Proteomic and Glycoproteomic Profilings Reveal That Post

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Proteomic and glycoproteomic profilings reveal that posttranslational modifications of toxins contribute to venom phenotype in snakes. Débora Andrade-Silva, André Zelanis, Eduardo S. Kitano, Inácio L. M. Junqueirade-Azevedo, Marcelo S. Reis, Aline S. Lopes, and Solange M.T. Serrano J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00217 • Publication Date (Web): 14 Jun 2016 Downloaded from http://pubs.acs.org on June 14, 2016

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Journal of Proteome Research

Proteomic and glycoproteomic profilings reveal that posttranslational modifications of toxins contribute to venom phenotype in snakes.

Débora Andrade-Silva1, André Zelanis1,2, Eduardo S. Kitano1, Inácio L. M. Junqueira-de-Azevedo1, Marcelo S. Reis1, Aline S. Lopes1,3, Solange M. T. Serrano1*

1

Laboratório Especial de Toxinologia Aplicada, Center of Toxins, Immune-

Response and Cell Signaling (CeTICS), Instituto Butantan, São Paulo, Brazil. 2

Instituto de Ciência e Tecnologia, Universidade Federal de São Paulo (ICT-

UNIFESP), São José dos Campos, Brazil. 3

Departamento de Ciências Exatas e da Terra, Universidade Federal de São

Paulo, Diadema, Brazil.

*Corresponding author: Solange M. T. Serrano Laboratório Especial de Toxinologia Aplicada Instituto Butantan Av. Vital Brasil 1500 05503-000, São Paulo, SP, Brazil Tel./Fax 55-11-3726-1024 E-mail: [email protected]

Running title: Glycoproteome and Bothrops venom phenotype

1 ACS Paragon Plus Environment


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Abstract Snake venoms are biological weapon systems composed of secreted proteins and peptides that are used for immobilizing or killing prey. Although posttranslational modifications are widely investigated because of their importance in many biological phenomena, we currently still have little understanding of how protein glycosylation impacts variation and stability of venom proteomes. To address these issues, here we characterized the venom proteomes of seven Bothrops snakes using a shotgun proteomics strategy. Moreover, we compared the electrophoretic profiles of native and deglycosylated venoms and, in order to assess their subproteomes of glycoproteins, we identified the proteins with affinity for three lectins with different saccharide specificities and their putative glycosylation sites. As proteinases are abundant glycosylated toxins, we examined the effect of N-deglycosylation in their catalytic activities and show that proteinases of the seven venoms were similarly affected by removal of Nglycans. Moreover, we prospected putative glycosylation sites of transcripts of a B. jararaca venom gland dataset and detected toxin family-related patterns of glycosylation. Based on our global analysis, we report that Bothrops venom proteomes and glycoproteomes contain a core of components that markedly define their composition, which is conserved upon evolution in parallel to other molecular markers that determine their phylogenetic classification.

Keywords: glycoproteome; lectin-affinilty chromatography; mass spectrometry; peptidome; proteome; snake venom; transcriptome.


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Abbreviations 5NCL:

5’-nucleotidase;

BPP:

bradykinin

potentiating

peptide;

ConA:

concanavalin A; CRISP: cysteine-rich secretory protein; CTL: C-type lectin; LAAO: L-amino acid oxidase; NGF: nerve growth factor; OT: other; PL: phospholipase;

PNA:

peanut

agglutinin;

SVMP:

snake

venom

metalloproteinase; SVSP: snake venom serine proteinase; VEGF: vascular endothelial growth factor; WGA: wheat germ agglutinin.



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Introduction Snake venoms are sophisticated biological weapon systems composed of secreted proteins and peptides that are used for immobilizing or killing prey and in defense against predators. They evolved on numerous occasions resulting in toxin mixtures capable of affecting, alone or synergistically, key physiological functions of prey such as those of the hemostatic, cardiovascular and nervous systems. As in all eukaryotic cells, the transcriptomes of snake venom gland tissues are highly complex, comprising a great number and diversity of multidomain proteins.1-3 Likewise, their synthesis products, i.e. the venom proteomes, are highly complex and their protein integrity control and maintenance of homeostasis within the gland lumen are crucial for the expression of the toxic activities that are essential for the survival of the snake in its particular environment. Despite the high complexity of viperid snake venoms, they are comprised of proteins from a limited number of toxin families; however, there are numerous homologous proteins within each category that share significant amounts of identical sequence information,2,4 a fact that cannot be easily explained only by intense gene paralogy. Another structural feature of snake venom proteins that contribute to proteome complexity is their variable glycosylation levels.5-7 This leads to significant variation in venom proteomes that has been extensively documented at several taxonomic levels.8-15 Protein glycosylation is one of the major post-translational modifications (PTMs) in viperid venoms, and, as in other eukaryotic proteomes, should significantly affect protein folding, conformation, stability, pharmacodynamics and activity, as well as increase the size of venom proteomes and diversify functions of toxins. Glycosylation can be classified based on the nature of the chemical linkage


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between specific acceptor residues in the protein and the carbohydrate, and the following linkages are known so far: N-linked and O-linked glycosylation, Cmannosylation, phosphoglycosylation, phosphoglycation, and glypiation.16 The N-glycosylation process occurs at the sequon Asn-X-Ser/Thr (often) and Asn-XC (very rare), where X is any amino acid, except Pro,17-19 of extracellular or secreted proteins. N-linked glycosylation plays fundamental roles in many biological processes ranging from important structural and functional roles for a protein, such as folding, stability, solubility,20 and protection against proteases,21 to cell adhesion, cell migration, and signal transduction.22 O-glycosylation occurs predominantly at the hydroxyl oxygen of Ser and Thr residues but it may also be found in Tyr, hydroxylysine (Hyl) and hydroxylproline (Hyp) of intracellular and extracellular proteins.23 The GalNAc-α-Ser/Thr linkage is present in a variety of glycoproteins of eukaryotes, and O-linked glycans play important roles in protein localization and trafficking, protein solubility, antigenicity and cell-cell interactions. Moreover, site-specific protein GalNAc Oglycosylation is emerging as a differentially regulated PTM that co-regulates the important pro-protein processing process.24 In snake venoms, glycan moieties have been described in purified toxins,25-28 however, few studies have reported on whole venom glycosylation profiles. Ten snake venoms (Bungarus multicinctus, B. fasciatus, Naja n. atra, Naja n. kaouthia, Ophiophagus hannah, Vipera russelli formosensis, V. r. siamensis, Trimeresurus mucrosquamatus, T. stejnegeri, and Deinagkistrodon acutus) were evaluated using fluorescein isothiocyanate-labeled lectin glycoconjugates and the findings indicated that all showed binding affinity to at least one type of lectin, however, each venom had different lectin-binding properties indicating



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that the glycoproteins of each snake venom contain different sugar moieties.5 In another high throughput study, eight fluorescein isothiocyanate-labeled lectins were used to show variable profiles of glycoproteins in elapid venoms of the genera

Notechis,

Pseudonaja,

Austrelaps,

Pseudechis,

Acanthophis,

Oxyuranus, Tropidechis, Rhinoplocephalus and Hoplocephalus7, however, the proteins that bound to the lectins were not identified. Most venomous snakes found in Central and South America belong to the Bothrops genus and venom proteomes of few species have so far been analyzed regarding their glycoproteomes and the function of glycosylation as a PTM contributing to interspecies variation. Snakes of Bothrops genus are responsible for the majority of the envenomation cases in Central and South America than any other group of venomous snakes.29 Venoms of Bothrops snakes contain multiple components that target the hemostatic system, the endothelial microcirculation, the extracellular matrix, and the cardiovascular system. Interestingly, the venom of B. jararaca undergoes significant ontogenetic variation, however, an analysis of the N-glycome of newborn and adult venom proteins showed that the main N-glycans identified in both venoms are similar and of the hybrid/complex type.30 The systematic morphologic, phylogenetic and taxonomic classification of bothropoid snakes has been the subject of intense analysis and in the last decade various studies have reported different results that reflect the great diversity of these snakes and their distribution in Central and South America.3133

They are diverse in their morphologic features and natural history, and inhabit

a wide array of habitat types. In a study published recently by Carrasco and colleagues,34 Bothrops (sensu stricto) was indicated to be paraphyletic,


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Bothrocophias was proposed as an independent genus, Bothrops was recognized

as

the

sister

clade

and

Bothriopsis,

Bothropoides

and

Rhinocerophis were considered synonymous. In a recent study on the proteomic characterization of the composition of venom from six species of snakes from the Bothrops complex, distributed in pairs from three distinct genera according to the classification by Fenwick and colleagues (2009)33 (B. atrox, B. jararacussu, B. jararaca, B. neuwiedi, R. alternatus, and R. cotiara),35 it was recognized that the phylogenetic classification per se was not directly linked to the venom composition. In another study, a detailed proteomic characterization of the venom from B. cotiara and B. fonsecai, which inhabit similar areas of Araucaria angustifolia forests in different geographical regions of Brazil and are morphologically very difficult to distinguish, revealed important compositional differences that defined a taxonomy signature that could be employed for their unambiguous differentiation independently of geographical and morphological factors.36 To investigate the mechanisms governing venom variation, Casewell and colleagues assessed the venom composition of six related snakes (Echis ocellatus, E. coloratus, E. pyramidum leakeyi, E. carinatus sochureki, Bitis arietans and Cerastes cerastes) regarding the interspecific changes in the number of toxin genes, their transcription and translation into proteins secreted in the venom and showed that multiple levels of regulation are responsible for generating variation in venom composition between closely related snake species.37 These and other studies have pointed out the significant variability that occurs in snake venoms, and the fact that it is largely attributed to differences in toxinencoding genes present in the genome of snakes; however, the role of the most



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frequent PTM that affects venom proteins, i.e. glycosylation, has not been evaluated in terms of its impact in defining the complexity of venom proteomes and the extent of the subproteomes of glycoproteins present in different venoms. In the recent past years, venom proteome research has experienced significant benefit from the recent advances in DNA/RNA sequencing and mass spectrometric technologies leading to a greater understanding of factors that influence snake venom proteome complexity. However, we currently still have little understanding of how protein glycosylation impacts venom variation and the stability of venom proteomes. Notably, it remains untested whether the glycosylated toxins have undergone the same evolutionary history of their nontoxin ancestors. To further investigate proteome venom variation and the mechanisms involved in the generation of different venoms by related snakes concerning protein glycosylation, in this investigation, we characterized the whole venom proteomes of seven Bothrops snakes (B. cotiara, B. insularis, B. jararaca, B. moojeni, B. neuwiedi, B. jararacussu, and B. erythromelas) and, in order to assess their subproteomes of glycoproteins, we identified proteins with affinity for three different lectins, and their putative glycosylation sites. As both SVMPs and SVSPs are abundant glycosylated toxins, we examined the effect of nondenaturing N-deglycosylation in their catalytic activities. Furthermore, we prospected putative glycosylation sites of transcript sequences of a B. jararaca venom gland dataset. Based on our global analysis, we now report for the first time that Bothrops venom proteomes are markedly defined by their content of glycoproteins, and that the total proteomes and glycoproteomes of these species cluster them similarly to their phylogenetic classification.


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Materials and Methods

Snake venoms Lyophilized venom composed of pooled samples from adult specimens of B. cotiara, B. insularis, B. jararaca, B. moojeni, B. neuwiedi, B. jararacussu, and B. erythromelas was from Instituto Butantan (São Paulo, Brazil). Pools were composed of venom extractions from at least ten individuals of each species.

Identification of venom proteins by LC-MS/MS For the identification of whole venom proteins, venom from B. cotiara, B. insularis, B. jararaca, B. moojeni, B. neuwiedi, B. jararacussu and B. erythromelas were submitted to trypsin digestion,38 in two independent experiments. Briefly, a solution of urea (in 100 mM Tris-HCl, pH 7.8) was added to a sample of 100 µg of protein from each venom sample to a final concentration of 6 M, followed by the addition of 1 mM dithiothreitol (DTT, final concentration). The mixture was incubated for 60 min at room temperature. Iodoacetamide (IAA) was then added to a final concentration of 4 mM and the samples were incubated for 60 min in the dark, at room temperature, after which 4 mM DTT was added to quench the excess of IAA. Samples were then diluted 10 times with deionized water, trypsin (Sigma) was added at a 1:50 enzyme-to-substrate ratio, and submitted to incubation at 37°C for 18h. For desalinization the samples were individually loaded in Sep-pak C18 cartridges (Waters, Milford, USA) previously conditioned with 0.1% TFA and eluted with 0.1% TFA in H20/acetonitrile (ACN) (50:50). The resulting peptide eluates were



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dried using a speedvac centrifuge and dissolved in 100 µL of 0.1% formic acid (solution A). Each peptide mixture (5 µL) was injected into a 2 cm C-18 trap column (100 µm I.D. × 360 µm O.D.; EASY II-nanoLC system, Proxeon) coupled to an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific). Chromatographic separation of tryptic peptides was performed on 10-cm long column (75 µm I.D. x 360 µm O.D.) packed in-house with 5 µm Aqua® C-18 beads (Phenomenex). Peptides eluted with a linear gradient of 335% acetonitrile in 0.1% formic acid (solution B) at 200 nL/min: 3-35% in 95 min, 35-85% B in 7 min, 85% B for 5 min, back to 3% in 3 min and 3%B for 10 min Spray voltage was set at 2.0 kV and the mass spectrometer was operated in data dependent mode, in which one full MS scan was acquired in the m/z range of 300-1,800 followed by MS/MS acquisition using collision induced dissociation of the ten most intense ions from the MS scan. MS spectra were acquired in the Orbitrap analyzer at 30,000 resolution (at 400 m/z) whereas the MS/MS scans were acquired in the linear ion trap. Isolation window, activation time and normalized collision energy were set to, respectively, 3 m/z, 30 ms and 35%. A dynamic peak exclusion was applied to avoid the same m/z of being selected for the next 90 seconds. LTQ-Orbitrap Velos raw data were converted to MGF format using the MS convert

tool

(ProteoWizard

version

3.0.3535,

http://proteowizard.sourceforge.net/) for database searching through Mascot server (version 2.2; Matrix Science, UK) against a target database restricted to the taxonomy ‘Serpentes’ (UniProt release 09_2015; 58,895 sequences, including the following numbers of sequences of Bothrops proteins: Bothrops 1295; B. jararaca, 109; B. neuwiedi, 53; B. jararacussu, 30; B. moojeni, 28; B.


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erythromelas, 21; B. insularis, 21; B. cotiara, 19) appended with 79 sequences derived from the translation of cDNAs encoding toxins in the B. jararaca venom gland, as described below), to which a set of reverse sequences was added (decoy dataset), with a parent tolerance of 10 ppm and fragment tolerance of 0.6 Da. Iodoacetamide derivatives of cysteine and oxidation of methionine were specified in Mascot, respectively, as fixed and variable modifications. The output of the search was loaded into Scaffold 4.3.4 (Proteome Software, Portland, OR) and filtered using at least 2 unique peptides per protein and with a False Discovery Rate (FDR) of 1% at peptide level. Moreover, to avoid redundancy in the output of identified proteins we used a parsimony method in which, for each venom, all proteins whose set of identified peptides was a subset of another set of peptides, were eliminated from the analysis.

Enzymatic deglycosylation of venom proteins For protein deglycosylation under denaturing conditions, 20 µg of venom samples (B. cotiara, B. insularis, B. jararaca, B. moojeni, B. neuwiedi, B. jararacussu and B. erythromelas) were incubated in 10% SDS for 1 min at 95 °C. After adding 0.02 M sodium phosphate buffer, 0.08% sodium azide, 0.01M EDTA, 2% Triton X-100, pH 7.0, incubation was prolonged for 2 min at 95 °C. After cooling on ice, 1 U of N-glycosidase F or 1 mU of O-glycosidase (Roche, Penzberg, Germany) was added, and the mixture was incubated for 18 h at 37 °C. As a negative control, venom samples were also submitted to the same procedure without N-glycosidase F or O-glycosidase. Alternatively, for Odeglycosylation under denaturing conditions, a sample of each venom (20 µg) was dissolved in 0.25 M sodium phosphate buffer, pH 7.0, and incubated in the



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denaturation solution of the Calbiochem Glycoprotein Deglycosylation Kit (Millipore, San Diego, CA), for 5 min at 95 °C. After cooling 0.15% Triton X-100 and 0.5 µL of each enzyme solution of the same kit containing the following amounts in enzyme units (Endo-α-N-acetylgalactosaminidase, 0.125 mU; α23,6,8,9-Neuraminidase,

0.5

mU;

β1,4-galactosidase,

0.3

mU;

β-N-

Acetylglucosaminidase, 5 mU) were added and the mixture was incubated for 16 h at 37 °C. As a negative control, the venom samples were submitted to the same procedure but without any glycosidase. The protein deglycosylation profiles were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions using 12% gels.39 For N-deglycosylation under non-denaturing conditions, 100 µg of venom samples (B. cotiara, B. erythromelas, B. insularis, B. jararaca, B. moojeni, B. neuwiedi and B. jararacussu) were incubated with 5 U of N-glycosidase F (Roche, Penzberg, Germany) in 0.02 M sodium phosphate buffer, 0.08% sodium azide, 0.01M EDTA, 2% Triton X-100, pH 7.0, for 18 h at 37 °C. After that, venom samples were submitted to assays of amidolytic and gelatinolytic activities.

Western blot analysis Western blot analysis was carried out only with PNA-binding proteins from B. insularis, B. jararaca, B. moojeni, B. neuwiedi and B. jararacussu venoms. The PNA-binding fractions of B. cotiara and B. erythromelas showed protein concentration below the quantification limit of the Bradford method. PNA-bound proteins (5 µg) were submitted to SDS-PAGE (12% gels)39 and immunostaining was carried out as described elsewhere using (i) anti-bothropasin rabbit


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polyclonal

antibody,8 raised against the P−III class metalloproteinase

bothropasin from B. jararaca,40 (ii) anti-MSP1/2 rabbit polyclonal antibody, raised against a mixture of the basic serine proteinases MSP1 and MSP2 from B. moojeni,41 (iii) anti-BthTX-I rabbit polyclonal antibody, raised against the basic PLA2 bothropstoxin-I from B. jararacussu (a kind gift from Dr. A. M. Moura-da-Silva, Instituto Butantan).42

Gelatin zymography Gelatin zymography was carried out as described elsewhere using 60 µg of B. cotiara, B. insularis, B. jararaca, B. moojeni, B. neuwiedi, B. jararacussu and B. erythromelas venoms (intact or N-deglycosylated under non-denaturing conditions).8

Amidolytic activity Amidolytic activity was determined on the substrate Bz-Arg-pNA (Merck) at 1 mM in 0.1 M Tris–HCl, pH 8.0, using 15 µg of B. cotiara, B. insularis, B. jararaca, B. neuwiedi and B. jararacussu venoms or 25 µg of B. moojeni and B. erythromelas venoms (intact or N-deglycosylated under non-denaturing conditions) in a final volume of 450 µL at 37 °C for 30 min. Reactions were stopped by adding 50 µL of 30% acetic acid. Release of p-nitroaniline was monitored at 405 nm and activity was calculated using a molar absorbance of 10,200 M-1 cm-1 for p-nitroaniline. Specific activity was expressed as nmol BzArg-pNA hydrolyzed per minute per mg protein.

Lectin affinity chromatography 13 ACS Paragon Plus Environment


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For affinity chromatography the following lectin resins were used: Concanavalin A-sepharose (ConA), Wheat Germ Agglutinin-agarose (WGA) and Peanut Agglutinin-agarose (PNA) (Sigma-Aldrich, St. Louis, USA). Before assays, the resins (1 mL) were conditioned using 5 mL of a solution containing 1 M NaCl, 5 mM MgCl2, 5 mM MnCl2 and 5 mM CaCl2. A volume of 1 mL of resin was mixed with 1 mL of venom sample (B. cotiara, B. insularis, B. jararaca, B. moojeni, B. neuwiedi, B. jararacussu, and B. erythromelas) at 10 mg/mL (resuspended in an equilibration buffer specific for each lectin analysis) and incubated at room temperature during 20 min in a 0.98 cm x 1 cm column. After that, 13 mL of 20 mM Tris-HCl, pH 7.4, containing 500 mM NaCl and 5 mM CaCl2 (equilibration buffer for ConA and WGA), or 10 mM HEPES buffer, pH 8.0 containing 150 mM NaCl and 2 mM CaCl2 (equilibration buffer for PNA), were applied for washing non-glycosylated or weakly-bound proteins. Binding proteins were eluted with 5 mL of equilibration buffer containing 0.5 M glucose for ConA, or 5 mL of equilibration buffer at pH 3.0 containing 0.5 M N-acetylglucosamine or 0.5 M galactose for WGA and PNA, respectively.

Identification of lectin-bound venom proteins by LC-MS/MS Before

identification

of

the

lectin-binding

proteins

by

tandem

mass

spectrometry, the chromatographic fractions were submitted to concentration and cleaning by precipitation with a mixture of acetone and methanol. In this procedure, ConA- and WGA-binding proteins were precipitated with 8 volumes of cold acetone plus 1 volume of cold methanol, and incubated at -80°C during 2 h. After that, samples were centrifuged at 14,000 g for 15 min at 4°C. The pellets were dissolved with 100 µL of 6 M urea (prepared in 100 mM Tris-HCl,


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pH 7.4) and submitted to quantification using the Bradford reagent (Sigma, St. Louis, MO) prior to protein digestion with trypsin. For the PNA-binding proteins, the same cleaning procedure was used, however, due to the presence of two phases, the higher density phase was diluted with 500 µL of 10 mM HEPES buffer, pH 8, following by the addition of 200 µL of phenol. This mixture was submitted to shaking for 10 min at 4°C, and centrifugation for 10 min at 10,000 g at 4°C. The organic phase was removed and submitted to protein precipitation by the acetone/methanol method, as described above. The protein pellet was washed with cold acetone (200 µL), dissolved in 200 µL of deionized water, quantified using the Bradford reagent (Sigma, St. Louis, MO) and submitted to trypsin digestion. The protein digestion with trypsin was carried out as described above, using 150 µg protein from the ConA- and WGA-bound fractions and 15 µg from the PNA-bound fraction. The resulting peptide mixtures were dried using a speedvac centrifuge, submitted to desalinization as described above, and dissolved in 50 µL of 0.1% formic acid. The LC-MS/MS analysis was carried out in a LTQ XLTM Linear Ion Trap (Thermo Scientific, California, USA) coupled to a nanoLC system (Eksigent, California, USA), and in a Q-TOF Ultima API (Waters, Milford, USA) coupled to a nanoUPLC System (Acquity, Waters, Milford, USA), using a pre-column nanoAcquity UPLC Symmetry C18 (180 µm x 20 mm x 5 µm) (Waters, Milford, USA) and an analytic column nanoAcquity UPLC Peptide BEH C18 (100 µm x 100 mm x 1.7 µm) (Waters, Milford, USA). For the LC-MS/MS analysis using the LTQ XL instrument, samples of 5 µL were injected and the chromatographic runs were carried out during 110 min at 300 nL/min using a gradient composed by 0.1% formic acid (solution A) and 0.1%



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formic acid in acetonitrile (solution B) as follows: 3-48% B from 0 to 70 min, 4880% B from 70 to 90 min, 80% B for 10 min, and then back to 3% B in 10 min. The LTQ XL instrument was operated in the top ten mode using 2.5 kV as source voltage and with 35% normalized collision energy. The mass range was fixed at 300-2,000 m/z for parent ions. The dynamic exclusion time was 60 s to 500 ions. For the LC-MS/MS analysis using the Q-TOF instrument, samples of 5 µL were injected and the chromatographic runs were carried out during 110 min at 600 nL/min using a gradient composed by 0.1% formic acid (solution A) and 0.1% formic acid in acetonitrile (solution B) as follows: 3-48% B from 0 to 70 min, 4880% B from 70 to 90 min, 80% B for 10 min, and then back to 3% B in 10 min. The Q-TOF instrument was operated in the top eight mode using 3.5 kV as source voltage, and the collision energy was automatically set according to the m/z and ion charge varying from 15 to 56 eV. The mass range was fixed at 3002,000 m/z for parent ions. The dynamic exclusion time was 90 s. The spectra peak lists (.mgf files for LTQ XL; .pkl files for Q-TOF) were submitted to searches using MASCOT 2.2.04 (Matrix Science, London, UK) against a target database restricted to the taxonomy ‘Serpentes’ (UniProt release 09_2015; 58,895 sequences, including the following numbers of sequences of Bothrops proteins: Bothrops 1295; B. jararaca, 109; B. neuwiedi, 53; B. jararacussu, 30; B. moojeni, 28; B. erythromelas, 21; B. insularis, 21; B. cotiara, 19) appended with 79 sequences derived from the translation of cDNAs encoding toxins in the B. jararaca venom gland, as described below), to which a set of reverse sequences was added (decoy dataset), using the following search parameters: oxidation of methionine as variable modification and


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carbamidomethylation of cysteine as fixed modification, up to two missed cleavages by trypsin, peptide charges of +2 and +3, and tolerance of respectively, 2.0 and 1.0 Da for MS and MS/MS events (for the LTQ-XL data) or 0.5 Da for both MS and MS/MS events (for Q-ToF data). The output of the search was loaded into Scaffold 4.3.4 (Proteome Software, Portland, OR) and filtered using at least 2 unique peptides per protein and with FDR of 1% at peptide level. Moreover, to avoid redundancy in the output of identified proteins we used a parsimony method in which, for each venom, all proteins whose set of identified peptides was a subset of another set of peptides, were eliminated from the analysis.

cDNAs encoding toxins in the B. jararaca venom gland The cDNA sequences encoding toxins of the B. jararaca venom gland were obtained from a previous transcriptomic analysis.

43

Briefly, total RNA from the

venom glands of a single specimen of B. jararaca was extracted and mRNA was isolated and used to generate the cDNA library, which was pyrosequenced in a GS Junior 454 Sequencing System (Roche Diagnostics) following the manufacturer protocols. Sequences were assembled with Newbler 2.7 (Roche Diagnostics), which removed adaptors and ribosomal RNA sequences in an initial step. A minimum overlap length of 80% of the read and a minimum identity of 98% in the overlap were set, with the other parameters set with the software's default values. Toxin annotation was performed by BlastX analysis against UniProt and by reciprocal Blast searches against a compiled set of snake toxin sequences.



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Prediction of N- and O-glycosylation sites in venom proteins Amino acid sequences of proteins identified in the venom lectin-bound fractions were obtained from the UniProt database (www.uniprot.org) and run through the web-based

glycosylation

prediction

(www.cbs.dtu.dk/services/NetNGlyc/)

tools and

NetNGlyc NetOGlyc

(www.cbs.dtu.dk/services/NetOGlyc/).44,45 cDNA sequences encoding toxins in the B. jararaca venom gland were translated and submitted to N- and Oglycosylation sites using the same prediction tools. All putative N- glycosylation sites were considered as such if they crossed the default threshold of 0.5 and thus represented a predicted glycosylated site (as long as it occurred in the required sequon Asn-Xaa-Ser/Thr, without Pro at Xaa), according to NetNGlyc (www.cbs.dtu.dk/services/NetNGlyc/). Likewise, all putative O-glycosylation sites were considered as such if they resulted from prediction scores higher than 0.5 according to NetOGlyc.

Computational methods The

identification

of

the

global

proteomes

and

of

the

lectin-bound

glycoproteomes produced lists of the protein accession codes, grouped by venom. Those lists were processed through an in-house program coded in Perl, which assessed the total number of unique proteins, that is, the size of the union of all protein accession codes that appeared at least once in all seven venoms. Using this Perl program, we generated occurrence matrices, in which the rows are the seven different venoms and the columns are all the unique proteins. Hence, for each pair (venom, protein), its respective value is binary, in which zero or one means, respectively, absence or presence of the pair


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(venom, protein). We also produced two additional occurrence matrices, in which each pair (venom, protein) ranges from zero to three or zero to four and were used for the z-normalization of, respectively, the lectin-binding subproteome and the union of whole proteome and lectin-binding subproteome. These two matrices account for the number of experiments in which a given pair (venom, protein) was observed. Clustering analyses were performed using MATLAB (MathWorks, Natick, MA), executing the “clustergram” function. For each occurrence matrix, we carried out two hierarchical clusterings, one for the venoms and other for the protein accession codes. In both clusterings, we employed an agglomerative method with single linkage and the Jaccard or the Euclidian distance as the distance metric of, respectively, binary and z-normalized matrices. Additionally, for both types of matrices, we also used the Hamming distance as the distance metric. Principal component analysis (PCA) was carried out executing the “pca” function in MATLAB. This analysis relied on the whole proteome binary matrix. We considered the seven venoms as the variables and the proteins as the data observations, and used the singular value decomposition algorithm to generate the principal components. 3-means clustering was also performed using MATLAB, through the execution of the “kmeans” function. The used distance metric was the squared Euclidean distance, which implies that for a given set, the centroid is the mean of the points that compose that set.

Results and Discussion



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Workflow and experimental design Studies on the venom composition of many species of the Bothrops complex revealed that despite the high complexity and variability of these proteomes, they are comprised of a limited number of protein and peptide families.1,2,35,36,46 On the other hand, the molecular basis of venom variability by a PTM such as glycosylation and the possibility of this being a selective, adaptive feature underlying evolutionary patterns in the Bothrops complex, are only poorly understood. The main objective of this study was to evaluate the profiles of the whole proteomes and of the glycoproteomes of seven Bothrops venoms in order to explore the relationship between phylogenetic classification and the composition of these venoms, and also to find out in what extent the subproteomes of glycoproteins impact the whole venom proteome composition. Moreover, we wanted to determine whether the use of multiple lectins could substantially increase venom glycoproteome coverage. For these purposes, we established an analytical workflow (Scheme 1) to compare the whole venom proteomes and the different subproteomes of glycoproteins with affinity for three lectins (ConA, WGA and PNA) by shotgun mass spectrometric analysis. In parallel,

we

examined

the

effect

of

deglycosylation

in

the

venoms

electrophoretic profiles and proteolytic activities. Finally, we analyzed the patterns of putative glycosylation sites present in transcripts encoding toxins of B. jararaca venom gland and correlate them with those of their paralogous genes.

Whole venom proteome analysis


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In order to further explore the variability among Bothrops venom proteomes, here we characterized the whole venom proteomes of seven of these venoms (B. cotiara, B. jararaca, B. insularis, B. moojeni, B. neuwiedi, B. jararacussu and B. erythromelas). For this purpose, venoms were separately submitted to in solution trypsin digestion and analyzed by LC-MS/MS using a LTQ-Orbitrap Velos mass spectrometer. To diminish risk of reporting false positive identifications and to assess the quality of obtained data, a False Discovery Rate (FDR) analysis was performed using a decoy database composed of the protein entries of the Uniprot database of the taxa Serpentes with reverseoriented sequences. After processing the resulting MS/MS spectra and performing database search, we were able to identify a variable number of toxins in the venoms (B. cotiara: 68; B. insularis: 75; B. jararaca: 100; B. moojeni: 69; B. neuwiedi: 87; B. jararacussu: 81; B. erythromelas: 75) which belong to the main classes of toxins found in viperid venoms (Supplemental table S1). The fact that a higher number of toxins were identified in the venom of B. jararaca is likely related to the higher number of protein and peptide sequences of this species present in the database used for database search. The determination of the whole proteome composition by this shotgun approach, which is based on in solution trypsin digestion of whole venom proteins followed by the identification of tryptic peptides by reversed-phase liquid chromatography separation and mass spectrometric analysis, allowed for a comprehensive appreciation of the venom composition including the identification of some minor toxins that are usually detected in venom gland transcriptomic studies but not by proteomic analysis. Figure 1A shows that a significantly higher number of SVMPs and SVSPs was identified in all seven



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venoms confirming the significant occurrence of proteolytic enzymes in the Bothrops genus.8,35,46,47 A somewhat lower and variable number of toxins identified as L-amino acid oxidase, phospholipase (A2 and B) and C-type lectin was detected in the venoms while other proteins (cysteine-rich secretory protein,

glutaminyl-peptide

cyclotransferase,

phosphodiesterase,

5'-

nucleotidase, vascular endothelial growth factor, nerve growth factor, and hyaluronidase) were clearly less abundant (Supplemental table S1) In general, similar results were obtained by Sousa and colleagues by the shotgun analysis of six venoms of the Bothrops complex.35 In our analysis, a total of 137 unique proteins were identified in the seven whole venom proteomes and their distribution, grouped by venom, is shown as a Venn diagram (Figure 1B). It was interesting to note that only 19 unique proteins were detected in all venoms while there are proteins that appeared only in one of the venoms. Most unique toxins that were detected in all venoms are SVMPs and SVSPs (Supplemental table S1). Regarding the venom of B. cotiara, in a previous report it was shown not to contain PLA2,36 however, in this study we identified three proteins that matched other snake venom PLA2 sequences indicating that they are present in B. cotiara venom, although at a very low abundance in comparison to other Bothrops species. Moreover, the profile of B. erythromelas venom showing a high content of SVMPs is similar to those recently reported for specimens from five different geographic populations within the Caatinga in the Northeast region of Brazil.48 The composition of whole venom proteomes was used to classify the venoms by hierarchical clustering of their unique proteins in a binary matrix assignment using the Jaccard index as the distance metric. Figure 2 shows the graphical visualization of the two hierarchical clusterings of the whole venom


Page 23 of 72

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proteome characterization considering for each pair (venom, protein) the presence or absence of a given unique protein. The conserved core of toxins in these Bothrops venoms is composed of 19 proteins that were detected as present in all venoms (1 phosphodiesterase, 1 PLB, 1 5NCL, 1 glutaminylpeptide cyclotransferase, 6 SVSPs and 9 SVMPs). According to this clustering, B. cotiara showed the most distinct venom composition, while B. erythromelas, B. insularis, and B. jararaca formed a cluster distinct from the other cluster composed by B. moojeni, B. neuwiedi and B. jararacussu venoms. Interestingly, the analysis of the unique proteins identified in the whole venom proteomes in a binary matrix assignment, in which the lines were z-normalized considering either the Euclidean distance or the Hamming distance metrics, resulted in a hierarchical clusterings where the venoms occupied similar positions as in Figure 2 (Supplemental figure S1A-D). To establish correlations between venoms and toxin families, which could give insights on the hierarchical clustering depicted in Figure 2, a Principal Component Analysis (PCA) was carried out. A plot of the first three principal component loadings against the toxin scores revealed that the second and third components could be used to associate groups of venoms to groups of toxins (Supplemental figures S2A-B). To this end, we performed a 3-mean clusterization of the toxin scores in the second and third components, hence partitioning the toxins into three sets (Supplemental figure S2C). For each set, we grouped the toxins by toxin family and verified the size of each group. SVMPs mostly populated sets 1-3 and therefore showed relevant contribution to venom clusterization (Supplemental figure S2D). Sousa and colleagues reported a lack of similarity between the venom proteomes of closely related



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Page 24 of 72

snakes and a high similarity between the venom proteomes of phylogenetically more distant snakes, and suggested little connection between taxonomic position and venom composition in six species of the Bothrops complex (Bothropoides jararaca, Bothropoides neuwiedi, Rhinocerophis alternatus, Rhinocerophis

cotiara,

Bothrops

jararacussu

and

Bothrops

atrox).35

Interestingly, in our study the placements of all seven venoms in the whole proteome clustering parallel those of the phylogeny cladograms of South American bothropoid snakes reported in recent studies on morphological and molecular data,33 which place them grouped in different clades: B. cotiara is in the Bothrops alternatus clade, B. jararaca, B. insularis, B. erythromelas and B. neuwiedi are in the Bothrops jararaca + Bothrops neuwiedi clade, and B. jararacussu and B. moojeni are in the Bothrops atrox clade.

Comparison of electrophoretic profiles of native and deglycosylated Bothrops venoms To get a general view of the glycosylation level of proteins present in the Bothrops venoms, we next submitted them to incubation with N- and Oglycosidase under denaturing conditions for deglycosylation, and compared their electrophoretic profiles with those of the native venoms by SDSpolyacrylamide gel electrophoresis (SDS-PAGE). The first observation from these experiments was a remarkable variation in the electrophoretic protein profile between these venoms in native, non-deglycosylated conditions (Figure 3A). Clear profile variation concerning presence/absence of bands and their intensities was visualized at molecular masses between ~10 kDa and ~80 kDa. Nevertheless, a large protein band of ∼50 kDa was observed in most venom


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profiles although with variable intensity, especially in the venom of B. neuwiedi, where it was less stained, and in B. jararacussu venom, where it was absent. In addition, all venoms showed a conserved protein band of ~20 kDa that however varied in intensity between them (Figure 3A). Significant shift in the molecular masses of intensely stained protein bands was observed in all venoms after N-deglycosylation under denaturing conditions, indicating the removal of carbohydrate moieties and suggesting the presence of a high content of N-glycosylated proteins (Figure 3A). Most noticeable molecular mass differences after N-deglycosylation were found in the molecular mass range of 25-50 kDa in all venoms. Interestingly, after N-deglycosylation all venoms showed a protein band of ~45 kDa while the protein band of ~20 kDa observed in the native venom profiles apparently did not change. Moreover, Ndeglycosylation clearly did not affect the electrophoretic mobility of protein bands of ~15 kDa and lower suggesting that these are likely not N-glycosylated (Figure 3A). It is notable that after N-deglycosylation the venoms showed less distinct electrophoretic profiles indicating that although their proteomes differ at the N-glycosylation level they are not so distinct regarding the length of polypeptide chains of N-glycosylated proteins. The same experiment was carried out using O-glycosidase but no significant change in the pattern of Bothrops venom protein migration was observed indicating that this glycosidase was not able to remove intact O-linked glycans from venom proteins (Figure 3A). Therefore, in order to further explore Oglycosylation in Bothrops toxins, the venoms were submitted to incubation with the

enzymes

α2-3,6,8,9-neuraminidase,


β-N-

acetylglucosaminidase and endo-α-N-acetylgalactosaminidase (O-glycosidase



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Page 26 of 72

from Streptococcus pneumoniae) under denaturing conditions, which resulted in changes in their electrophoretic profiles. Mainly proteins of molecular masses between 25 kDa and 60 kDa were affected by O-deglycosylation as they showed lower intensity or disappeared upon removal of O-glycans (Figure 3B). These results indicate that contrarily to what we have previously reported on protein glycosylation in B. jararaca venom,9 it appears to contain O-glycosylated proteins. Birrell and colleagues tested a combination of neuraminidase and Oglycosidase to O-deglycosylate the venom proteins from the elapid snake Pseudonaja textilis and because their electrophoretic pattern was identical to that observed with neuraminidase alone, they concluded that O-linked sugars are absent in Pseudonaja textilis venom proteins.49 Here, the clear change in the molecular mass profile of Bothrops venom proteins upon O-deglycosylation indicates that the use of multiple enzymes is essential to assess the presence of O-glycans in venom proteins.

Analysis of the glycoprotein sub-proteomes of Bothrops venoms Glycoproteins can be isolated by affinity to immobilized glycan-binding proteins such as lectins. Certain lectins possess affinity for particular oligosaccharide moieties, and various lectins bind to different structures of glycans in glycoconjugates, thus they may reveal subtle differences in complex glycoprotein profiles such as those of snake venoms. An important advantage of the lectin-affinity chromatography to enrich the glycoprotein fraction of a complex proteome is the fact that binding components can be recovered for characterization. In the case of snake venoms, it is not known which lectins or combinations of lectins are best for glycoproteome profiling or if specific lectins


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show preferential association with particular glycosylation sites. In order to get a comprehensive view of the glycoproteomes of Bothrops venoms, in this study three lectins were selected for their different glycan structures specificities: Concanavalin A (ConA), which binds to mannose, Wheat Germ Agglutinin (WGA), which binds to sialic acid and N-acetyl-glucosamine, and Peanut Agglutinin (PNA), which binds to terminal galactose and N-acetylgalactosamine groups. Supplemental figure S3 shows notably diverse electrophoretic profiles of the ConA-, WGA-, and PNA-bound venom protein fractions indicating a clear variable content of glycoprotein types in the analyzed Bothrops venoms and hence the presence in these venoms of different subproteomes of glycoproteins. Considering the number of protein bands and their intensities, one interesting observation is that the content of proteins with affinity for ConA is in general somewhat higher than that of proteins with affinity for WGA. This suggests that the repertoire of Bothrops venom glycoproteins is richer in glycans containing mannose than N-acetyl-glucosamine, or sialic acid. However, it should be considered that in some cases the same proteins may be present in both the ConA- and WGA-bound fractions, since the hybrid-type Nglycan present in the glycoproteins containing N-acetylglucosamine or sialic acid may show affinity for ConA using the antennae of mannose present in the N-glycan.30 The subproteomes of proteins with affinity for ConA showed notable variability between the venoms (Supplemental figure S3). B. cotiara, B. moojeni, B. neuwiedi, B. jararacussu and B. erythromelas showed more complex electrophoretic profiles containing protein bands in the molecular mass range of



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Page 28 of 72

~10 kDa to ~50 kDa, while those of B. insularis and B. jararaca were more simple and showed a main band of ~45 kDa accompanied by a few weakly stained bands. In contrast, the subproteomes of proteins with affinity for WGA showed less complexity and variability among the venoms. Overall, all venoms showed only a few protein bands of ~35-70 kDa and in addition, a band of ~10 kDa with variable intensity, which, however, was clearly more intense in B. neuwiedi and B. jararacussu venoms (Supplemental figure S3). The subproteomes of proteins with affinity for PNA showed similar electrophoretic profiles among the venoms, however they were rather different from those of the ConA- and WGA-binding proteins (Supplemental figure S3). Notably, the amount of protein present in the PNA-binding fractions of B. cotiara and B. erythromelas was very low and did not allow for their visualization on the SDS-PAGE profile (not shown). Nevertheless, the PNA-bound fractions of B. insularis, B. jararaca, B. moojeni, B. neuwiedi, and B. jararacussu showed mainly protein bands between ~ 35 kDa and ~10 kDa, and the overall band profiles among the venoms were variable (Supplemental figure S3). A general observation from these experiments is that the ConA- and WGAbinding proteins of these Bothrops venoms vary from 10 kDa to 70 kDa while the PNA-bound fraction is composed of lower molecular mass proteins (10-35 kDa). Hence, all electrophoretic profiles of lectin-binding venom proteins showed the unexpected presence of protein bands of ~10-15 kDa with variable intensities, which typically correspond to components that are not glycosylated such as phospholipase A2 and C-type lectin.

Identification of lectin-binding proteins by LC-MS/MS


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In order to identify the toxin classes to which the ConA-, WGA- and PNAbinding proteins belong, these were analyzed by in solution trypsin digestion followed by LC-MS/MS analysis, and only those with at least two peptides were considered as reliable identifications. The lists of identified ConA-, WGA- and PNA-binding proteins are shown on Supplemental tables 2, 3, and 4, respectively, and the summary of these results is presented in Figure 4. The identified proteins belong to five main classes: SVMP, SVSP, LAAO, PL, and CTL. Some of these venom protein classes are known to be glycosylated: many SVMPs, especially those of the P-III class, and SVSPs are glycosylated and contain a variable number of putative N-glycosylation sites;50,51 CTLs are often not glycosylated but have a carbohydrate recognition domain (CRD);52 LAAOs are frequently glycosylated proteins, but the precise role of their carbohydrates moieties is not known.53 The two major classes of Bothrops toxins that bound to ConA and possibly contain mannose as sugar moiety in their structure are the SVMPs and SVSPs (Figure 4; Supplemental table S2). This profile was observed in all venoms analyzed and SVMPs and SVSPs together accounted for more than 60% of the total proteins that showed affinity for ConA. In particular, SVSP is an interesting toxin class in which glycosylation seems to constitute an important factor of molecular mass variability.50,54 They usually show 232-234 amino acid residues and similar primary structures; however, experimental molecular mass determination often reveals higher values than those corresponding to their theoretical molecular masses due to their variable carbohydrate chains. An unexpected observation was that in all venoms proteins identified as phospholipase A2 and B were among the proteins with affinity for ConA (Figure



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Page 30 of 72

4; Supplemental table S2). As the primary structure of phospholipases A2 usually does not show any putative glycosylation site, the presence of these proteins in the ConA-bound fraction suggests that they may have unspecifically interacted with this lectin via an exosite different from the CRD. Phospholipases B, on the other hand, often contain putative N- glycosylation sites and may have been captured by ConA via its CRD. Moreover, considering that the chromatography was carried out in non-denaturing conditions, it is plausible to suggest that non-glycosylated proteins may have been indirectly captured by the lectin via the interaction with some of the CTLs, which typically contain a CRD. Although ConA and WGA show affinity for different carbohydrates, the subproteome of Bothrops proteins with affinity for WGA showed a similar profile to that of the ConA-binding proteins and also contained SVMPs and SVSPs as major proteins, and PLA2 and CTLs as minor components (Figure 4; Supplemental table S3). Interestingly, no CTL was detected in the Con-A and WGA-bound fractions of B. cotiara venom. The affinity of SVSPs, SVMPs and LAAO for WGA indicates that these toxins contain sialic acid or N-acetyl glucosamine in their carbohydrate chains. Recently, acutobin, a SVSP with specific fibrinogenolytic activity from Deinagkistrdon acutus venom, was shown to contain hybrid- and complex-type N-glycans with a terminal disialyl cap composed of eight sialic acids evenly distributed on a tetra-antennary core structure.55 The presence of sialic acid at the terminal end of carbohydrate chains in proteins confers negative charge(s) and may influence their isoelectric point (pI). Therefore, depending on the number of occupied putative glycosylation


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sites and sialic acid molecules added to the sugar chains of venom glycoproteins, these might show variable molecular masses and isoelectric points, as has been observed for instance for some viperid SVSPs and LAAOs suggesting that this PTM may be used as a molecular fine-tuning to diversify the toxin arsenal upon evolution to deal with different types of prey.56-60 Here we tested the effect of N-deglycosylation on the isoeletric points of B. jararaca venom proteins by 2-D electrophoresis and observed that upon removal of Nglycans many protein spots show a shift towards the basic region of the gel indicating

the

removal

of

carbohydrate

chains

containing

sialic

acid

(Supplemental figure S4). Furthermore, sialic acid-containing carbohydrate chains are hydrophilic and hence they may increase protein solubility by protecting hydrophobic amino acid residues at the protein surface. Therefore, the fact that a large amount of glycosylated toxins showed affinity for a sialic acid specific lectin suggests that their sugar moieties in viperid venoms might contribute to overall protein solubility and even to the typical acidic pH of the venom. As glycoproteins containing sialic acid such as many cell surface receptors play a significant role in the regulation of function of cells in the innate and adaptive immune system, another aspect to be considered about the presence of sialic acid-containing glycans in snake venom proteins is their potential role in the interaction of toxins with their molecular targets in the prey, which might interfere with and trick the immune system of snakebite victims. In comparison to the subproteomes of the ConA- and WGA-bound fractions, the PNA-binding proteins of these Bothrops venoms showed a clear diverse profile. The number of unique proteins that showed affinity for PNA was smaller than



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Page 32 of 72

that of those that bound to ConA or WGA, and they diverged in terms of the toxin classes and their relative proportions (Figure 4; Supplemental table S4). Phospholipases were present in the PNA-bound fractions of all venoms and constitute the major class of proteins with affinity to PNA in B. jararaca, B. moojeni,

B.

neuwiedi

and

B.

jararacussu

venoms.

Although

both

phospholipases A2 and B were identified in the ConA- and WGA-bound fractions, interestingly, in all venoms only phospholipases A2 showed affinity for this lectin (Supplemental table S4). In order to further analyze the affinity of venom toxins to PNA, we next carried out a Western blot analysis of the PNA-binding protein fractions from these venoms, except for those from B. cotiara and B. erythromelas since the amount of recovered protein was not enough to perform the experiment. Using specific anti-SVMP, anti-SVSP and anti-PLA2 antibodies, we further probed for the presence of these toxins in the PNA-bound fraction. In accordance to the fact that just a few SVMPs were detected in the PNA-bound fraction of most tested venoms, the immunostaining of these proteins with the anti-bothropasin (P-III SVMP from B. jararaca) antibody revealed just a few weakly recognized protein bands (Supplemental figure S5A). A variable number of SVSPs was detected in the PNA-bound fractions and, accordingly, the anti-SVSP antibody (antiMSP1/2, a mixture of polyclonal antibodies against the B. moojeni serine proteinases MSP1/2) recognized various protein bands in the venom of B. insularis, B. jararaca, B. moojeni, B. neuwiedi and B. jararacussu (Supplemental figure S5B). Phospholipases A2 were identified in the PNA-bound fractions of these venoms, and, accordingly, the anti-BthTX-I antibody, raised against the basic, K49 PLA2 bothropstoxin-I from B. jararacussu, intensely immunostained


Page 33 of 72

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protein bands of ~14 kDa, which correspond to the typical molecular mass of this type of toxin. However, no cross-reactivity of this antibody was observed with the PNA-bound proteins in the venom of B. insularis indicating an unexpected structural diversity in this toxin class in such closely related species as B. insularis and B. jararaca (Supplemental figure S5C), which is in accordance to previous transcriptomic studies that showed the presence of transcripts of K49 PLA2 in B. jararaca but not in B. insularis venom glands.1,43 An overall conclusion of these experiments was that the use of multiple lectins substantially increased the coverage of the subproteomes of glycoproteins and highlighted the fact that in venom proteomes not only glycoproteins display affinity for lectins, since non-glycosylated toxins may bind to them due to interaction properties that do not involve protein-glycan recognition.

Comparison of the subproteomes of lectin-binding toxins of Bothrops venoms A total of 118 unique proteins were identified in the ConA-, WGA- and PNAbound fractions of B. cotiara, B. jararaca, B. insularis, B. moojeni, B. neuwiedi, B. jararacussu and B. erythromelas venoms and their distribution, grouped by venom, is shown as a Venn diagram (Figure 5A; Supplemental tables S2, S3 and S4). As occurred with the analysis of the whole venom proteomes, a low number of unique proteins (15) were detected in the lectin-bound fraction of all venoms while there are proteins that appeared only in one venom. Among the unique proteins, 22 appeared only in the ConA-bound fraction (yellow), 26 appeared only in the WGA-bound (blue) fraction, and 18 appeared only for the PNA-bound fraction (orange) (Figure 5B). Moreover, a higher number of unique



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proteins were shared by the ConA- and WGA-bound fractions (30) than that of shared proteins between the PNA-bound fraction and the ConA- and WGAbound fractions (5 and 3, respectively). The composition of lectin-binding subproteomes was used to classify the venoms by hierarchical clustering of their unique proteins in a binary matrix assignment considering the Jaccard index as the distance metric. Figure 6 shows the graphical visualization of the two hierarchical clusterings of the lectinbinding subproteomes characterization, considering for each pair (venom, protein) the presence or absence of a given unique protein in at least one of the lectin-binding subproteomes. According to this clustering, similarly as it was obtained in the analysis of the whole venom proteomes (Figure 2), B. cotiara and B. erythromelas showed the most distinct venom composition, while B. moojeni, B. jararacussu and B. neuwiedi clustered together, and B. insularis and B. jararaca venoms showed similar profiles but different from the others (Figure 6). As in the analysis of the whole venom proteomes, a group of lectinbinding proteins was identified as shared by the seven venoms (1 PLB, 1 PLA2, 1 5NCL, 2 LAAO, 1 glutaminyl-peptide cyclotransferase, 3 SVSPs, and 6 SVMPs). The combination of all unique proteins identified in the analyses of both the whole proteomes (137) and the lectin-binding subproteomes (118) in a Venn diagram revealed that 84 of them were shared, indicating that lectin-binding proteins are major components of Bothrops venoms (Figure 7A). Among the proteins that were identified exclusively in the analysis of the whole proteomes, there are 36 putatively glycosylated proteins, according to the prediction tools NetNGlyc and NetOglyc, indicating that these proteins may be glycosylated but


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their carbohydrate moieties do not interact with the three lectins used for the affinity chromatography. Moreover, among the proteins that were identified exclusively in the lectin-binding subproteomes, there are 18 putatively glycosylated proteins, which are likely very low abundant in the venom and were only identified due to the fact that they were enriched in the lectin-binding fraction. Likewise, 16 non-glycoproteins that were only identified in the lectinbinding subproteomes are probably minor components that unspecifically bound to the lectins or interacted with (bound) glycosylated toxins and, therefore, were also co-eluted (Figure 4; Figure 7A; Supplemental tables S2, S3 and S4). Taken together, these data indicate that the enrichment of the glycoproteome by lectinaffinity chromatography can indeed improve the identification of snake venom proteins, however, the use of lectins with different specificities is essential for achieving a thorough identification of glycoproteins in a given snake venom. Furthermore, Figure 7B shows that the hierarchical clustering of the 171 unique proteins that were identified in both the analyses of whole proteomes and lectinbinding subproteomes resulted very similar to that obtained by their individual analyses (Figures 2 and 6). In addition, the removal of the putatively nonglycosylated proteins that were identified in the lectin-binding venom fractions did not significantly affected the positions of the venoms in the hierarchical cluster of the whole venom proteins combined with lectin-binding proteins (Figure 7C). In contrast, when only the non-glycoproteins present in the lectinbinding fractions (PLA2 and CTL) were considered for clustering, the profile obtained diverge from all of the others described here (Figure 7D). Moreover, the analysis of the unique proteins present in the clusters of Figures 7B-D, after z-normalization of the rows in an occurrence matrix, resulted in similar



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hierarchical clusterings (Supplemental figure S6A-C), and, in particular, the cluster of the non-glycoproteins confirmed the lack of similarity between the venom proteomes of closely related snakes, such as B. jararaca and B. insularis and, therefore, once more evidenced that they do no play a role in molding the proteome of these venoms. Taken together, these data suggest that the glycoproteins are responsible for shaping the proteome signature of the Bothrops species sampled in this study, and that their clustering according to species mirrors that of the taxonomic organization. In this respect, despite the variable abundance of individual toxins and toxin families in these venoms, their contents of glycoproteins seem to be robust traits that stand above venom variation and reflect the phylogenetic relationships among these species.

Deglycosylation of Bothrops venom components As proteolytic enzymes are abundant glycosylated components of Bothrops venoms that play a major role in the envenomation process, we evaluated the role of the carbohydrate moieties in their stability and functional activities. To this end, we incubated the venoms with N-glycosydase F under non-denaturing conditions. N-glycosydase F is an enzyme that removes hybrid, complex and high mannose type N-linked oligosaccharides from proteins. The effect of Ndeglycosylation of B. cotiara, B. jararaca, B. insularis, B. moojeni, B. neuwiedi, B. jararacussu and B. erythromelas venoms was evaluated by an assay of gelatinolytic

activity

by

zymography,

which

showed

that

the

partial

deglycosylation of these venoms promoted a shift of apparent molecular masses of proteinases active on gelatin. Except for B. erythromelas venom,


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which did not display any clear zone of lysis against the blue background of the gel, all other deglycosylated venoms showed bands of gelatinase activity of molecular masses lower than those of the intact venoms (Supplemental figure S7A), indicating that both SVMPs and SVSPs may have been affected by deglycosylation. We also examined the effect of N-deglycosylation on the amidolytic activity of the venoms using the SVSP substrate Bz-Arg-pNA. Supplemental figure S7B shows that this activity significantly varied between the venoms, indicating that their content of SVSPs is variable as demonstrated by their whole proteomic analyses (Figure 1A). Moreover, although at different extensions, the amidolytic activity of all of them was affected by the partial Ndeglycosylation, indicating that the carbohydrate moieties of SVSPs are important for proper substrate catalysis. Despite the variable whole proteome content of these venoms and the variable hydrolytic potencies of their proteolytic enzymes, these were similarly affected by partial N-deglycosylation, indicating that overall the carbohydrate moieties of SVSPs and SVMPs of Bothrops venoms may be important to ensure structure stability and integrity. However, experimental evidence is needed to confirm that all proteolytic enzymes are susceptible to instability upon deglycosylation.

Analysis of putative glycosylation sites in cDNAs encoding toxins in the B. jararaca venom gland In view of the diversity of venom proteins that showed affinity for the lectins used for chromatography in this study, which included proteins that are putatively glycosylated or not, we examined the translated amino acid sequences of a B. jararaca venom gland cDNA library in order to assess the



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putative glycosylation sites present in toxin transcripts (Supplemental file 1).43 Using NetNGlyc and NetOglyc as prediction tools, the 79 identified full-length transcripts showed diverse putative glycosylation levels depending on the toxin class (Figure 8; Supplemental table S5).44,45 Notably, despite the finding that Ctype lectins and phospholipases A2 were detected among the lectin-binding proteins of B. jararaca venom, none of the transcripts encoding these proteins showed putative glycosylation sites. The fact that these proteins show comparatively short polypeptide chains and contain many disulfide bonds, which may play a role in the stabilization their tertiary structures, suggests that they do not require glycosylation as a factor to contribute to their structural integrity. Another interesting outcome of this analysis was the high number of Oglycosylation sites predicted for four transcripts encoding the precursor of bradykinin potentiating peptides and C-type natriuretic peptide. This type of precursor protein in the B. jararaca venom gland is predicted to be processed to release seven BPPs, a poly-His/poly-Gly (pHpG) peptide and a natriuretic peptide in the venom.9,47,61 Interestingly, three of the putative O-glycosylation sites detected in the transcripts present in the cDNA library are located in the short sequences between BPP 2, 3, 4 and 5, and twelve are found in the long intervening sequence that links the 7th BPP and the pHpG peptide (Supplemental figure S8). Whether these glycosylation sites are occupied and play a role in the folding of the nascent precursor protein and its processing to release the bioactive peptides remains to be evaluated. Among the transcripts for proteolytic enzymes found in the B. jararaca venom gland transcriptome, the SVSP sequences showed only a low number (1-3) of


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putative N-glycosylation sites, which, however, do not occupy conserved positions along their amino acid sequences (Figure 8; Supplemental table S5). Likewise, most SVSPs detected in the Bothrops venom lectin-binding fractions show only putative N-glycosylation sites (Supplemental tables S2, S3 and S4). The SVSP family of toxins is considered to have evolved from glandular kallikrein,62,63 where the ancestral physiological activity of releasing kinin from kininogen was preserved in some proteinases while others display varied specific biological activities (e.g. conversion of fibrinogen into fibrin; activation of platelets to aggregation; activation of protein C; activation of Factor V; activation of plasminogen). Notably, sequons for O-glycosylation in glandular kallikreins are rare,64 and it is clear that even if this enzyme is the putative common ancestor of SVSPs as previously suggested,62 their amino acid sequences have been nonetheless diversified upon accelerated evolution to exhibit exquisite specificity towards different macromolecular substrates.50,65 Interestingly, in a recent study on the comparative transcriptomic survey on body tissues from B. jararaca, no transcript encoding a protein similar to glandular kallikrein was identified, and instead, trypsin was the closest paralog.43 These observations suggest the possibility that SVSPs may have been originated from other type of serine proteinase. In any case, the fact that the number, type and position of putative N-glycosylation sites vary between B. jararaca venom serine proteinases suggests that once most glycosylation occurs at solvent accessible regions, the glycans might play a role in the interaction of these proteinases with their molecular targets in the prey. Differently from the SVSPs, a total of 21 transcripts of SVMPs (of P-I, P-II and P-III classes) showed a high number of putative O-glycosylation sites and most



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of them occupy conserved positions respectively in the catalytic, disintegrin or disintegrin-like/cysteine-rich domains. In contrast, in the SVMP transcripts the putative N-glycosylation sites are clearly less abundant and most of them do not occupy conserved positions (Figure 8). Interestingly, among these SVMP transcripts, seven of the P-III class were identified both in the venom gland and in other organs of B. jararaca (pancreas and kidney).43 Moreover, three transcripts encoding B. jararaca ADAMs and ADAMTSs (kidney, brain and heart) also showed a similar pattern of various putative N- and O-glycosylation sites (Supplemental table S6). The evolution of venoms in viperids suggests that SVMPs derived from an ADAM ancestor and were recruited into the venom gland near the base of the advanced snake radiation and prior to substantial gene duplication and diversification.63,66,67 Events of gene duplication, domain loss, and positive selection resulted in generation of the P-I and P-II classes of SVMP in viperids, which lack the cysteine-rich domain which is present only in enzymes of the P-III class.51,66-69 The evolutionary history of SVMPs and their hypothetical ancestors reveals progressive alterations in the amino acid composition

and

structural

characteristics

of

ADAMs/SVMPs

through

evolutionary time and the consequent generation of the large number of functionally-diverse SVMP isoforms that are observed in venoms.67 The finding of the presence of both putative N- and O-glycosylation sites in transcripts encoding P-III SVMPs in the venom gland and in non-venom gland tissues (pancreas and kidney) and in their non-toxin paralogous transcripts, (i.e. ADAM/ADAMTS), indicates the conservation of glycosylation as a PTM that might be crucial for the expression of functional activities and stability. Sitespecific O-glycosylation has been proposed as playing a role as an important


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co-regulator of limited proteolytic processing events in various types of protein families, including the ADAMs.24 Likewise, the extracellular N-terminal tail of the 7TM GPCR β1 adrenergic receptor is O-glycosylated at four residues whose Oglycans protect against processing by a member of the ADAM family.70 Intriguingly, some of the conserved putative O-glycosylation sites found in the SVMP transcripts are located at the N- and C-terminal portions of the prodomain, which is processed to generate the active form of the proteinase, and also at the N-terminal region of the disintegrin or disintegrin-like domains, which similarly are processed to release the disintegrin mature form or in the autolysis process of some P-III class SVMPs (Figure 8).51,71 It remains to be evaluated if these O-glycosylation sites serve similar role of regulation of limited proteolytic processing events of SVMPs. An overall observation of the analysis of putative glycosylation sites in the transcripts of venom proteolytic enzymes is that in the processes of evolution and neofunctionalization of these genes, the overall glycosylation profile was conserved in regard to their ancestral genes, with SVSPs showing only sites of N-glycosylation and SVMPs showing both N- and O-type glycosylation sites.

Conclusions Protein glycosylation is a PTM that requires a complex machinery of enzymes, the presence of sequence motifs that direct the nascent proteins along the endoplasmic reticulum/Golgi apparatus route, that demands energy at various adding and trimming steps to compose the diverse glycan chains and that must be in full action upon protein synthesis in the snake venom gland, especially after venom gland draining upon milking or prey capture. Various types of



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glycosyltransferases are responsible for the modification of carbohydrate chains upon protein synthesis and consequently the glycan families co-developed upon the evolution of multicellularity,72 and indeed, the protein glycosylation category was one of the most enriched gene ontology biological processes in the venom gland of B. jararaca in comparison to other organs.43 It will be interesting to compare the composition of the repertoire of glycosyltransferases in the venom glands of different snake families in order to understand how the overall glycosylation process is controlled and evolved with regard to enzymes and substrates. It has been suggested that gene duplication facilitates rapid evolution and divergence in the venom composition among species, and that these processes may be associated with evolutionary responses to diet, habitat and predatorprey interactions. However, in the case of the Bothrops species sampled in this study, what is observed is that in their adult lives they do not considerably differ in terms of prey/diet and that the outcome of the envenomation described in human accidents is quite similar. This suggests that despite various evolutionary pressure factors, their venom whole proteomes and subproteomes of glycoproteins contain a core of components as protein signatures that define their composition which is conserved upon evolution in parallel to other molecular markers that determines their phylogenetic classification. In a recent study on the characterization of the N-glycoproteomes of various whole

organisms

(Arabidopsis

thaliana,

Schizosaccharomyces

pombe,

Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and Danio rerio), it was found that extracellular N-glycosylated proteins are significantly more conserved within the same phylogenetic lineage than


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extracellular non-N-glycosylated proteins, indicating that the majority of glycoproteins developed after species divergence to acquire important functions which were preserved in evolution.73 In this line, it is reasonable to hypothesize that glycosylation of specific venom components might have provided an evolutionary novelty for venom adaptation to deal with different inhibitors present in different prey types in their respective ecological niches.

Supporting Information Supplementary Figures Supplemental figure S1. Bothrops venom clusterings according to the composition of whole proteome. Supplemental figure S2. Principal Component Analysis (PCA) on wholeproteome data of Bothrops venoms. Supplemental

figure

S3.

Lectin-bound

fractions

of

Bothrops

venoms.

Electrophoretic profiles of Bothrops venom proteins that bound to ConASepharose, WGA-agarose and PNA-agarose. Supplemental figure S4. Effect of N-deglycosylation upon the electrophoretic profile of newborn (top) and adult B. jararaca (bottom) venom proteins. Supplemental figure S5. Western blot analysis of PNA-bound proteins using anti-metalloproteinase, anti-serine proteinase, and anti-phospholipase A2 antibodies. Supplemental figure S6. Unique proteins identified both in the whole proteome and lectin binding subproteomes of Bothrops venoms. Supplemental figure S7. Effect of N-deglycosylation of Bothrops venom proteins under non-denaturing conditions.



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Supplemental figure S8. Alignment of amino acid sequences of four cDNAs encoding precursors of bradykinin potentiating peptides/poly-His-poly-Gly peptide/C-type natriuretic peptide. Supplementary Tables Supplemental table S1. Identified Bothrops venom proteins by LC-MS/MS and database search. Supplemental table S2. Identified venom proteins in the ConA-bound fraction by LC-MS/MS and database search. Supplemental table S3. Identified venom proteins in the WGA-bound fraction by LC-MS/MS and database search. Supplemental table S4. Identified venom proteins in the PNA-bound fraction by LC-MS/MS analysis and database search. Supplemental table S5. B. jararaca venom gland transcript sequences and their putative N- and O-glycosylation sites according to NetNGlyc and NetOGlyc prediction tools. Supplemental table S6. Translated amino acid sequences of transcripts encoding ADAM-like sequences in B. jararaca. Supplementary Files Supplemental file 1. Translated amino acid sequences of transcripts encoding toxins in B. jararaca venom gland.

Acknowledgments We thank Dr. Otávio A. V. Marques of the Laboratório de Ecologia e Evolução, Instituto Butantan, for helpful discussions. This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP


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2013/07467-1; 2013/13548-4), fellowships from FAPESP to D.A.S., E.S.K, A.S.L. and A.Z., and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) to D.A.S and S.M.T.S.

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P-III metalloproteinase from the venom of Bothrops jararaca. Toxicon 2003, 41, 217-227. 41. Serrano, S.M.; Matos, M.F.; Mandelbaum, F.R.; Sampaio, C.A. Basic proteinases from Bothrops moojeni (caissaca) venom--I. Isolation and activity of two serine proteinases, MSP 1 and MSP 2, on synthetic substrates and on platelet aggregation. Toxicon 1993, 31(4), 471-481. 42. Moura-da-Silva, A.M.; Cardoso, D.F.; Tanizaki, M.M.; Mota, I. Neutralization of myotoxic activity of Bothrops venoms by antisera to purified myotoxins and to crude venoms. Toxicon 1991, 29(12), 1471-1480. 43. Junqueira-de-Azevedo, I.L.; Bastos, C.M.; Ho, P.L.; Luna, M.S.; Yamanouye, N.; Casewell, N.R. Venom-related transcripts from Bothrops jararaca tissues provide novel molecular insights into the production and evolution of snake venom. Mol. Biol. Evol. 2015, 32(3), 754-766. 44. Gupta, R.; Brunak, S. Prediction of glycosylation across the human proteome and the correlation to protein function. Pac Symp Biocomput. 2002, 310–322. 45. Steentoft, C.; Vakhrushev, S.Y.; Joshi, H.J.; Kong, Y.; Vester-Christensen, M.B.; Schjoldager, K.T.; Lavrsen, K.; Dabelsteen, S.; Pedersen, N.B.; Marcos-Silva, L.; et al. Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology. EMBO J. 2013, 32(10), 1478-1488. 46. Calvete, J.J.; Sanz, L.; Pérez, A.; Borges, A.; Vargas, A.M.; Lomonte, B.; Angulo, Y.; Gutiérrez, J.M.; Chalkidis, H.M.; Mourão, R.H.; et al. Snake population

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atrox:

Paedomorphism along its transamazonian dispersal and implications of



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geographic venom variability on snakebite management. J. Proteomics 2011, 74(4), 510-27. 47. Tashima, A.K.; Zelanis, A.; Kitano, E.S.; Ianzer, D.; Melo, R.L.; Rioli, V.; Sant'anna, S.S.; Schenberg, A.C.; Camargo, A.C.; Serrano, S.M. Peptidomics of three Bothrops snake venoms: insights into the molecular diversification of proteomes and peptidomes. Mol. Cell. Proteomics 2012, 11(11), 1245-1262. 48. Jorge, R.J.; Monteiro, H.S.; Gonçalves-Machado, L.; Guarnieri, M.C.; Ximenes, R.M.; Borges-Nojosa, D.M.; Luna, K.P.; Zingali, R.B.; CorrêaNetto, C.; Gutiérrez, J.M.; et al. Venomics and antivenomics of Bothrops erythromelas from five geographic populations within the Caatinga ecoregion of northeastern Brazil. J. Proteomics 2015, 114, 93-114. 49. Birrell, G.W.; Earl, S.; Masci, P.P.; Jersey, J.; Wallis, T.P.; Gorman, J.J.; Lavin, M.F.

Molecular diversity in venom from the Australian Brown

snake, Pseudonaja textilis. Mol. Cell. Proteomics 2006, 5, 379-389. 50. Serrano, S.M.; Maroun, R.C. Snake venom serine proteinases: sequence homology vs. substrate specificity, a paradox to be solved. Toxicon 2005, 45(8), 1115-1132. 51. Fox, J.W.; Serrano, S.M. Insights into and speculations about snake venom metalloproteinase

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formation and their contribution to venom complexity. FEBS J. 2008, 275(12), 3016-3030. 52. Morita, T. Structures and functions of snake venom CLPs (C-type lectin-like proteins) with anticoagulant-, procoagulant-, and platelet-modulating activities. Toxicon 2005, 45(8), 1099-1114.


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53. Guo, C.; Liu, S.; Yao, Y.; Zhang, Q.; Sun, M.Z. Past decade study of snake venom L-amino acid oxidase. Toxicon 2012, 60(3), 302-311. 54. Serrano, S.M. The long road of research on snake venom serine proteinases. Toxicon 2013, 62, 19-26. 55. Lin, C.W.; Chen, J.M.; Wang, Y.M.; Wu, S.W.; Tsai, I.H.; Khoo, K.H. Terminal disialylated multiantennary complex-type N-glycans carried on acutobin define the glycosylation characteristics of the Deinagkistrodon acutus venom. Glycobiology 2011, 21(4), 530-42. 56. Hayes, M.B.; Wellner, D. Microheterogeneity of L-amino acid oxidase. Separation

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electrofucusing. Biol. Chem. 1969, 244(24), 6636-6644. 57. Serrano, S.M.; Hagiwara, Y.; Murayama, N.; Higuchi, S.; Mentele, R.; Sampaio, C.A.; Camargo, A.C.; Fink, E. Purification and characterization of a kinin-releasing and fibrinogen-clotting serine proteinase (KN-BJ) from the venom of Bothrops jararaca, and molecular cloning and sequence analysis of its cDNA. Eur. J. Biochem 1998, 251(3), 845-853. 58. Serrano, S.M.; Sampaio, C.A.; Mentele, R.; Camargo, A.C.; Fink, E. A novel fibrinogen-clotting enzyme, TL-BJ, from the venom of the snake Bothrops jararaca: purification and characterization. Thromb. Haemost. 2000, 83(3), 438-44. 59. Mandal, S.; Bhattacharyya, D. Two L-amino acid oxidase isoenzymes from Russell's viper (Daboia russelli russelli) venom with different mechanisms of inhibition by substrate analogs. FEBS J. 2008, 275(9), 2078-2095. 60. Yamashiro, E.T.; Oliveira, A.K.; Kitano, E.S.; Menezes, M.C.; Junqueira-deAzevedo, I.L.; Paes Leme, A.F.; Serrano, S.M. Proteoforms of the



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platelet-aggregating enzyme PA-BJ, a serine proteinase from Bothrops jararaca venom. Biochim. Biophys. Acta. 2014, 1844(12), 2068-2076. 61. Murayama, N.; Hayashi, M.A.; Ohi, H.; Ferreira, L.A.; Hermann, V.V.; Saito, H.; Fujita, Y.; Higuchi, S.; Fernandes, B.L.; Yamane, T.; et al. Cloning and sequence analysis of a Bothrops jararaca cDNA encoding a precursor of seven bradykinin-potentiating peptides and a C-type natriuretic peptide. Proc. Natl. Acad. Sci. USA 1997, 94(4), 1189-93. 62. Itoh, N.; Tanaka, N.; Funakoshi, I.; Kawasaki, T.; Mihashi, S.; Yamashina, I. Organization of the gene for batroxobin, a thrombin-like snake venom enzyme. Homology with the trypsin/kallikrein gene family. J. Biol. Chem. 1988, 263(16), 7628-7631. 63. Fry, B.G.; Scheib, H.; Van der Weerd, L.; Young, B.; McNaughtan, J.; Ramjan, S.F.; Vidal, N.; Poelmann, R.E.; Norman, J.A. Evolution of an arsenal: structural and functional diversification of the venom system in the advanced snakes (Caenophidia). Mol. Cell. Proteomics 2008, 7(2), 215-246. 64. Guo, S.; Skala, W.; Magdolen, V.; Brandstetter, H.; Goettig, P. Sweetened kallikrein-related peptidases (KLKs): glycan trees as potential regulators of activation and activity. Biol. Chem. 2014, 395(9), 959-976. 65. Deshimaru, M.; Ogawa, T.; Nakashima, K.; Nobuhisa, I.; Chijiwa, T.; Shimohigashi, Y.; Fukumaki, Y.; Niwa, M.; Yamashina, I.; Hattori, S.; Ohno, M. Accelerated evolution of crotalinae snake venom gland serine proteases. FEBS Lett. 1996, 397(1), 83-88. 66. Casewell, N.R.; Wagstaff, S.C.; Harrison, R.A.; Renjifo, C.; Wüster, W. Domain loss facilitates accelerated evolution and neofunctionalization of


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duplicate snake venom metalloproteinase toxin genes. Mol. Biol. Evol. 2011, 28, 2637–2649. 67. Casewell, N.R. On the ancestral recruitment of metalloproteinases into the venom of snakes. Toxicon 2012, 60, 449–454. 68. Juárez, P.; Comas, I.; González-Candelas, F.; Calvete, J.J. Evolution of snake venom disintegrins by positive Darwinian selection. Mol. Biol. Evol. 2008, 25(11), 2391-407. 69. Serrano, S.M.; Oliveira, A.K.; Menezes, M.C.; Zelanis, A. The proteinaserich proteome of Bothrops jararaca venom. Toxin Reviews 2014, 33(4), 169-184. 70. Hakalahti, A.E.; Vierimaa, M.M.; Lilja, M.K.; Kumpula, E.P.; Tuusa, J.T.; Petäjä-Repo, U.E. Human beta1-adrenergic receptor is subject to constitutive and regulated N-terminal cleavage. J. Biol. Chem. 2010, 285(37), 28850-28861. 71. Moura-da-Silva, A.M.; Della-Casa, M.S.; David, A.S.; Assakura, M.T.; Butera, D.; Lebrun, I.; Shannon, J.D.; Serrano, S.M.; Fox, J.W. Evidence for heterogeneous forms of the snake venom metalloproteinase jararhagin: a factor contributing to snake venom variability. Arch. Biochem. Biophys. 2003, 409(2), 395-401. 72. Varki, A. et al. Evolution of glycan diversity. In: Essentials of glycobiology, 2nd Ed., Varki, A.; Cummings, R.D.; Esko, J.D.; Freeze, H.H.; Stanley, P.; Bertozzi, C.R.; Hart, G. W.; Etzler, M.E., Eds., Cold Spring Harbor Laboratory Press ,Cold Spring Harbor (NY), 2009, Chapter 19. 73. Zielinska, D.F.; Gnad, F.; Schropp, K.; Wiśniewski, J.R.; Mann, M. Mapping N-glycosylation sites across seven evolutionarily distant species reveals



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a divergent substrate proteome despite a common core machinery. Mol. Cell. 2012, 46(4), 542-548.

Figure legends Scheme 1. Schematic overview of the strategies applied for analyzing the whole proteomes and glycoproteomes of the venom from seven Bothrops species. Figure 1. Bothrops venom whole proteome characterization. (A) Graphical overview of the distribution of toxin classes identified in seven Bothrops venom proteomes by trypsin digestion and LC-MS/MS analysis. Values on the y axis indicate the number of unique proteins. (B) Venn diagram of the distribution of unique proteins identified in the whole venom proteomes, grouped by venom. 19 unique proteins are present in all seven venoms, while there are proteins that appeared in one venom only. Two-two to six-six venom comparisons are not shown. Figures were generated using the data of Supplemental Table S1. Figure 2. Bothrops venom clustering according to the composition of whole proteomes. Graphical visualization of the two hierarchical clusterings of the venom proteome characterization by trypsin digestion and LC-MS/MS analysis. For each venom, a given protein is either present (red) or absent (black). Figure 3. Glycoproteomic analysis of Bothrops venoms. (A) SDS-PAGE (12% gel) profiles of venoms (20 µg) incubated in the presence and in the absence of N-glycosidase F and O-Glycosidase. Proteins were stained with colloidal Coomassie blue. (B) SDS-PAGE (12% gel) profiles of Bothrops venoms (12 µg) incubated in the presence and in the absence of α2-3,6,8,9-neuraminidase,


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and

β-N-acetylglucosaminidase

endo-α-N-

acetylgalactosaminidase. Proteins were stained with silver. Figure 4. Venom toxin classes identified in the ConA-bound, WGA-bound and PNA-bound fractions. Values on the y axis indicate the number of unique proteins identified in each lectin-bound fraction. CTL: C-type lectin; LAAO: Lamino acid oxidase; PL: phospholipase; SVMP: metalloproteinase; SVSP: serine proteinase. Figure 5. Venn diagrams of the distribution of the 118 unique proteins identified in the ConA-, WGA- and PNA-bound fractions by trypsin digestion and LCMS/MS analysis. (A) The unique proteins are grouped by venom. 15 proteins are present in all seven venoms, while there are proteins that appeared in one venom only. (B) Venn diagram illustrating shared and uniquely identified proteins in the ConA-, WGA- and PNA-bound fractions of Bothrops venoms. The unique proteins are grouped by lectin type. 22 proteins appeared only in the ConA-bound fraction (yellow), 26 proteins appeared only in the WGA-bound fraction (blue) and 18 proteins appeared only in the PNA-bound (orange). 14 proteins appeared in all lectin-bound fractions. The diagram was generated using the data of Supplemental Tables S2, S3 and S4. Figure 6. Bothrops venom clustering according to the composition of lectinbinding subproteomes. Graphical

visualization of

the two

hierarchical

clusterings of the unique proteins identified in the ConA-, WGA- and PNAbound fractions by trypsin digestion and LC-MS/MS analysis. For each pair (venom, protein), a given protein is either present (red) or absent (black). Figure 7. Unique proteins identified both in the whole proteome and lectinbinding subproteomes of Bothrops venoms. (A) Venn diagram of the distribution 57 ACS Paragon Plus Environment


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of the unique proteins identified in the whole venom proteome and lectin-binding subproteome characterization. Numbers inside small circles correspond to nonglycoproteins. (B) Graphical visualization of the hierarchical clustering of the 171 proteins identified both in the whole venom proteome and lectin-binding subproteome characterizations. (C) Graphical visualization of the hierarchical clustering of the 119 glycoproteins present on panel B. (D) Graphical visualization of the hierarchical clustering of the 52 non-glycoproteins present on panel B. Figure 8. Schematic representing some B. jararaca venom gland transcripts and their putative N- and O-glycosylation sites according to NetNGlyc and NetOGlyc prediction tools, as detailed in Supplemental Table S5. P: signal peptide; red diamonds: N-glycosylation; green diamonds: O-glycosylation. In the case of SVMPs and SVSPs, only two representative transcripts are shown.


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A

40 35 30 25 20

15 10 5 0

Figure 1 ACS Paragon Plus Environment


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Figure 2

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 N-glycosidase F 13 O-glycosidase 14 kDa 15 16 17 68 18 50 19 43 20 21 22 23.5 23 24 25 14 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

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A

B. cotiara

-

+ -

+

B. insularis

-

+ -

+

B. jararaca

-

+ -

+

B. moojeni B. neuwiedi B. jararacussu

B. erythromelas

-

-

+ -

+

-

+ -

kDa

+

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+ -

+ kDa

68 50

68 50

43

43

23.5

23.5

14

14


+ -

+

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B -

+

-

+

-

+

-

+

-

kDa 68

68

50

50

43

43

23.5

23.5

14

14


+

-

+

-

+


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

ConA 20 15 10 5 0

WGA 20 15 10 5 0

PNA 20 15 10 5 0


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A

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Figure 6

B. cotiara

B. erythromelas

B. insularis

B. jararaca

B. neuwiedi

B. jararacussu

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B. moojeni



SVMP SVMP LAAO OT SVMP OT SVSP OT SVMP OT OT SVMP SVMP PL SVSP SVMP LAAO SVMP CTL OT SVSP SVMP SVMP SVMP PL SVSP SVMP LAAO PL SVSP SVMP PL PL SVMP SVMP SVSP SVSP PL CTL SVSP SVSP SVSP SVSP PL SVSP SVMP CTL SVSP PL SVSP OT OT PL LAAO LAAO SVSP SVSP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVSP SVMP OT SVMP OT SVMP PL LAAO LAAO LAAO PL PL SVSP SVSP SVMP SVMP SVMP PL LAAO CTL SVSP PL LAAO CTL CTL SVMP SVMP SVMP SVMP SVMP PL CTL CTL OT CTL PL PL SVSP PL CTL SVMP SVMP SVSP OT CTL PL OT CTL CTL SVMP CTL CTL CTL

-

VM1BI_BOTMO R4NNL0_VIPAA,VM3VA_MACLB BJAR454LAO2 F2Q6E5_CROHD,F2Q6E6_CROHD VM1B1_BOTBA U3TBU1_OVOOK,U3TDI3_OVOOK BJAR454SVSP01 T1DNX8_CROHD BJAR454SVMPPIII06 A0A077L7M9_PROFL,T2HRS9_PROFL BJAR454NGF1,NGFV_BOTJR VM3B4_BOTJA VM2JC_BOTJA I2DAL4_BOTDP BJAR454SVSP05 BJAR454SVMPPII02 BJAR454LAO1 VM3BE_BOTER BJAR454CTL06 A0A0B8RNS9_BOIIR T1E3B5_CROHD BJAR454SVMPPIII11 BJAR454SVMPPII04 BJAR454SVMPPII05 PA2A_BOTIN BJAR454SVSP07 BJAR454SVMPPIII04 OXLA_CROAD,T1DP54_CROOH PA2H3_BOTAS VSPTL_BOTAL BJAR454SVMPPIII07 PA2B2_BOTJR PA2H1_BOTPI BJAR454SVMPPIII01 A0A0C4ZNF1_GLOIT T1DP86_CROOH VSPF_BOTAT L7VGC1_BOTAN BJAR454CTL10,LECG_BOTIN BJAR454SVSP06,VSP_BOTIN BJAR454SVSP_AB004067,VSP2_BOTJA BJAR454SVSP10 VSPA_BOTJA PA2H2_BOTMO BJAR454SVSP08 VM3H3_BOTJA LECG_BOTPA BJAR454SVSP03 A0A0F7Z632_CROAD,PLB_CROAD VSP1_BOTJA QPCT_BOTJA BJAR4545NUCL1 PA2H_BOTPA OXLA_BOTPA OXLA_BOTMO BJAR454SVSP04 BJAR454SVSP01,VSP14_BOTJA BJAR454SVMPPIII12 BJAR454SVMPPIII10 BJAR454SVMPPIII09 BJAR454SVMPPIII05 BJAR454SVMPPIII03 BJAR454SVMPPIII02,VM36A_BOTIN E3UJL2_BOTNU Q7T1T5_BOTJR VSPL_BOTAS E3UJL9_BOTNU BJAR454DIESTER2 VM3BP_BOTJA J3SEA2_CROAD E3UJL8_BOTNU T1DLW3_CROHD OXLA_BOTLC OXLA_BOTJR R4FJP5_9SAUR PA2H1_BOTPISV=1 PA2BD_BOTLC BJAR454SVSP02 VSPF5_MACLB VM3LB_BOTLC E3UJL5_BOTNU VM1LA_BOTLC PA2B3_BOTAS OXLA_CERCE LECG_TRIST VSP12_BOTJA PA2HB_ATRNM OXLA_DEMVE LECG_BOTJR BJAR454CTL02 VM31_BOTAT E3UJL1_BOTNU VM33_BOTAT BJAR454SVMPPII06,E3UJM0_BOTNU E3UJL4_BOTNU BJAR454PLA21,PA2B1_BOTJR SL9B_BOTJA SLEB_BOTJA BJAR454CRISP1,F2Q6G1_DEIAC LECG_BOTPI PA2H1_BOTMO BJAR454PLA22 VSP3_BOTJA BJAR454PLA24 BJAR454CTL15,M1V359_BOTJA BJAR454SVMPPIII08 BJAR454SVMPPI02,VM1B_BOTIN BJAR454SVSP09 BJAR454CRISP1 SL1A_BOTJA PA2A_BOTMO A0A077L6N8_PROEL,A0A0F7ZD91_CROAD BJAR454CTL08 BJAR454CTL14,SLAA_BOTJA,SLA_BOTIN BJAR454SVMPPIII13 BJAR454CTL18 SL1B_BOTJA BJAR454CTL07

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A

Figure 7



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B

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Figure 7


B. cotiara

B. erythromelas

B. insularis

B. jararaca

B. neuwiedi

C B. moojeni

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B. jararacussu

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Figure 7

SVSP SVSP SVMP SVMP OT SVMP OT OT OT OT SVMP SVSP OT OT SVMP SVMP SVMP OT LAAO OT SVMP LAAO SVMP SVMP SVMP SVSP SVMP LAAO SVMP LAAO SVMP SVSP SVSP LAAO SVMP OT SVMP SVMP SVMP SVMP OT OT SVMP SVMP SVSP SVSP SVMP SVMP SVMP SVMP SVMP OT SVMP SVMP SVSP OT SVSP SVMP SVSP LAAO LAAO LAAO OT SVSP SVMP SVSP SVSP SVMP OT SVSP SVMP SVSP SVSP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVSP OT SVMP LAAO OT SVSP SVSP SVMP SVSP SVMP SVMP SVSP SVMP SVMP SVMP SVMP SVMP SVMP SVSP SVMP SVMP SVMP LAAO SVMP SVMP SVMP SVSP LAAO SVSP SVSP SVSP SVMP SVSP SVMP SVMP OT OT SVSP

-

VSPBH_BOTAL VSP_LACST VM3B2_BOTJA R4NNL0_VIPAA,VM3VA_MACLB U3TBU1_OVOOK,U3TDI3_OVOOK E9KJZ5_ECHOC,E9KJZ6_ECHOC,Q2UXQ2_ECHOC T1DNX8_CROHD V8NS94_OPHHA F2Q6E5_CROHD,F2Q6E6_CROHD CRVP_HELAG VM3L_BOTLC BJAR454SVSP01 A0A077L7M9_PROFL,T2HRS9_PROFL BJAR454NGF1,NGFV_BOTJR VM3VA_CROAT V5Z141_DEIAC BJAR454SVMPPII06,E3UJM0_BOTNU BJAR454CRISP1,F2Q6G1_DEIAC OXLA_DEMVE NGFV_BOTJR VM1BI_BOTMO OXLA_CERCE VM11_BOTAT VM1_BOTPI VM3LB_BOTLC VSPF5_MACLB A0A077L6L9_PROEL R4FJP5_9SAUR VM1B2_BOTJR OXLA_BOTJR J3S831_CROAD BJAR454SVSP05 VSPH_BOTJR A0A068EPZ2_GLOIT,OXLA_GLOBL E3UJL4_BOTNU BJAR454VEGF1,TXVE_BOTJA VM3B4_BOTJA E3UJL0_BOTNU VM2JC_BOTJA VM1B1_BOTBA BJAR454HYALU1 A0A0B8RNS9_BOIIR E3UJL1_BOTNU VM3BA_BOTAS VSP_CRODD BJAR454SVSP09 VMXJ4_BOTJA VM3B3_BOTJA VM33_BOTAT BJAR454SVMPPIII04 A0A0F7Z792_CROAD J3SEA2_CROAD VM3BE_BOTER VM31_BOTAT BJAR454SVSP07 BJAR4545NUCL1 BJAR454SVSP_AB004067,VSP2_BOTJA E3UJL8_BOTNU F8S115_CROAD,J3S3W7_CROAD,J3SDX1_CROAD OXLA_BOTLC OXLA_BOTMO OXLA_BOTPA QPCT_BOTJA T1E3B5_CROHD VM2IA_BOTIN VSP1_BOTJA VSPL_BOTAS VM3JA_BOTJA BJAR454DIESTER2 BJAR454SVSP04 BJAR454SVMPPII05 BJAR454SVSP03 BJAR454SVSP01,VSP14_BOTJA BJAR454SVMPPIII02,VM36A_BOTIN BJAR454SVMPPIII03 BJAR454SVMPPIII12 BJAR454SVMPPIII05 BJAR454SVMPPIII11 BJAR454SVMPPIII10 BJAR454SVMPPIII08 BJAR454SVMPPIII09 BJAR454SVSP06,VSP_BOTIN F2Q6G1_DEIAC BJAR454SVMPPIII07 BJAR454LAO2 BJAR454CRISP1 BJAR454SVSP10 VSPA_BOTJA E3UJL2_BOTNU BJAR454SVSP02 Q7T1T5_BOTJR E3UJL9_BOTNU BJAR454SVSP08 BJAR454SVMPPIII13 VM3BP_BOTJA BJAR454SVMPPI02,VM1B_BOTIN BJAR454SVMPPII03 BJAR454SVMPPII04 A0A0C4ZNF1_GLOIT T1DP86_CROOH E3UJM0_BOTNU VM1LA_BOTLC E3UJL5_BOTNU BJAR454LAO1 BJAR454SVMPPII02 BJAR454SVMPPIII06 BJAR454SVMPPIII01 VSPTL_BOTAL OXLA_CROAD,T1DP54_CROOH J3S3W4_CROAD,T1DE97_CROOH VSPF_BOTAT VSP12_BOTJA VM3H3_BOTJA VSP3_BOTJA BJAR454SVMPPI01 BJAR454SVMPPII01 TXVE_BOTIN A0A077L6N8_PROEL,A0A0F7ZD91_CROAD VSP41_BOTPI


Page 70 of 72

B. cotiara

Figure 7 B. moojeni

B. erythromelas

B. insularis

B. jararaca

D

B. neuwiedi

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

B. jararacussu


PL PL PL PL PL PL CTL PL PL CTL CTL PL PL PL PL PL CTL CTL PL PL CTL CTL CTL CTL CTL CTL CTL CTL PL PL PL PL CTL PL PL CTL PL CTL CTL PL CTL CTL CTL PL PL CTL CTL CTL PL CTL CTL CTL


-

PA2A2_BOTAS PA2BD_BOTLC PA2H1_BOTPISV=1 PA2A_BOTJR PA2B2_BOTJR BJAR454PLA21,PA2B1_BOTJR SL9B_BOTJA I2DAL6_BOTDP PA2H1_BOTMO SLEB_BOTJA LECG_BOTPI BJAR454PLA21,PA2B1_BOTJR,PA2H_BOTPA PA2H1_BOTPI PA2H3_BOTAS L7VGC1_BOTAN PA2B3_BOTAS LECG_BOTJR LECG_TRIST PA2HB_ATRNM I2DAL4_BOTDP BJAR454CTL07 BJAR454CTL18 LECG_BOTPA SL1B_BOTJA BJAR454CTL04 BJAR454CTL02 SLB_BOTIN SLEA_BOTJA A0A0F7Z632_CROAD,PLB_CROAD PA2H_BOTPA BJAR454PLB1 T1DLW3_CROHD SLA_BOTIN PA2A_BOTIN PA2H2_BOTMO BJAR454CTL10,LECG_BOTIN PA2A_BOTMO SL1A_BOTJA BJAR454CTL16 BJAR454PLA24 BJAR454CTL06 BJAR454CTL05 BJAR454CTL01,M1VNP5_BOTJA PA2A_BOTJA BJAR454PLA22 BJAR454CTL15,M1V359_BOTJA BJAR454CTL20 BJAR454CTL21 BJAR454PLA23 BJAR454CTL03,BJAR454CTL09 BJAR454CTL08 BJAR454CTL14,SLAA_BOTJA,SLA_BOTIN

Page 71 of 72

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42


P-I SVMP

P

Pro

Proteinase

P-I SVMP

P

Pro

Proteinase

P-II SVMP

P

Pro

Proteinase

Disintegrin

P-II SVMP

P

Pro

Proteinase

Disintegrin

P-III SVMP

P

Pro

Proteinase

Disintegrin-like

Cys-rich

P-III SVMP

P

Pro

Proteinase

Disintegrin-like

Cys-rich

SVSP

P

SVSP

P

LAAO

P

PLB

P

PLA2

P

CTL

P



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

For TOC only


Page 72 of 72

Proteomic and Glycoproteomic Profilings Reveal That Post

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