil) immunorecognized trimu-crotoxin and neutralized the lethal activity ofP. mucrosquamatusvenom [107]. The corollary of this research is that the bivalent

antivenom can be improved through enrichment of the immuniza-tion mixture with a trimucrotoxin cross-immunoreacting PLA2molecule, such as the South American rattlesnake crotoxin.

Expert commentary

The increasing availability of comprehensive venomic, transcrip-tomics and genomic databases for studying the interplay betweenophidian phylogeny, biogeography and organismal biology offersan unprecedented opportunity to address fundamental questionsabout the natural history of snakes and their venoms such as:How did snakes evolve and what processes govern the generationof venom convergences and divergences we see today? How dosnakes come to be adapted to their environment? Why do differ-ent snakes use distinct venom formulations to subdue similarprey? Like the story of the blind men who described differentlywhat an elephant looked like because each one touched a differentpart of the elephants body, separately eachomicsapproach revealsonly part of the system under study. Nothing in biology makes

sense except in the light of evolutionis a 1973 essay by the evolu-

tionary biologist Theodosius Dobzhansky [108]. The central issueof this essay criticizing antievolution creationism and espousingtheistic evolution, applies seemless to all fields of biology includ-ing venomics. The fact that evolution occurs explains the interre-latedness of the various facts of biology, and thus understandingthe molecular bases that have shaped the variation in venomoussnakes requires a joint analysis of the different omics data setswithin an evolutionary framework. The realization that conver-gence in venom composition between the two marine snake line-ages is driven by their parallel specialization for a fish-specific dietexplains the remarkable cross-reactivity between sea snake anti-venom and sea krait venom [109]. On the other hand, the com-

monly occurring compositional variability of venoms from widelydistributed and highly adaptable terrestrial snake species is of par-ticular medical concern, for allopatric venom variability representsa source of diversity of the pathological effects of envenoming.The environmental and molecular mechanisms generating thisdiversity remain elusive, though a robust knowledge of the venomarsenal within an accurate phylogeny and a detailed biogeographi-cal pattern, along with the recognition of convergent and diver-gent trends along the venomsevolutionary history, are of appliedimportance for selecting the most suitable species and specimensfor the production of improved therapeutic antivenoms. The evo-lutionary hypothesis represents the Rosetta Stone to bridge thegap between the inventory of toxins and biology.

Five-year view

The recent sequencing of the King cobra [18] and Burmesepython [19]in conjunction with the publication of the genomes of

Anolis carolinensis[110,111]and the American alligator,Alligator mis-sissippiensis, the gharial Gavialis gangeticusand the saltwater croco-dileCrocodylus porosus [112], has barely begun to clarify aspects ofthe biology of Archosauria (birds and crocodilians) and Squamata.

With over 9000 species among 61 families, squamate reptiles (liz-ards and snakes) are one of the most diverse group of vertebrates.Squamates offer outstanding model systems in ecology and

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evolution. As recent advances in sequencing technologies havegreatly reduced the time, cost and difficulty of generating novelgenomes, the next 5 years will undoubtedly witness the establish-ment of significant genomic resources for more nonavian reptilespecies including venomous and nonvenomous snakes. Compara-tive genomics between birds, reptiles and mammals will provideinsights into over 300 million years evolution of amniotes [113]including a key perspective on the implication of retroelements inthe genesis, genomic organization and evolution of venomous sys-tems during the last 200 million years [2,114]. The explosion ofsequencing and annotation of complete nonavian genomes willmake the coming years exciting for evolutionary toxinologists.

Genetic variation is the driving force of evolution, and the nat-ural selection acting on the organisms phenotype, the primarymechanism for adaptation of the heritable variation that results infitness differences. Venom represents a trophic adaptive trait, cru-cial to the foraging success of the snake. Bridging the gap betweengenotype and phenotype requires both, a well-resolved phylog-

eny[115]and an understanding of the mechanisms controlling theaccelerated evolution of venom toxins [116], and the regulatorycomponents of the venom secretory system determining the pat-tern of transcript abundance and translation. miRNA-mediatedpost-transcriptional modulation of the venom transcriptome hasbeen hypothesized to contribute to venom evolvability withoutlarge-scale alterations of the underlying gene expression machin-ery[117]. The next 5 years will provide evidence to substantiate orrefute this hypothesis. This will require same species tissue-specifictranscriptomic and small RNA deep sequencing.

Deconstructing complex molecular phenotypes, such as snakevenoms, is within the reach of current omictechnologies. Para-doxically, the increased capabilities (and shorter duty cycle) of

mass spectrometers have led to high-throughput proteomicsincreasingly functioning as a black box [118]. The increasing

availability of full-length genomic and transcriptomic databaseswithin the next 5 years will contribute to the explosion of venomproteomics. Genus-wide venomics, antivenomics and bio-geographical studies will reveal the chemical and immunologicalspace of venoms at different taxonomic levels. This informationis relevant for venomics to meet the challenge of snakebite enve-noming in tropical and subtropical regions of Africa, Asia, Latin

America and Oceania. During the next years, next-generationsnake venomics research developed in close collaboration in theSpanish Instituto de Biomedicina de Valencia and the CostaRican Instituto Clodomiro Picado [14,15,25,94,96,99,103107,119] willcontinue to serve the interests of the Global Snakebite Initiative.Hopefully, results of these multicentric collaborative studies willcontribute to improving the therapeutic management of snake-bite and may ultimately reduce the current snakebite mortalityand improve the quality of life of thousands of men, women andchildren in the worlds poorest communities.

AcknowledgementsFunding for the research described in this paper was provided by grants

BFU2010-17373 from the Ministerio de Ciencia eInnovacio n (currently,

Ministerio de Econom a y Competitividad), Madrid; PROMETEO/2010/

005 from the Generalitat Valenciana; CRUSA-CSIC (2009CR0021) and

CYTED project BIOTOX P211RT0412.

Financial & competing interests disclosure

The author has no relevant affiliations or financial involvement with

any organization or entity with a financial interest in or financial con-

flict with the subject matter or materials discussed in the manuscript.

This includes employment, consultancies, honoraria, stock ownership or

options, expert testimony, grants or patents received or pending, or

royalties.No writing assistance was utilized in the production of this manuscript.

Key issues

Though initially conceived as a technological platform, next-generation snake venomics also represents a conceptual framework for the

comprehensive analysis of venoms.

The availability of species-specific full-length venom gland transcript sequences as a reference database has greatly enhanced the efforts

of mass spectrometry-based venom proteomics, circumventing the need for de novo mass spectrometry sequencing.

Full proteome coverage at locus-specific resolution is within the reach of currentomics technologies, but requires integrating hypothesis-

driven and technology-driven approaches. The need to decomplex the venom proteome represents an opportunity to quantitate the rel-

ative abundances of the different venom components.

An interesting derivation of next-generation snake venomics is the possibility of using the proteomics data to proof checking the

accuracy of the assembly and translation of the transcriptome database.

The recent publications of the King cobra and the Burmese python genomes provide insights into the biology and the evolution of

venom toxin genes at the genome structural level and represent the foundation of comparative snake genomics.

Developing the full potential of venom research requires the integration of data across the biological system within the frame of an

evolutionary hypothesis.

Bridging the gap between genotype and phenotype requires an understanding of the mechanisms controlling the accelerated evolution

of venom toxins and the regulatory components of the venom secretory system.

Genus-wide venomics, antivenomics and biogeographical studies will reveal the chemical and immunological space of venoms at

different taxonomic levels. This information is relevant for venomics to meet the challenge of snakebite envenomings.

Understanding evolutionary trends across venoms represents the Rosetta Stone for generating broad-ranging polyspecific antivenoms.




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