Bothrops jararaca venom gland transcriptome: Analysis of the gene expression pattern

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Toxicon 48 (2006) 437–461 www.elsevier.com/locate/toxicon

Bothrops jararaca venom gland transcriptome: Analysis of the gene expression pattern Daniela A.P. Cidadea,, Tatiana A. Sima˜oa, Alberto M.R. Da´vilab, Glauber Wagnerb, Ina´cio de L.M. Junqueira-de-Azevedoc, Paulo Lee Hoc, Cassian Bond, Russolina B. Zingalie, Rodolpho M. Albanoa, a

Departamento de Bioquı´mica, Universidade do Estado do Rio de Janeiro, CEP 20551 013 Rio de Janeiro, RJ, Brasil b Departamento de Bioquı´mica e Biologia Molecular, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil, 4365 CEP 21045 900 Rio de Janeiro, RJ, Brasil c Centro de Biotecnologia, Instituto Butantan, CEP 05503-900 Sa˜o Paulo, SP, Brasil d Centre National de la Recherche Scientifique 3, rue Michel-Ange 75794, Paris, France e Instituto de Bioquı´mica Me´dica, Rede Proteoˆmica do Rio de Janeiro, Universidade Federal do Rio de Janeiro, CEP 21941-590 Rio de Janeiro, RJ, Brasil Available online 7 July 2006

Abstract Bothrops jararaca is a pit viper responsible for the majority of snake envenoming accidents in Brazil. As an attempt to describe the transcriptional activity of the venom gland, ESTs of a cDNA library constructed from B. jararaca venom gland were generated and submitted to bioinformatics analysis. The results showed a clear predominance of transcripts coding for toxins instead of transcripts coding for proteins involved in cellular functions. Among toxins, the most frequent transcripts were from metalloproteinases (52.6%), followed by serine-proteinases (28.5%), C-type lectins (8.3%) and bradykinin-potentiating peptides (BPPs) (6.2%). Results were similar to that obtained from the transcriptome analysis of B. insularis, a phylogenetically close sister of B. jararaca, though some differences were observed and are pointed out, such as a higher amount of the hypotensive BPPs in B. insularis transcriptome (19.7%). Another striking difference observed is that PIII and PII-classes of metalloproteinases are similarly represented in B. jararaca in contrast to B. insularis, in which a predominance of PIII-class metalloproteinase, which present a more intense hemorrhagic action, is observed. These features may, in part, explain the higher potency of B. insularis venom. The results obtained can help in proteome studies, and the clones can be used to directly probe the genetic material from other snake species or to investigate differences in gene expression pattern in response to factors such as diet, aging and geographic localization. r 2006 Elsevier Ltd. All rights reserved. Keywords: Snake venom; Viperidae; Toxins; cDNA library; Expressed sequence tags

1. Introduction Corresponding author. Tel.: +55 21 2587 6428;

fax: +55 21 25876136. E-mail addresses: [email protected] (D.A.P. Cidade), [email protected] (R.M. Albano). 0041-0101/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2006.07.008

Bothrops genus includes more than 30 species and subspecies distributed between Central and South America. Members of this genus are responsible for approximately 90% of venomous snakebite

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accidents in Brazil. Bothrops jararaca is the most common species that occurs in the southeast region of Brazil, accounting for the majority of accidents (Franc- a and Malaque, 2003; Ribeiro and Jorge, 1997). Clinically, patients bitten by B. jararaca usually present edema, systemic bleeding, thrombocytopenia and prolongation of whole blood clotting time (Sano-Martins et al., 1997; Santoro and SanoMartins, 2004). These symptoms result from the 3 main activities of bothropic venom: proteolytic, with local inflammatory edema at the snakebite site; hemorrhagic, with endothelium damage and systemic bleeding; and procoagulant, leading to the consumption of coagulant factors and disrupting the equilibrium of blood coagulation (Matsui et al., 2000; Varanda and Giannini, 1999). The principal classes of toxins involved in these actions are lectins, prothrombin activating toxins, hemorrhagins, disintegrins and serine-proteinases among others (Markland, 1998; White, 2005). In this work we generated and analyzed 2318 expressed sequence tags (ESTs) from a cDNA library of the venom gland of B. jararaca, aiming to describe its protein contents. Similar transcriptome approach have been conducted with other snakes such as B. insularis (Junqueira-de-Azevedo and Ho, 2002), Bitis gabonica (Franscischetti et al., 2004) and B. jararacussu (Kashima et al., 2004). This kind of study is a good source of information about venom composition, eventually leading to new toxin discovery. B. jararaca is a close sister taxa of B. insularis (Werman, 1992), an endemic snake from Ilha da Queimada Grande, located in Sa˜o Paulo State (Southeast of Brazil). The geographical isolation and special diet (based exclusively on birds and some invertebrates available on the island) to what B. insularis has been submitted for thousands of years may have caused some changes in its venom composition (Daltry et al., 1996). In this work, we adopted the bioinformatic parameters to construct the EST database as close as possible to those used for B. insularis transcriptome (Junqueirade-Azevedo and Ho, 2002), in order to allow comparative studies between these two pit vipers. Therefore, we were able to infer similarities and differences in their venom composition, and correlate it with the potency and clinical symptoms of both venoms. In our transcriptome analysis, since we have sequenced a larger number of sequence tags, we were able to characterize even not abundant

transcripts besides the most frequent ones, providing a set of sequences from a very specialized secretory tissue that can be used for comparative studies and homology searches, and are available at EST section of the NCBI GenBank (http://www. ncbi.nlm.nih.gov/dbEST). Moreover, an EST database can also be used as an auxiliary reference in proteome studies, leading to easier and faster protein identification (Mathesius et al., 2001). 2. Materials and methods 2.1. cDNA library construction and EST technology The first procedure of an EST project is to obtain mRNA from the tissue of interest, transform it into double-stranded cDNA and clone it into a suitable vector to create a cDNA library. With this intent, poly(A)-rich RNA was prepared from total RNA of the venom gland by oligo(dT)-cellulose chromatography. A directional cDNA library was constructed using a plasmid cloning kit (Superscript plasmid system, Life Technologies Inc.), as described previously (Arocas et al., 1997). The cDNA fragments, representing transcripts for the genes that were expressed at the moment of RNA extraction, were ligated into the pT7T3D EcoRI/ NotI/BAP phagemid vector (Pharmacia LKB Biotechnology Inc.) and used to transform Epicurian coli KL1 Blue MRF supercompetent cells (Stratagene), producing a library of about 2
105 independent colonies. An important quality assessment of the library is to determine the average length of the cDNAs that were cloned in the vector and, for this, isolated colonies were randomly chosen and grown in small-scale cultures for plasmid preparation. Plasmid DNA extraction was performed according to Sambrook et al., 1989, and PCR reactions were performed using a pair of primers that flank the multiple cloning site of the vector (T7 upstream and T3 downstream). Amplification conditions were 95 1C for 5 min and 30 cycles of 95 1C for 45 s, 50 1C for 45 s and 72 1C for 4 min, with a final extension step of 72 1C for 5 min. The length of cDNAs was estimated by agarose gel electrophoresis analysis of PCR amplified products. The next step is the large-scale sequencing of the cDNAs and, for this, aliquots of the plasmid DNA library were used to transform Escherichia coli XL1-Blue by electroporation, and plated on Circle Grow medium (Bio 101 Systems, USA) containing

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ampicilin (100 mg/ml), isopropyl-b-D-thiogalactopyranoside (IPTG) (120 mg/ml) and 5-bromo4-chloro-3-indolyl-b-D-galactopyranoside (X-Gal) (400 mg/ml) to allow blue/white selection of the colonies. White colonies, which are the ones that contain vectors in which cDNA molecules have been cloned, were randomly selected and plasmid DNA was prepared by standard methods (Sambrook et al., 1989). After quantification, 400 ng of template DNA, corresponding to a single colony, were added to sequencing reactions based on the dideoxy-chain termination method (Sanger et al., 1977), with the Dyenamic ET dye terminators Kit (Amersham-Biosciences) and T7 primer. The sequencing reactions were analyzed on the MEGABACE 1000 automated DNA sequencer (AmershamBiosciences). The next step is to analyze the sequence chromatograms and remove low quality bases and vector sequences. With this intent, the chromatograms were analyzed with Phred/Phrap software (http://www.phrap.org/phredphrapconsed.html). With the aid of this software, nucleotide sequences were subjected to vector sequence removal and low quality 50 and 30 ends were also trimmed. Only good quality (Phred425) sequences, longer than 150 bp, were considered for annotation. At this point we had a collection of good quality sequences free of any vector contamination, which had to be organized into clusters. A cluster corresponds to a group of sequences that encode the same transcript. The clusterization of the sequences was performed using CAP3 software (Huang and Madan, 1999). A minimum overlap of 50 bp, with at least 98% identity, was required for any assembly, in order to avoid misassembly of paralogous sequences, considering the eventual presence of many isoforms of toxins in the venom gland. Once the clusters have been formed, the next step is their careful annotation. The annotation step involves a thorough search for related sequences in several databases of DNA and protein sequences, protein motifs, etc. Our first action was to search the clustered sequences against the protein NR GenBank NCBI database by the use of the Blast tool. The expectation values considered to allow a putative identification were e-values o10-05. In the cases where no matches were found, Blast was performed against the nucleotide NR GenBank NCBI database. The 3rd option in searching for homologies was to perform Blast against the EST section of the NCBI nucleotide database, where

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most of the B. insularis EST sequences have been deposited (Junqueira-de-Azevedo and Ho, 2002). Conserved domain searches were also performed using the CDD tool of NCBI. As all of the information generated by the ESTs and their annotation had to be organized in an user-friendly format, the GARSA system (Davila et al., 2005) was used to perform all above-mentioned bioinformatics analysis and to store all information as a data bank. 3. Results and discussion 3.1. Quality assessment of the cDNA library The length of cloned cDNAs was estimated by agarose gel electrophoresis analysis of PCR amplified products. The results showed a distribution between 400 and 1600 bp, with an average of 800 bp (data not shown). After sequencing, the 3327 chromatograms were submitted to bioinformatics analysis to remove vector and poor quality sequences, resulting in 2318 good quality sequences. After clusterization 1154 clusters were formed, from which 855 were singlets. This high number of clusters resulted from the very stringent parameters used for clusterization, in order to avoid misassembling of protein isoforms. All of the 1154 clusters were deposited on the EST division of GenBank (http://www.ncbi.nlm.nih.gov/dbEST) under accession numbers DW712660 to DW713813. Additionally, clusters, reads and similarity analysis results are available trough the GARSA system at http:// www.biowebdb.org/garsa. 3.2. Cluster identification by homology searches After performing the Blast searches against the protein, nucleotide and EST databases of NCBI GenBank and considering a minimum e-value of 1005 for identification, we found 434 clusters (549 reads) with no database match (Table 1). This accounted for 23.7% of the reads, a value that is consistent with other transcriptome studies, which show values that vary from 10% to 47% of no hits (Davey et al., 2001; Valenzuela et al., 2002; Francischetti et al., 2002 and 2004; Ribeiro et al., 2004). These sequences may represent a source of new information about venom composition, coding for proteins not yet described. In addition to the sequences with no database match, there are 83 clusters (115 reads) possessing homology with proteins that have been already described but with

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no functional assessment, hereby named unknown proteins. The identified proteins were separated in two groups: toxins and cellular proteins. From the matching clones, toxins account for 77.6% of the transcripts, while cellular proteins represent 22.4% (Table 1). In the analysis of the B. insularis transcriptome, the toxin representation over matching clones was 67%, with the cellular proteins being responsible for 33% of the transcripts (Junqueira-de-

Azevedo and Ho, 2002). The same pattern was observed for B. jararaca, where 71.6% of matching clones codes for toxins and 28.4% codes for cellular proteins (Kashima et al., 2004). It becomes clear that cDNA for toxins are highly represented in snake venom gland transcriptomes, suggesting that these molecules are preferentially expressed over proteins related with cellular functions. The toxin clusters are listed on Table 2 and the cellular protein clusters are listed on Table 3.

Table 1 Representation of the ESTs from B. jararaca venom glands Category

Clusters (% over total)

Clones (% over total)

Redundancy (clones/ clusters)

Representation over matching clones (%)

No database match Unknown proteins Matching sequences Similar to toxins Similar to cellular proteins

434 (37.6) 83 (7.2) 637 (55.2) 409 (35.5) 228 (19.7)

549 (23.7) 115 (5.0) 1654 (71.3) 1283 (55.3) 371 (16.0)

1.3 1.4 2.6 3.1 1.6

— — 100.0 77.6 22.4

Table 2 Identification of putative toxin-encoding clusters from B. jararaca venom Cluster

N

Putative identification

e-value

Metalloproteinases JARO001001E01 JARO001002D11 JARO001001E09 JARO001001C04 JARO001001H12 JARO001003B09 JARO001013A02 JARO001005A03 JARO001002H10 JARO001006H07 JARO001012C05 JARO001016B04 JARO001003D11 JARO001006B09 JARO001010B06 JARO001010E11 JARO001012A02 JARO001014A01 JARO001003D12 JARO001004C08 JARO001013H03 JARO001014H07 JARO001015D06 JARO001023G05 JARO001043B04 JARO001001C06

137 103 77 29 15 14 13 11 10 10 06 06 05 05 05 05 05 05 04 04 04 04 04 04 04 03

Bothrops jararaca bothrostatin precursor Bothropasin/jararhagin [Bothrops jararaca] Bothropasin/jararhagin [Bothrops jararaca] Bothropasin precursor [Bothrops jararaca] Metalloproteinase/disintegrin ussurin precursor [Gloydius ussuriensis] Bothrops jararaca bothrostatin precursor Hemorrhagic metalloproteinase HF3 [Bothrops jararaca] Bothropasin/jararhagin [Bothrops jararaca] Bothrops jararaca bothrostatin precursor Bothrops jararaca bothrostatin precursor Metalloprotease BOJUMET II [Bothrops jararacussu] Bothropasin precursor [Bothrops jararaca] Metalloprotease BOJUMET II [Bothrops jararacussu] Bothropasin precursor [Bothrops jararaca] Bothrops jararaca bothrostatin precursor Hemorrhagic metalloproteinase HF3 [Bothrops jararaca] Vascular apoptosis-inducing protein [western diamond back rattlesnake Bothrops jararaca bothrostatin precursor Hemorrhagic metalloproteinase HF3 [Bothrops jararaca] Bothropasin/jararhagin [Bothrops jararaca] Jararhagin precursor [Bothrops jararaca] Crotalus atrox prepro-hemorrhagic toxin c, atrolysin c Hemorrhagic metalloproteinase HF3 [Bothrops jararaca] Metalloproteinase-disintegrin-like protein [Agkistrodon contortrix laticinctus] Bothrops jararaca bothrostatin precursor Metalloproteinase precursor [Bothrops insularis]

e-131 0.0 3e-44 0.0 1e-33 e-180 e-109 7e-14 e-137 6e-36 e-122 e-163 e-103 e-124 e-160 2e-17 e-166 e-106 6e-83 e-114 e-149 4e-80 7e-28 1e-08 e-123 5e-97

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Table 2 (continued ) Cluster

N

Putative identification

e-value

JARO001001G09 JARO001002B05 JARO001009G02 JARO001010A02 JARO001010B05 JARO001010F02 JARO001010G12 JARO001013G05 JARO001023A06 JARO001041C11 JARO001002A04 JARO001003B12 JARO001003C11 JARO001003F09 JARO001004D08 JARO001008B04 JARO001008E06 JARO001010B03 JARO001010C06 JARO001010C10 JARO001010E10 JARO001012B03 JARO001014G10 JARO001016G03 JARO001032G05 JARO001034B08 JARO001035B06 JARO001040E08 JARO001041A06 JARO001041B03 JARO001045D04 JARO001052D06 JARO001053A12 JARO001001E05 JARO001002B06 JARO001004C09 JARO001005A04 JARO001005B03 JARO001005B07 JARO001005E08 JARO001005F05 JARO001005H04 JARO001005H11 JARO001006A12 JARO001006C09 JARO001006H09 JARO001006H12 JARO001008A08 JARO001008D05 JARO001008F11 JARO001008G06 JARO001008H03 JARO001009H07 JARO001009H08 JARO001010E04 JARO001010F04 JARO001010F09 JARO001010H11

03 03 03 03 03 03 03 03 03 03 02 02 02 02 02 02 02 02 02 02 02 02 02 02 02 02 02 02 02 02 02 02 02 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01

Bothropasin precursor [Bothrops jararaca] Disintegrin jararacin [Bothrops jararaca] Bothropasin precursor [Bothrops jararaca] Bothrops insularis cluster BITM02A (metalloproteinase precursor) Bothrops jararaca bothrostatin precursor Disintegrin jararacin [Bothrops jararaca] Bothropasin/jararhagin [Bothrops jararaca] Bothropasin precursor [Bothrops jararaca] Metalloproteinase-disintegrin-like protein[Agkistrodon contortrix laticinctus] Disintegrin jararacin [Bothrops jararaca] Deinagkistrodon acutus metalloproteinase MD1 precursor Bothropasin precursor [Bothrops jararaca] Bothrops jararaca bothrostatin precursor Metalloproteinase/disintegrin saxin precursor [Gloydius saxatilis] Metalloproteinase [Gloydius halys brevicaudus] Bothropasin/jararhagin [Bothrops jararaca] Disintegrin jararacin [Bothrops jararaca] Bothropasin precursor [Bothrops jararaca] Bothrops jararaca bothrostatin precursor Bothropasin precursor [Bothrops jararaca] Berythractivase [Bothrops erythromelas] Zinc metalloproteinase jerdonitin precursor [Trimeresurus jerdonii] Bothropasin/jararhagin [Bothrops jararaca] Bothropasin precursor [Bothrops jararaca] Hemorrhagic metalloproteinase HF3 [Bothrops jararaca] Bothrops jararaca bothrostatin precursor Jararhagin precursor [Bothrops jararaca] Bothropasin precursor [Bothrops jararaca] Hemorrhagic metalloproteinase HF3 [Bothrops jararaca] Metalloprotease BOJUMET II [Bothrops jararacussu] Deinagkistrodon acutus metalloproteinase MD2 precursor Bothrops jararaca bothrostatin precursor Hemorrhagic metalloproteinase HF3 [Bothrops jararaca] Bothropasin precursor [Bothrops jararaca] Bothropasin precursor [Bothrops jararaca] Bothrops insularis cluster BITM02A (metalloproteinase precursor) Bothropasin precursor [Bothrops jararaca] Deinagkistrodon acutus metalloproteinase MD2 precursor Metalloproteinase [Gloydius halys brevicaudus] Metalloproteinase precursor [Bothrops insularis] Insularinase and insularin precursor [Bothrops insularis] Crotalus scutulatus scutulatus GP-IV metalloproteinase precursor-like protein Bothropasin precursor [Bothrops jararaca] Bothrops jararaca bothrostatin precursor Bothropasin/jararhagin [Bothrops jararaca] Bothrops jararaca bothrostatin precursor Crotalus atrox prepro-hemorrhagic toxin d, atrolysin d Bothrops jararacussu metalloprotease BOJUMET II Bothropasin precursor [Bothrops jararaca] Bothrops jararaca bothrostatin precursor Bothropasin precursor [Bothrops jararaca] Bothrops jararaca bothrostatin precursor Bothrops jararaca bothrostatin precursor Bothrops insularis cluster BITM06A (metalloproteinase precursor) Bothropasin precursor [Bothrops jararaca] Bothrops jararacussu metalloprotease BOJUMET II Bothropasin/jararhagin [Bothrops jararaca] Disintegrin jararacin [Bothrops jararaca]

8e-97 1e-15 0.0 e-163 e-117 1e-23 5e-08 e-146 1e-39 3e-31 5e-98 e-176 1e-94 7e-52 1e-54 3e-31 7e-06 3e-59 2e-93 0.0 1e-77 1e-75 4e-48 2e-74 6e-06 e-108 e-137 e-127 7e-37 2e-92 e-114 e-130 1e-47 2e-80 3e-44 0.0 0.0 1e-112 7e-91 2e-82 2e-24 7e-06 1e-156 5e-69 7e-38 3e-33 1e-147 0.0 1e-178 2e-64 4e-66 1e-19 9e-83 0.0 0.0 1e-107 4e-26 9e-28

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442 Table 2 (continued ) Cluster

N

Putative identification

e-value

JARO001012G06 JARO001013A11 JARO001013C11 JARO001013F04 JARO001013H04 JARO001014A02 JARO001014A05 JARO001014A09 JARO001014B12 JARO001014F08 JARO001014H02 JARO001015B03 JARO001015F10 JARO001015G09 JARO001017D01 JARO001017F10 JARO001017F12 JARO001023B11 JARO001023C01 JARO001023D12 JARO001023F01 JARO001023H11 JARO001031A08 JARO001032H05 JARO001033H10 JARO001034A08 JARO001035H12 JARO001036A07 JARO001036B03 JARO001036D03 JARO001036F06 JARO001036F11 JARO001036G01 JARO001036G11 JARO001036H01 JARO001040A03 JARO001040E12 JARO001041A05 JARO001041C01 JARO001041E12 JARO001041F03 JARO001042H12 JARO001043D02 JARO001043D09 JARO001044E02 JARO001044F06 JARO001045E08 JARO001046A07 JARO001046E04 JARO001047A01 JARO001047A11 JARO001047B11 JARO001047G03 JARO001047H08 JARO001048A03 JARO001048C06 JARO001048C08 JARO001048D02

01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01

Bothrops insularis cluster BITM02A (metalloproteinase precursor) Bothropasin/jararhagin [Bothrops jararaca] Bothrops jararaca bothrostatin precursor Hemorrhagic metalloproteinase HF3 [Bothrops jararaca] Bothrops insularis cluster BITM02A (metalloproteinase precursor) Vascular apoptosis-inducing protein [Trimeresurus flavoviridis] Bothrops jararaca bothrostatin precursor Bothropasin/jararhagin [Bothrops jararaca] Insularinase and insularin precursor [Bothrops insularis] Bothropasin precursor [Bothrops jararaca] Bothropasin/jararhagin [Bothrops jararaca] Bothropasin/jararhagin [Bothrops jararaca] Bothropasin precursor [Bothrops jararaca] Bothropasin/jararhagin [Bothrops jararaca] Bothropasin precursor [Bothrops jararaca] Atrolysin A—western diamondback rattlesnake Bothropasin/jararhagin [Bothrops jararaca] Bothrops jararaca bothrostatin precursor Bothrops jararaca hemorrhagic metalloproteinase HF3 Bothrops jararacussu metalloprotease BOJUMET III Bothrops jararaca bothrostatin precursor Bothrops jararaca bothrostatin precursor Metalloprotease [Gloydius halys] Bothrops insularis cluster BITM02A (metalloproteinase precursor) Bothropasin precursor [Bothrops jararaca] Jararhagin precursor [Bothrops jararaca] Bothrops jararaca bothrostatin precursor Bothropasin precursor [Bothrops jararaca] Hemorrhagic metalloproteinase HF3 [Bothrops jararaca] Bothrops jararaca bothrostatin precursor Bothrops jararaca bothrostatin precursor Bothropasin precursor [Bothrops jararaca] Hemorrhagic metalloproteinase HF3 [Bothrops jararaca] Bothrops jararaca bothrostatin precursor Bothropasin precursor [Bothrops jararaca] Bothrops jararaca bothrostatin precursor Western diamondback rattlesnake metalloproteinase preprometalloproteinase;(atrolysin e) Bothrops insularis cluster BITM02A (metalloproteinase precursor) Bothropasin precursor [Bothrops jararaca] Bothropasin/jararhagin [Bothrops jararaca] Deinagkistrodon acutus acurhagin precursor Bothropasin/jararhagin [Bothrops jararaca] Bothropasin/jararhagin [Bothrops jararaca] Bothrops jararaca bothrostatin precursor Bothrops jararaca bothrostatin precursor Bothrops jararaca bothrostatin precursor Hemorrhagic metalloproteinase HF3 [Bothrops jararaca] Bothropasin/jararhagin [Bothrops jararaca] Bothropasin/jararhagin [Bothrops jararaca] Deinagkistrodon acutus acurhagin precursor Bothropasin/jararhagin [Bothrops jararaca] Bothrops jararaca bothrostatin precursor Bothropasin precursor [Bothrops jararaca] Bothropasin precursor [Bothrops jararaca] Bothropasin/jararhagin [Bothrops jararaca] Bothropasin precursor [Bothrops jararaca] Hemorrhagic toxin a [Crotalus atrox] Bothrops jararaca bothrostatin precursor

4e-06 7e-14 1e-122 e-114 8e-08 1e-89 1e-59 1e-63 2e-21 0.0 8e-27 5e-24 7e-97 2e-11 1e-109 4e-07 3e-73 1e-110 1e-174 2e-51 1e-106 1e-122 4e-52 1e-129 e-101 e-110 3e-41 2e-35 1e-18 1e-108 4e-42 0.0 3e-74 1e-106 2e-39 3e-89 1e-48 1e-155 3e-98 5e-34 1e-162 4e-06 1e-59 7e-49 1e-85 1e-65 9e-76 7e-10 1e-18 1e-35 4e-50 1e-128 5e-83 1e-160 4e-19 5e-61 6e-15 1e-68

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Table 2 (continued ) Cluster

N

Putative identification

e-value

JARO001048E07 JARO001048F04 JARO001048G03 JARO001049A12 JARO001049B08 JARO001049C10 JARO001049E07 JARO001050C06 JARO001050G02 JARO001051D01 JARO001051G02 JARO001052B03 JARO001052B07 JARO001052C06 JARO001052D07 JARO001052E12 JARO001052H10 JARO001053A09 JARO001053D01 JARO001053D07 JARO001053D09 JARO001054C08 JARO001054C11 JARO001054D02 JARO001055D07 JARO001056C03 JARO001056D07 JARO001056E06 JARO001057A02 JARO001057B04

01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01

Bothropasin precursor [Bothrops jararaca] Bothrops jararaca bothrostatin precursor Bothropasin/jararhagin [Bothrops jararaca] Bothropasin precursor [Bothrops jararaca] Bothrops jararaca bothrostatin precursor Bothropasin precursor [Bothrops jararaca] Bothrops jararaca bothrostatin precursor Bothropasin precursor [Bothrops jararaca] Agkistrodon contortrix contortrix contortrostatin precursor Metalloproteinase precursor [Bothrops insularis] Bothropasin precursor [Bothrops jararaca] Hemorrhagic metalloproteinase HF3 [Bothrops jararaca] Bothrops jararaca bothrostatin precursor Bothropasin precursor [Bothrops jararaca] Bothropasin/jararhagin [Bothrops jararaca] Bothrops jararaca bothrostatin precursor Bothrops jararaca bothrostatin precursor Bothropasin precursor [Bothrops jararaca] Bothropasin/jararhagin [Bothrops jararaca] Bothropasin/jararhagin [Bothrops jararaca] Bothrops insularis cluster BITM06A (metalloproteinase precursor) Atrolysin A—western diamondback rattlesnake Bothrops jararaca bothrostatin precursor Bothrops jararaca bothrostatin precursor Metalloproteina-se precursor [Bothrops insularis] Bothropasin/jararhagin [Bothrops jararaca] Bothropasin precursor [Bothrops jararaca] Bothropasin/jararhagin [Bothrops jararaca] Bothropasin precursor [Bothrops jararaca] Bothrops jararaca bothrostatin precursor

1e-35 1e-18 2e-27 1e-137 8e-24 2e-39 2e-63 2e-83 4e-48 9e-12 1e-150 1e-83 2e-89 1e-155 7e-10 1e-117 4e-86 7e-89 e-108 8e-14 9e-96 1e-79 6e-71 1e-38 5e-20 2e-34 1e-118 5e-65 2e-57 2e-73

Serine-proteinases JARO001002G10 JARO001001F08 JARO001004E09 JARO001002E07 JARO001001G01 JARO001001E03 JARO001005B12 JARO001010A10 JARO001012H07 JARO001002B10 JARO001005E06 JARO001006E08 JARO001032H12 JARO001002C08 JARO001002C09 JARO001004B09 JARO001012D05 JARO001013E02 JARO001041H08 JARO001004A10 JARO001004C10 JARO001006A11 JARO001010A06 JARO001017E10 JARO001033G09 JARO001035B07

32 23 23 13 11 09 08 07 07 06 06 06 06 05 05 05 05 05 05 04 04 04 04 04 04 04

Macrovipera lebetina serine alpha-fibrinogenase precursor Macrovipera lebetina serine alpha-fibrinogenase precursor Bothrops jararaca mRNA for KN-BJ2 Serine proteinase precursor [Bothrops insularis] Bothrops jararaca mRNA for KN-BJ2 Bothrops jararaca mRNA for protease A Macrovipera lebetina serine alpha-fibrinogenase precursor Macrovipera lebetina serine alpha-fibrinogenase precursor Trimeresurus gramineus mRNA for serine protease Macrovipera lebetina serine alpha-fibrinogenase precursor Bothrops jararaca mRNA for KN-BJ2 Bothrops jararaca mRNA for KN-BJ2 Macrovipera lebetina serine alpha-fibrinogenase precursor Macrovipera lebetina serine alpha-fibrinogenase precursor Bothrops jararaca mRNA for KN-BJ2 Serine protease catroxase II precursor [Crotalus atrox] Macrovipera lebetina serine alpha-fibrinogenase precursor Bothrops jararaca mRNA for KN-BJ2 Bothrops jararacussu serine protease Macrovipera lebetina serine alpha-fibrinogenase precursor Bothrops jararacussu serine protease Bothrops jararaca mRNA for KN-BJ2 Bothrops jararaca mRNA for KN-BJ2 Macrovipera lebetina serine alpha-fibrinogenase precursor Bothrops jararaca mRNA for KN-BJ2 Macrovipera lebetina serine alpha-fibrinogenase precursor

0.0 2e-31 2e-78 e-111 1e-36 0.0 2e-31 4e-31 0.0 3e-07 e-151 8e-22 3e-36 2e-31 7e-28 1e-15 2e-31 2e-62 0.0 1e-31 0.0 2e-64 e-128 9e-29 e-104 2e-26

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444 Table 2 (continued ) Cluster

N

Putative identification

e-value

JARO001043F02 JARO001005A05 JARO001005H09 JARO001008E04 JARO001008H09 JARO001009A11 JARO001045G06 JARO001001C07 JARO001001F11 JARO001001G04 JARO001002D06 JARO001002D07 JARO001003E11 JARO001005B11 JARO001008F03 JARO001009B02 JARO001009G08 JARO001010G07 JARO001012F02 JARO001013E10 JARO001031B04 JARO001031B08 JARO001034G10 JARO001040C05 JARO001041G06 JARO001043E08 JARO001044F02 JARO001046A06 JARO001001B05 JARO001001C08 JARO001001E06 JARO001001E12 JARO001002B07 JARO001002E08 JARO001002F12 JARO001005C07 JARO001005E03 JARO001006A08 JARO001006C08 JARO001008B11 JARO001008C06 JARO001008H05 JARO001009D11 JARO001009G01 JARO001010A07 JARO001010B11 JARO001012B07 JARO001014A08 JARO001014B07 JARO001014C08 JARO001015A05 JARO001015A06 JARO001015B06 JARO001017C05 JARO001017D12 JARO001023D03 JARO001023E07 JARO001023F12

04 03 03 03 03 03 03 02 02 02 02 02 02 02 02 02 02 02 02 02 02 02 02 02 02 02 02 02 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01

Macrovipera lebetina serine alpha-fibrinogenase precursor Macrovipera lebetina serine alpha-fibrinogenase precursor Bothrops jararaca mRNA for KN-BJ2 Macrovipera lebetina serine alpha-fibrinogenase precursor Bothrops jararaca mRNA for KN-BJ2 Macrovipera lebetina serine alpha-fibrinogenase precursor Acubin [Deinagkistrodon acutus] Macrovipera lebetina serine alpha-fibrinogenase precursor Bothrops atrox batroxobin gene Macrovipera lebetina serine alpha-fibrinogenase precursor Venom serine proteinase A precursor Serine protease [Bothrops jararacussu] Macrovipera lebetina serine alpha-fibrinogenase precursor Bothrops jararaca mRNA for KN-BJ2 Bothrops jararaca mRNA for KN-BJ2 Macrovipera lebetina serine alpha-fibrinogenase precursor Bothrops jararaca mRNA for KN-BJ2 Bothrops jararaca mRNA for KN-BJ2 Macrovipera lebetina serine alpha-fibrinogenase precursor Macrovipera lebetina serine alpha-fibrinogenase precursor Acubin2 [Deinagkistrodon acutus] Bothrops jararaca mRNA for KN-BJ2 Macrovipera lebetina serine alpha-fibrinogenase precursor Bothrops jararaca mRNA for KN-BJ2 Macrovipera lebetina serine alpha-fibrinogenase precursor JH3C12F Snake Bothrops insularis serine proteinase Macrovipera lebetina serine alpha-fibrinogenase precursor Serine protease catroxase II precursor [Crotalus atrox] Macrovipera lebetina serine alpha-fibrinogenase precursor Macrovipera lebetina serine alpha-fibrinogenase precursor Bothrops jararaca mRNA for KN-BJ2 Bothrops insularis cluster BITS01A serine proteinase precursor Bothrops jararaca mRNA for KN-BJ2 JH3C12F Snake Bothrops insularis serine protease Bothrops jararaca mRNA for KN-BJ2 Bothrops jararaca mRNA for KN-BJ2 Bothrops insularis cluster BITS01A serine proteinase precursor Macrovipera lebetina serine alpha-fibrinogenase precursor Serine protease catroxase II precursor [Crotalus atrox] ] Bothrops insularis cluster BITS01A serine proteinase precursor Macrovipera lebetina serine alpha-fibrinogenase precursor Macrovipera lebetina serine alpha-fibrinogenase precursor Macrovipera lebetina serine alpha-fibrinogenase precursor Macrovipera lebetina serine alpha-fibrinogenase precursor Macrovipera lebetina serine alpha-fibrinogenase precursor Macrovipera lebetina serine alpha-fibrinogenase precursor Macrovipera lebetina serine alpha-fibrinogenase precursor Macrovipera lebetina serine alpha-fibrinogenase precursor Macrovipera lebetina serine alpha-fibrinogenase precursor Macrovipera lebetina serine alpha-fibrinogenase precursor Bothrops jararaca mRNA for protease A Venom serine proteinase A precursor Bothrops jararaca mRNA for KN-BJ2 Trimeresurus flavoviridis mRNA for serine protease Macrovipera lebetina serine alpha-fibrinogenase precursor Bothrops jararacussu serine protease Serine protease [Bothrops jararacussu] Bothrops jararaca mRNA for protease A

1e-34 1e-31 9e-15 1e-31 3e-36 1e-28 5e-57 1e-31 1e-24 1e-34 7e-43 2e-80 4e-34 1e-50 6e-09 3e-22 e-106 5e-54 1e-31 3e-30 5e-44 3e-10 2e-09 2e-50 2e-29 3e-23 4e-18 2e-77 9e-07 1e-27 2e-10 1e-130 1e-104 2e-05 0.0 2e-14 1e-106 1e-31 4e-53 2e-94 4e-27 9e-23 3e-06 5e-20 1e-05 5e-11 1e-24 3e-26 4e-15 6e-39 1e-84 6e-36 3e-62 1e-131 8e-32 1e-139 3e-40 0.0

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445

Table 2 (continued ) Cluster

N

Putative identification

e-value

JARO001023H05 JARO001031C05 JARO001031D07 JARO001031E02 JARO001031H12 JARO001033F12 JARO001034D01 JARO001034G01 JARO001035B09 JARO001035C09 JARO001035G10 JARO001036C12 JARO001036D01 JARO001036F10 JARO001040G05 JARO001041C06 JARO001041D12 JARO001041H03 JARO001042A12 JARO001042D07 JARO001042F12 JARO001042H08 JARO001043B07 JARO001043E01 JARO001043F06 JARO001044E06 JARO001044F05 JARO001045E05 JARO001046A04 JARO001046D10 JARO001046E11 JARO001046F04 JARO001046H06 JARO001047E10 JARO001048B03 JARO001048D12 JARO001049A02 JARO001049C12 JARO001050G01 JARO001050G10 JARO001051A10 JARO001051B02 JARO001051C04 JARO001051C12 JARO001051D05 JARO001051E03 JARO001051E11 JARO001052F01 JARO001052G04 JARO001052G07 JARO001053A05 JARO001053G04 JARO001054D11 JARO001055E10 JARO001056B12 JARO001056H07 JARO001057B12

01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01

Bothrops jararacussu serine protease Macrovipera lebetina serine alpha-fibrinogenase precursor Macrovipera lebetina serine alpha-fibrinogenase precursor Macrovipera lebetina serine alpha-fibrinogenase precursor Bothrops jararaca mRNA for KN-BJ2 Bothrops jararaca mRNA for KN-BJ2 Bothrops jararaca mRNA for protease A Macrovipera lebetina serine alpha-fibrinogenase precursor Bothrops jararaca mRNA for protease A Venom serine proteinase A precursor JH3C12F Snake Bothrops insularis serine protease Trimeresurus gramineus mRNA for serine protease Bothrops jararaca mRNA for KN-BJ2 Macrovipera lebetina serine alpha-fibrinogenase precursor Bothrops jararaca mRNA for KN-BJ2 Macrovipera lebetina serine alpha-fibrinogenase precursor Macrovipera lebetina serine alpha-fibrinogenase precursor Macrovipera lebetina serine alpha-fibrinogenase precursor Macrovipera lebetina serine alpha-fibrinogenase precursor Bothrops insularis cluster BITS01A serine proteinase precursor Bothrops jararaca mRNA for KN-BJ2 Bothrops jararaca mRNA for protease A Macrovipera lebetina serine alpha-fibrinogenase precursor Bothrops jararaca mRNA for KN-BJ2 Bothrops jararaca mRNA for KN-BJ2 Macrovipera lebetina serine alpha-fibrinogenase precursor Bothrops jararaca mRNA for protease A Macrovipera lebetina serine alpha-fibrinogenase precursor Bothrops jararaca mRNA for KN-BJ2 Macrovipera lebetina serine alpha-fibrinogenase precursor Bothrops jararaca mRNA for KN-BJ2 Macrovipera lebetina serine alpha-fibrinogenase precursor Bothrops jararaca mRNA for KN-BJ2 Bothrops jararaca mRNA for KN-BJ2 Bothrops jararaca mRNA for KN-BJ2 Bothrops jararaca mRNA for protease A Bothrops jararacussu serine protease Macrovipera lebetina serine alpha-fibrinogenase precursor Bothrops jararaca mRNA for KN-BJ2 Macrovipera lebetina serine alpha-fibrinogenase precursor JH3C12F Snake Bothrops insularis serine proteinase Macrovipera lebetina serine alpha-fibrinogenase precursor Bothrops jararaca mRNA for KN-BJ2 Macrovipera lebetina serine alpha-fibrinogenase precursor Bothrops insularis cluster BITS01A serine proteinase precursor Bothrops jararaca mRNA for protease A Bothrops jararaca mRNA for KN-BJ2 Bothrops jararaca mRNA for KN-BJ2 Macrovipera lebetina serine alpha-fibrinogenase precursor Macrovipera lebetina serine alpha-fibrinogenase precursor Macrovipera lebetina serine alpha-fibrinogenase precursor Macrovipera lebetina serine alpha-fibrinogenase precursor Macrovipera lebetina serine alpha-fibrinogenase precursor Trimeresurus gramineus mRNA for serine protease Trimeresurus gramineus mRNA for serine protease Macrovipera lebetina serine alpha-fibrinogenase precursor Bothrops insularis cluster BITS01A serine proteinase precursor

1e-178 2e-14 2e-16 6e-39 1e-134 0.0 5e-57 3e-16 0.0 4e-19 3e-25 1e-177 2e-67 1e-14 3e-30 3e-31 1e-18 9e-11 3e-29 1e-152 1e-64 2e-80 5e-32 2e-44 8e-35 2e-16 0.0 1e-33 3e-35 1e-16 1e-121 8e-29 4e-39 0.0 1e-14 0.0 1e-152 6e-27 2e-95 1e-21 1e-13 7e-11 5e-52 1e-15 1e-169 1e-104 2e-76 8e-81 4e-26 1e-21 1e-27 1e-31 2e-27 1e-158 0.0 8e-11 1e-78

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446 Table 2 (continued ) Cluster

N

Putative identification

e-value

C-Type lectins JARO001004F10 JARO001005B10 JARO001009F12 JARO001015B10 JARO001005B08 JARO001006G10 JARO001002C10 JARO001005F08 JARO001006B10 JARO001012D04 JARO001013F07 JARO001017D09 JARO001013E08 JARO001044G03 JARO001001D10 JARO001005G05 JARO001010D04 JARO001014B11 JARO001014E06 JARO001015C03 JARO001031D04 JARO001031H02 JARO001033A01 JARO001035E08 JARO001036B08 JARO001036D04 JARO001040C04 JARO001040D10 JARO001041G05 JARO001042E12 JARO001043F04 JARO001046G07 JARO001047B07 JARO001049E08 JARO001049F10 JARO001050C08 JARO001050C09 JARO001050F08 JARO001054F10 JARO001054F12 JARO001056B10

16 13 09 08 07 04 03 03 03 03 03 03 02 02 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01

Platelet glycoprotein Ib-binding protein alpha subunit Platelet glycoprotein Ib-binding protein beta subunit Chain A Coagulation Factor Ix-Binding Protein ACF 1/2 B-chain [Deinagkistrodon acutus] bothojaracin chain A precursor [Bothrops jararaca] Platelet glycoprotein Ib-binding protein beta subunit Botrocetin beta chain [Bothrops jararaca] Bothojaracin chain B precursor [Bothrops jararaca] bothojaracin chain A precursor [Bothrops jararaca] bothojaracin chain B precursor [Bothrops jararaca] bothojaracin chain A precursor [Bothrops jararaca] Platelet glycoprotein Ib-binding protein beta subunit Mamushigin beta [Agkistrodon blomhoffi] Bothojaracin chain A precursor [Bothrops jararaca] Flavocetin-A beta chain [Trimeresurus flavoviridis] Botrocetin alpha chain [Bothrops jararaca] Bothojaracin chain B precursor [Bothrops jararaca] Bothojaracin chain A precursor [Bothrops jararaca] Bothojaracin chain A precursor [Bothrops jararaca] precursor Stejaggregin-A alpha chain [Trimeresurus stejnegeri] Bothojaracin chain B precursor [Bothrops jararaca] Bothojaracin chain A precursor [Bothrops jararaca] Bothojaracin chain B precursor [Bothrops jararaca] mucrosquamatus] Trimeresurus stejnegeri factor IX/X binding protein beta chainPlatelet glycoprotein Ib-binding protein beta subunit Coagulation factor IX/X-binding protein: A chain Platelet glycoprotein Ib-binding protein beta subunit Bothojaracin chain B precursor [Bothrops jararaca]] Coagulation factor IX-binding protein chain A [Gloydius halys] Botrocetin beta chain [Bothrops jararaca] Factor IX/factor X binding protein B chain [Trimeresurus flavoviridis] Bothojaracin chain A precursor [Bothrops jararaca] Bothojaracin chain A precursor [Bothrops jararaca] ACF 1/2 B-chain [Deinagkistrodon acutus] Trimeresurus stejnegeri factor IX/X binding protein alpha chain Platelet glycoprotein Ib-binding protein beta subunit Gloydius halys coagulation factor IX-binding protein chain B Trimeresurus flavoviridis flavocetin-A alpha chain C-type lectin [Deinagkistrodon acutus] Coagulation factor IX/X-binding protein: A chain Platelet glycoprotein Ib-binding protein alpha subunit

1e-73 3e-69 6e-55 2e-61 8e-65 2e-66 1e-15 1e-75 2e-78 6e-55 2e-78 3e-17 1e-50 2e-27 4e-21 7e-26 3e-65 6e-50 6e-25 2e-63 1e-42 8e-52 7e-06 6e-43 2e-11 1e-32 1e-10 8e-15 6e-43 9e-18 5e-59 3e-30 1e-56 8e-36 8e-31 2e-61 7e-18 2e-29 1e-42 3e-29 4e-28

peptide precursors B. insularis cluster BITB08 bradykinin-potentiating proteinprecursor Bothrops jararaca bradykinin-potentiating/C-type natriuretic peptide Bothrops jararaca bradykinin-potentiating/C-type natriuretic peptide Agkistrodon blomhoffi mRNA for BPP-CNP precursor Bothrops jararaca bradykinin-potentiating/C-type natriuretic peptide Bradykinin-potentiating peptides and C-type natriuretic peptide precursor [Bothrops jararaca] Bothrops jararaca bradykinin-potentiating/C-type natriuretic peptide Bothrops jararaca mRNA for bradykinin-potentiating peptide and C-type natriuretic peptide precursor Bradykinin-potentiating peptides and C-type natriuretic peptide precursor [Bothrops jararaca] Bradykinin-potentiating peptides and C-type natriuretic peptide precursor [Bothrops jararaca] Bothrops jararaca bradykinin-potentiating/C-type natriuretic peptide Bothrops jararaca bradykinin-potentiating/C-type natriuretic peptide Bradykinin-potentiating/C-type natriuretic peptide [Bothrops jararaca] Bothrops insularis cluster BITB08 bradykinin-potentiating protein precursor

0.0 0.0 e-119 e-110 1e-47 3e-82 e-104 0.0 6e-53 2e-34 1e-94 5e-98 6e-48 1e-108

Bradykinin-potentiating JARO001009B06 16 JARO001013A07 14 JARO001001A07 09 JARO001014H12 09 JARO001003G09 06 JARO001047D04 03 JARO001002E11 02 JARO001010H03 02 JARO001014E08 02 JARO001045B02 02 JARO001002C11 01 JARO001006F09 01 JARO001008A10 01 JARO001009H05 01

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447

Table 2 (continued ) Cluster

N

Putative identification

e-value

JARO001012E09 JARO001023A11 JARO001023D07 JARO001031F10 JARO001031G01 JARO001033G07 JARO001040E07 JARO001047G12 JARO001052C04 JARO001053F02 JARO001054E08

01 01 01 01 01 01 01 01 01 01 01

Bradykinin-potentiating peptides and C-type natriuretic peptide precursor [Bothrops jararaca] Bothrops insularis cluster BITB08 bradykinin-potentiating protein precursor Bothrops jararaca mRNA for bradykinin-potentiating peptide and C-type natriuretic peptide precursor Bothrops jararaca bradykinin-potentiating/C-type natriuretic peptide Bothrops insularis cluster BITB08 bradykinin-potentiating protein precursor Bothrops jararacussu bradykinin-potentiating/C-type natriuretic peptide Bothrops insularis bradykinin-potentiating/c-type natriuretic protein precursor Bothrops jararaca bradykinin-potentiating/C-type natriuretic peptide Bradykinin-potentiating peptides and C-type natriuretic peptide precursor [Bothrops jararaca Bothrops jararaca bradykinin-potentiating/C-type natriuretic peptide Bothrops jararaca bradykinin-potentiating/C-type natriuretic peptide

3e-54 1e-116 0.0 2e-99 1e-116 1e-103 1e-31 4e-73 1e-23 1e-178 8e-27

Phospholipases A2 JARO001034D12 JARO001001G11 JARO001006F07 JARO001015A02 JARO001033F11 JARO001045H10 JARO001050F01 JARO001053F03

02 01 01 01 01 01 01 01

Snake BP-II gene for phospholipase A2 Snake BP-II gene for phospholipase A2 Myotoxic phospholipase A2-like [Bothrops jararacussu] Vipera berus berus phospholipase A2 gene Gloydius shedaoensis acidic phospholipase A2 Trimeresurus gramineus gene for phospholipase A2 Vipera berus berus phospholipase A2 gene Phospholipase A2 precursor [Canis familiaris]

3e-11 1e-90 2e-68 5.0e-06 6e-65 2e-25 5e-06 3e-11

Phospholipase A2 inhibitors JARO001031A02 03 Phospholipase JARO001009C01 02 Phospholipase JARO001005C10 01 Phospholipase JARO001009A03 01 Phospholipase JARO001010C09 01 Phospholipase JARO001043A01 01 Phospholipase JARO001045C10 01 Phospholipase JARO001052F12 01 Phospholipase JARO001055F03 01 Phospholipase

A2 A2 A2 A2 A2 A2 A2 A2 A2

inhibitor [Trimeresurus flavoviridis] inhibitory protein Annexin A1 inhibitor [Trimeresurus flavoviridis] inhibitor [Trimeresurus flavoviridis] inhibitor [Trimeresurus flavoviridis] inhibitor [Trimeresurus flavoviridis] inhibitor [Trimeresurus flavoviridis] inhibitor [Elaphe quadrivirgata] inhibitor [Trimeresurus flavoviridis]

4e-07 5e-34 2e-14 7e-11 6e-15 2e-26 2e-42 2e-20 2e-31

Cysteine-rich venom protein precursors JARO001006G08 08 Trimeresurus stejnegeri cysteine-rich secretory protein JARO001006E12 05 Catrin [Crotalus atrox] JARO001009A05 01 Trimeresurus stejnegeri cysteine-rich secretory protein JARO001009B05 01 Trimeresurus stejnegeri cysteine-rich secretory protein JARO001036C10 01 Ablomin [Agkistrodon blomhoffi] JARO001045A07 01 Catrin [Crotalus atrox] JARO001046B09 01 Piscivorin [Agkistrodon piscivorus piscivorus] JARO001049G12 01 Trimeresurus mucrosquamatus prepro-cysteine-rich venom protein JARO001054F11 01 Catrin [Crotalus atrox]

0.0 6e-99 3e-89 8e-96 7e-16 2e-33 1e-74 4e-39 8e-42

Other JARO001034A06 JARO001040H08 JARO001034F04 JARO001003E08

2e-89 3e-47 1e-05 6e-21

01 01 04 03

Vascular endothelial growth factor [B. erythromelas] Vascular endothelial growth factor [B. insularis] L-amino acid oxidase [Trimeresurus stejnegeri] FAD-containing L-amino acid oxidase Apoxin 1 [Crotalus atrox]

3.3. Analysis of toxin clusters The identification of some of the toxin-related clusters was not possible at the amino acid level, and could only be accomplished by a Blast search

against the nucleotide database (Blast N). This is due to the particularly long 30 untranslated region (UTR) of toxins, in particular of the serine proteinases, of which most of the clusters were identified by Blast N. The representation of each

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448

Table 3 Identification of putative cellular protein clusters from B. jararaca venom Cluster

N

Putative identification

e-value

General metabolism JARO001015D08 JARO001034A03 JARO001043G05 JARO001002G05 JARO001004D09 JARO001010H05 JARO001013H07 JARO001032A10 JARO001033C09 JARO001002E09 JARO001003D07 JARO001003D08 JARO001004B08 JARO001005E12 JARO001009F07 JARO001010B02 JARO001017A03 JARO001023C02 JARO001031A01 JARO001031G07 JARO001031H09 JARO001033E12 JARO001033G12 JARO001034B02 JARO001035C06 JARO001042C12 JARO001043D05 JARO001043D08 JARO001044B01 JARO001044B02 JARO001046C04 JARO001048G12 JARO001050C10 JARO001052C02 JARO001052H05 JARO001053A11 JARO001053C05 JARO001053F11 JARO001054D06 JARO001055B05 JARO001055C10 JARO001055E12

03 03 03 02 02 02 02 02 02 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01

Cytochrome oxidase subunit II [Dinodon semicarinatus] Mannosyl-oligosaccharide 1,2-alpha-mannosidase IC [Gallus gallus] UDP-N-acetylhexosamine pyrophosphorylase Asparagine synthase (glutamine-hydrolysing) [Gallus gallus] Cytochrome b [Crotalus viridis] Propionyl Coenzyme A carboxylase, beta polypeptide [Rattusnorvegicus] Cytochrome C oxidase copper chaperone [Ophiophagus hannah] Malate dehydrogenase, [Gallus gallus] Alpha 3 glucosyltransferase [Gallus gallus] Creatine kinase [Zaocys dhumnades] UDP-glucose pyrophosphorylase [Gallus gallus] Methionine aminopeptidase 2 Cytochrome oxidase subunit III [Dinodon semicarinatus] Glutaminyl cyclase [Bothrops jararaca] ATPase subunit 6 [Crotalus viridis] Tyrosine 3/tryptophan 5 -monooxygenase activation protein [Homo sapiens] Cytochrome b [Crotalus viridis] Ornithine-oxo-acid aminotransferase [Gallus gallus] Cytochrome oxidase subunit I [Dinodon semicarinatus] Cytochrome c oxidase subunit VIb [Tarsius syrichta] Creatine kinase [Zaocys dhumnades] Glyceraldehyde-3-phosphate dehydrogenase [Pelodiscus sinensis] Glutaminyl cyclase [Bothrops jararaca] Creatine kinase [Zaocys dhumnades] GDP-mannose 4,6-dehydratase [Gallus gallus] ADP-ribosylation factor 1 [Homo sapiens] Glutathione peroxidase [Gallus gallus] Glyoxylate reductaseHomo sapiens] Inorganic pyrophosphatase (Pyrophosphate phospho-hydrolase) Aspartate aminotransferase, cytoplasmic (Transaminase A) Adenylate kinase 3 [Gallus gallus] Aldehyde dehydrogenase [Gallus gallus] Aldehyde dehydrogenase [Gallus gallus] Tyrosine phosphatase protein [Macaca fascicularis] Iron storage protein H-ferritin [Trichosurus vulpecula] Alpha 3 glucosyltransferase [Gallus gallus] Transitional endoplasmic reticulum ATPase ATPase subunit 9 NADH dehydrogenase subunit 1 [Naja nivea] Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, theta polypeptide ATPase subunit 6 [Crotalus viridis] Dipeptidylpeptidase 4a [Gloydius blomhoffi brevicaudus]

3e-51 e-104 2e-07 e-113 e-140 5e-11 8e-25 9e-41 8e-59 5e-79 e-104 4e-57 3e-70 8e-21 3e-14 2e-29 1e-117 2e-64 5e-26 8e-31 1e-47 3e-84 5e-72 2e-47 1e-13 7e-25 1e-36 6e-07 5e-84 6e-93 7e-59 2e-30 5e-35 1e-10 3e-53 3e-59 8e-60 1e-18 1e-58 1e-37 6e-23 4e-07

DNA transcription and RNA translation JARO001002G08 05 Ribosomal protein L27a [Mus musculus] JARO001015G02 05 Ribosomal protein S3 [Xenopus tropicalis] JARO001002E10 04 Ribosomal protein 10 [Mus musculus] JARO001013E01 04 Ribosomal protein L45 [Mus musculus] JARO001023H03 04 Eukaryotic translation initiation factor 3 subunit 2 [Gallus gallus] JARO001001B02 03 Ribosomal protein S12 [Gallus gallus] JARO001003F12 03 Poly(A) binding protein [Homo sapiens] JARO001014C04 03 40S ribosomal protein S16 [Homo sapiens JARO001032G07 03 Heterogeneous nuclear ribonucleoprotein U isoform b [Gallus gallus] JARO001001D01 02 Eukaryotic translation elongation factor 1 alpha 1 [Gallus gallus] JARO001005G08 02 RNA helicase [Homo sapiens] JARO001010F03 02 Eukaryotic translation elongation factor 1 gamma [Homo sapiens]

3e-65 e-119 e-121 2e-41 e-133 6e-71 3e-28 1e-77 2e-85 6e-57 9e-74 6e-39

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449

Table 3 (continued ) Cluster

N

Putative identification

e-value

JARO001012H09 JARO001014G11 JARO001017E04 JARO001017G06 JARO001032C04 JARO001040G03 JARO001042D04 JARO001051A12 JARO001052G06 JARO001001A08 JARO001001D09 JARO001001G10 JARO001001H07 JARO001003E09 JARO001004B10 JARO001006D09 JARO001009G04 JARO001010D06 JARO001012B09 JARO001012H01 JARO001014A04 JARO001014D04 JARO001017A06 JARO001017C07 JARO001031C12 JARO001031D03 JARO001032A06 JARO001032B08 JARO001033B11 JARO001033F02 JARO001041A04 JARO001041H11 JARO001042E10 JARO001043B03 JARO001043D04 JARO001043D11 JARO001043E03 JARO001043H10 JARO001044D12 JARO001045C08 JARO001045F04 JARO001046D07 JARO001046F06 JARO001050B05 JARO001051D08 JARO001053B05 JARO001055A08 JARO001055D01 JARO001055F06 JARO001055H10 JARO001056B08 JARO001056E08 JARO001057C01 JARO001057C03 JARO001057G04 JARO001057H04

02 02 02 02 02 02 02 02 02 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01

40S ribosomal protein S8 [Gallus gallus] 40S ribosomal protein S14 [Ictalurus punctatus] Ribosomal protein S3a [Ophiophagus hannah] Ribosomal protein L30 [Gallus gallus] 60S acidic ribosomal protein P1 [Gallus gallus] Ophiophagus hannah ribosomal protein L30 Eukaryotic translation initiation factor 3, subunit 7 (zeta) [Danio rerio] Poly-A binding protein [Gallus gallus] Ribosomal protein L19 [Macaca fascicularis] Ribosomal protein L4 [Gallus gallus] Eukaryotic translation elongation factor 1 alpha 1 [Gallus gallus] Ribosomal protein [Gallus gallus] 40S ribosomal protein S3 [Gallus gallus] High mobility group protein B1[Gallus gallus] Ribosomal protein L23 [Pan troglodytes] Ribosomal protein L18a [Homo sapiens] TATA-box binding protein[Trimeresurus gramineus] Splicing factor U2AF 65 kDa subunit 60S ribosomal protein L27 60S acidic ribosomal protein P1 Ribosomal protein L45 [Mus musculus] Ribosomal protein L23 [Pan troglodytes] Smu-1 suppressor of mec-8 and unc-52 homolog [Homo sapiens] Eukaryotic translation initiation factor 3 subunit 2 [Gallus gallus] 60S ribosomal protein L10 [Homo sapiens] Eukaryotic translation initiation factor 3, subunit 9 [Homo sapiens] Ribosomal protein L10a [Rattus norvegicus] Gallus gallus ribonucleoprotein Ribosomal protein S27 [Pan troglodytes] 60S ribosomal protein L28 [Hippocampus comes Helicase 1 (matrix associated, actin-dependent regulator of chromatin subfamily A) Eukaryotic translation initiation factor 3 [Danio rerio] Homo sapiens poly(A) binding protein Eukaryotic translation elongation factor 1 gamma [Homo sapiens] 40S ribosomal protein S5 [Rattus norvegicus] Small nuclear ribonucleoprotein polypeptide A’ [Homo sapiens] DNA directed RNA polymerase II polypeptide K [Homo sapiens] Elongation factor 2 Homo sapiens nucleolin H3 histone family, member I [Rattus norvegicus] 60S ribosomal protein L36a Ribosomal protein S8 [Scyliorhinus canicula] Ribosomal protein S4 Ribosomal protein S27 [Homo sapiens] Branchiostoma belcheri ribosomal protein L22 60S ribosomal protein L13 [Pan troglodytes] Small nuclear ribonucleoprotein polypeptide G [Homo sapiens] Homo sapiens eukaryotic translation initiation factor 1A RNA binding protein (signal recognition particle 14kDa) Bcl-2-associated transcription factor shortform [Gallus gallus] Gallus gallus ribonucleoprotein Splicing factor [Mus musculus] Ribosomal protein S11 [Gallus gallus] DNA dependent ATPase and helicase [Homo sapiens] Ribosomal protein [Homo sapiens] HIV TAT specific factor 1 [Homo sapiens]

e-114 2e-70 1e-88 8e-53 6e-31 2e-09 2e-33 9e-49 1e-65 7e-23 1e-94 2e-29 6e-09 3e-26 5e-21 3e-58 5e-06 7e-13 9e-31 6e-25 3e-38 5e-62 9e-50 1e-15 4e-08 2e-31 4e-18 1e-95 6e-38 5e-25 1e-58 3e-33 9e-18 2e-44 1e-80 2e-19 5e-25 5e-91 3e-07 3e-41 5e-52 2e-35 1e-20 2e-38 5e-06 4e-41 1e-29 1e-51 2e-26 7e-95 1e-106 e-110 3e-78 6e-86 6e-70 3e-28

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450 Table 3 (continued ) Cluster

N

Putative identification

e-value

Pos-translational processing and sorting JARO001001E02 34 Protein disulfide-isomerase JARO001002H12 10 Protein disulfide isomerase A4 precursor [Gallus gallus] JARO001010B08 07 Protein disulfide-isomerase isomerase A6 precursor [Gallus gallus] JARO001005F10 05 JH4B01R Snake Bothrops insularis calreticulin precursor JARO001001A01 04 Protein disulfide-isomerase precursor JARO001014D01 03 JH4B01R Snake Bothrops insularis calreticulin precursor JARO001008F08 02 Protein disulfide isomerase JARO001010D07 02 Translocation protein SEC63 homolog [Gallus gallus] JARO001014G05 02 S100 calcium binding protein A6 (calcyclin) [Gallus gallus] JARO001014H04 02 Sec61 alpha subunit homolog [Gallus gallus] JARO001023B10 02 Protein disulfide-isomerase JARO001032C08 02 ER-associated dnaJ protein 3 [Gallus gallus] JARO001033B08 02 Protein disulfide isomerase A4 precursor [Gallus gallus] JARO001050D01 02 JH4B01R Snake Bothrops insularis calreticulin precursor JARO001001B04 01 Glucose regulated thiol oxido-reductase protein precursor [Gallus gallus] JARO001005G02 01 Protein disulfide-isomerase JARO001009F04 01 Chaperonin subunit 8 (theta) [Mus musculus] JARO001012D02 01 Clathrin-associated/assembly/adapter protein JARO001013A01 01 JH4B01R Snake Bothrops insularis calreticulin precursor JARO001023B05 01 GH053F Snake Bothrops insularis disulfide-isomerase JARO001023D06 01 Coatomer zeta-1 subunit [Rattus norvegicus] JARO001023G12 01 Protein disulfide isomerase A4 precursor [Gallus gallus] JARO001032D12 01 Calreticulin [Gallus gallus] JARO001034E09 01 Kinectin (kinesin-binding protein) JARO001035A08 01 GH053F Snake Bothrops insularis disulfide-isomerase JARO001035F07 01 GH053F Snake Bothrops insularis disulfide-isomerase JARO001042G01 01 GH053F Snake Bothrops insularis disulfide-isomerase JARO001045A02 01 GH011R Snake Bothrops insularis calcium binding protein JARO001046A08 01 Protein disulfide-isomerase [Cricetulus griseus] JARO001047D11 01 Protein disulfide-isomerase JARO001047G06 01 Protein disulfide-isomerase JARO001047G07 01 GH053F Snake Bothrops insularis disulfide-isomerase JARO001048H08 01 Plasma glutathione peroxidase precursor [Rattus norvegicus] JARO001049B07 01 GH053F Snake Bothrops insularis disulfide-isomerase JARO001051B11 01 Vesicle-fusing ATPase protein JARO001054B08 01 JH4D03F Snake Bothrops insularis Calreticulin precursor JARO001054H12 01 Transport protein SEC61 gamma subunit JARO001056E09 01 Glutathione reductase 1 [Mus musculus]

4e-77 2e-62 4e-60 e-142 7e-87 1e-90 3e-44 8e-30 1e-18 7e-92 4e-24 e-110 1e-79 e-129 1e-34 4e-63 5e-06 2e-68 1e-116 5e-11 7e-84 3e-58 1e-19 2e-59 3e-44 4e-09 3e-48 1e-128 4e-22 4e-28 7e-73 3e-13 4e-52 3e-84 5e-29 2e-74 1e-10 4e-41

Polypeptide degradation JARO001009B03 03 JARO001014G07 03 JARO001032A08 02 JARO001008G08 01 JARO001009B11 01 JARO001034F11 01 JARO001042F08 01 JARO001043H09 01 JARO001052E04 01

SMT3 suppressor of mif two 3 homolog 2 [Homo sapiens] Proteasome beta 3 subunit [Homo sapiens] Dipeptidylpeptidase 4a [Gloydius blomhoffi brevicaudus] Ubiquitin carboxyl-terminal hydrolase [Gallus gallus] ubiquitin [Oncorhynchus mykiss] Ubiquitin-like protein/ribosomal protein S30 Proteasome subunit alpha type 1 Ubiquitin specific protease 7 [Mus musculus] Proteasome 26S subunit, ATPase 3 [Mus musculus]

6e-49 e-107 2e-09 1e-43 1e-46 7e-21 3e-20 1e-09 4e-43

Structural functions JARO001012H05 JARO001003C08 JARO001003F07 JARO001004G08 JARO001005B01 JARO001009C11 JARO001013C03

Beta-actin [Tupaia belangeri] Myosin light chain [Oxyuranus scutellatus scutellatus] Actin-related protein 6 Mus musculus microtubule-actin crosslinking factor 1 Gallus gallus alpha-actinin Daboia russellii vimentin Gallus gallus myosin heavy chain

1e-25 1e-92 8e-90 4e-22 5e-12 0.0 7e-05

03 02 02 01 01 01 01

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451

Table 3 (continued ) Cluster

N

Putative identification

e-value

JARO001014B09 JARO001033H11 JARO001036E08 JARO001042B07 JARO001047H09 JARO001051B07 JARO001052D04 JARO001055C07

01 01 01 01 01 01 01 01

Homo sapiens capping protein (actin filament) Actin-related protein 6 Spectrin alpha chain [Gallus gallus] Homo sapiens profilin 2 phosphatase and actin regulator 2 [Gallus gallus] Homo sapiens spectrin SH3 Myosin light chain smooth muscle isoform [Meleagris gallopavo] Keratin 18 [Homo sapiens]

3e-10 3e-63 1e-23 2e-71 7e-30 3e-06 2e-62 5e-30

Cell regulation and JARO001010D11 JARO001001D11 JARO001002H09 JARO001023B02 JARO001003B11 JARO001013B04 JARO001013G12 JARO001033F01 JARO001041F11 JARO001002F11 JARO001005B02 JARO001005F01 JARO001006B12 JARO001008D08 JARO001009D08 JARO001010F07 JARO001012D01 JARO001013B08 JARO001013B12 JARO001014F02 JARO001015B12 JARO001016D08 JARO001023B08 JARO001023E11 JARO001032B06 JARO001032D11 JARO001032E04 JARO001032F04 JARO001034B07 JARO001034F03 JARO001035D06 JARO001035E12 JARO001040D03 JARO001040F09 JARO001041B07 JARO001041B10 JARO001042D03 JARO001043A05 JARO001043C11 JARO001043E05 JARO001043F12 JARO001044D01 JARO001045E01 JARO001045E10 JARO001045F10 JARO001046G03 JARO001047E05 JARO001048C09

other 07 06 03 03 02 02 02 02 02 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01 01

functions Selenoprotein M precursor (SelM protein) ARMET protein precursor (Arginine-rich protein) Superoxide dismutase [Cu-Zn] Heat shock protein 108 [Gallus gallus] Transposase [Pleuronectes platessa] Homo sapiens calmodulin 1 (phosphorylase kinase, delta) Zinc transporter-similar to Dri 27/ZnT4 protein [Gallus gallus] Signal sequence receptor delta[Xenopus laevis] HA008F Snake Bothrops insularis retrotransposon Calcium/calmodulin-dependent protein kinase I [Pan troglodytes] Interleukin 25; lymphocyte antigen 6 complex, locus E ligand [Gallus gallus] Selenoprotein H Zinc finger protein 91 protein [Mus musculus] Selenoprotein [Xenopus laevis] Voltage-dependent anion-selective channel protein 2 (Outermitochondrial membrane protein porin) Proliferating cell nuclear antigen (PCNA) Transposase [Pleuronectes platessa] Adenine phosphoribosyl-transferase Zinc ion transporter 27/ZnT4 protein [Gallus gallus] Zinc finger protein- cellular nucleic acid binding protein Signal sequence receptor—delta subunit Translocation associated membrane protein 1 [Gallus gallus] Mus musculus RAS-related C3 botulinum substrate 1 Iron storage protein H-ferritin [Trichosurus vulpecula] Caspase-7 [Gallus gallus] Heat shock 70 kDa protein 8 [Bos taurus] MAPHomo sapiens] Signal sequence receptor, beta precursor [Homo sapiens] Signal sequence receptor, beta precursor [Homo sapiens] Arginine-rich, mutated in early stage tumors [Homo sapiens] HA008F Snake Bothrops insularis retrotransposon Voltage-dependent anion channel 3 [Sus scrofa] glycolipid transfer protein—pig Translocation associated membrane protein 1 [Gallus gallus] Gallus gallus clone 9 MHC class II antigen Cell division control protein Septin 10, isoform 1 [Homo sapiens] Microvascular endothelial differentiation protein [Homo sapiens] Signal sequence receptor, delta [Mus musculus] Reverse transcriptase [schistosoma mansoni] THO complex subunit 3 (Tho3) [Gallus gallus] Endonuclease/reverse transcriptase [Mus musculus] DNA repair protein complementin G XP-A cells homolog Signal sequence receptor beta subunit [Canis familiaris] Heat shock cognate 70 protein [Spodoptera frugiperda] Pol (reverse transcriptase-RNase H-integrase) [Tricholoma matsutake] Cell division cycle 5 protein [Pan troglodytes] epithelial glycoprotein [Gallus gallus] Homo sapiens profilin 2 (PFN2)

9e-31 1e-32 1e-30 1e-70 8e-24 2e-79 2e-14 5e-77 1e-16 5e-73 4e-53 5e-13 5e-14 3e-07 9e-13 2e-16 7e-26 7e-27 2e-13 2e-29 2e-47 3e-81 1e-09 2e-53 3e-91 3e-34 7e-31 3e-26 1e-21 5e-16 2e-08 8e-64 1e-23 9e-52 7e-23 4e-38 7e-17 1e-35 8e-06 1e-12 4e-19 5e-29 8e-29 8e-53 2e-08 7e-74 2e-34 7e-11

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452 Table 3 (continued ) Cluster

N

Putative identification

e-value

JARO001051E09 JARO001051G05 JARO001052H07 JARO001053A02 JARO001054A09 JARO001054F04 JARO001055A09 JARO001055D03

01 01 01 01 01 01 01 01

Nuclear protein Ytm1 [Mus musculus] Interleukin 17 receptor C isoform 1 precursor [Homo sapiens Troponin T3 [Xenopus tropicalis] Transmembrane protein precursor [Mus musculus] Ras-associated protein RAB8[Gallus gallus] Homo sapiens profilin 2 (PFN2) Thioredoxin-dependent peroxidereductase [Gallus gallus] Transmembrane 4 superfamily member 15

4e-15 2e-36 7e-59 5e-17 3e-08 3e-10 8e-44 6e-59

Table 4 Representation of the putative toxin transcripts from B. jararaca venom glands according to their structural types, as well as their redundancy (clones/clusters) Toxins

Clusters (%) Clones (%) Redundancy

Metalloproteinase Serine proteinase C-type lectin BPP precursor PLA2 PLA2 inhibitor Cysteine-rich protein L-Amino acid oxidase SvVEGF

172 (42.1) 141 (34.5) 41 (10.0) 25 (6.1) 08 (1.9) 09 (2.2) 09 (2.2) 02 (0.5) 02 (0.5)

681 (53.1) 366 (28.5) 106 (8.3) 80 (6.2) 09 (0.7) 12 (0.9) 20 (1.6) 07 (0.5) 02 (0.2)

4.0 2.6 2.6 3.2 1.1 1.3 2.2 3.5 1.0

BPP—bradykinin-potentiating peptide; PLA2-phospholipase A2; svVEGF-snake venom vascular endothelium growth factor.

class of protein over the total number of toxin transcripts is shown in Table 4, as well as their redundancy (clones/clusters). 3.3.1. Metalloproteinases/disintegrins The most abundant toxins in our EST database are the metalloproteinases (53.1% of toxin transcripts). Most of these enzymes present hemorrhagic activity through degradation of proteins of the basement membrane in the blood vessel wall, and can be divided into four classes (PI–PIV) depending on the domain organization of their precursor that would be classified NI–NIV, as follows: NI comprise precursors with only a metalloproteinase domain, NII members have a metalloproteinase domain followed by a disintegrin domain, NIII members present metalloproteinase, disintegrin-like and cysteine-rich domains and the hypothetical NIV precursor would contain an additional lectin-like domain that remains linked by disulfide bonds to the metalloproteinase domain on the venom (Takeya et al., 1992; Hite et al, 1994; Jia et al.,

1996; Markland, 1998). Concerning our database, 198 clones (29.1%) could be characterized as NII metalloproteinases. This class of metalloproteinases presented the highest redundancy of all transcripts (14.1), including the most representative transcript in the whole library (137 clones), identified as a B. jararaca metalloproteinase bothrostatin homolog (Fernandez et al., 2005). In addition, 318 (46.7%) out of 681 metalloproteinase clones could be assigned as NIII precursors. These clones were grouped in 49 clusters, resulting in a redundancy of 6.5, which is lower than that observed for the NII class. The most abundant NIII clusters showed similarity with bothropasin (Mandelbaum et al., 1982) and/or jararhagin (Paine et al., 1992), which are PIII metalloproteinases isolated from B. jararaca venom presenting high-sequence homology. They show some differences in the metalloproteinase domain but are identical in their disintegrinlike and cysteine-rich domains. Thus, in some cases where the homology occurred at these domains, it was not possible to determine to which of them the B. jararaca cluster is more similar, being then classified as a bothropasin/jararhagin homolog. These proteins act by cleaving fibrinogen which leads to the enhancement of plasma fibrinolysis, resulting on local and systemic hemorrhage (Paine et al., 1992). The high expression of toxins similar to bothropasin/jararhagin observed in B. jararaca venom gland is compatible with the abundant bleeding observed in patients bitten by this snake. In addition, 2 clusters (JARO001012A02 [5 reads] and JARO001014A02 [1 read]) presented homology with the vascular apoptosis-inducing protein (VAIP), which is a PIII metalloproteinase presenting the metalloproteinase, disintegrin-like and cysteine-rich domains characteristic of this Zn2+dependent enzyme. VAIP is able to degrade fibrinogen and induce apoptosis in vascular endothelial cells (Masuda et al., 2000).

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Among the metalloproteinases, 24.2% of the clones could not be precisely identified through their domain composition because the clusters comprise only part of the metalloproteinase domain or because the homology occurred only at the 30 UTR. Concerning the metalloproteinases which could have their classes determined, in our study we found molecules that belong to NII and NIII classes of precursors, which is similar to what was found for Bitis gabonica venom gland (Franscischetti et al., 2004). In B. jararaca (Kashima et al., 2004) and B. insularis (Junqueira-de-Azevedo and Ho, 2002), the NI class was also identified while the hypothetical NIV metalloproteinase precursor was never identified. Looking at the clusters containing the disintegrin region, we could find 34 clusters (348 reads) coding for disintegrin (RGD-containing) or disintegrin-like (XCD-containing) peptides. Among the 348 reads, 178 (51.1%) code for RGD disintegrins contained in NII metalloproteinase precursors and 165 (47.4%) code for ECD disintegrin-like peptides contained in NIII metalloproteinase precursors. The most representative disintegrin domain-containing cluster (JARO001001E01–137 reads) presents 100% identity with the B. jararaca disintegrin jararacin (Scarborough et al., 1993), and the second one (JARO001002D11–103 reads) presents 100% identity with the B. jararaca disintegrin-like domain of the bothropasin precursor (Mandelbaum et al., 1982). Considering that the venom composition may be linked to adaptation to the environment (Okuda et al., 2001; Daltry et al., 1996), it is interesting to note that B. jararaca transcriptome contains, proportionally, much more ‘‘true’’ (RGD-containing) disintegrins, which are released from PII metalloproteinases (Kini and Evans, 1992), than the transcriptome of its close sister, B. insularis, in which just one cluster (02 reads) presented a true disintegrin motif, over a total of 33 metalloproteinases clusters (144 reads) (Junqueira-de-Azevedo and Ho, 2002). A dominance of NIII metalloproteinases in B. insularis, in contrast to the similar proportions of NII and NIII precursors observed in B. jararaca, is consistent with the activity of their venoms. Hemorragins of the PIII class present the most potent activity (Gutie´rrez et al., 2005; Markland, 1998), which may be due to the presence of the cysteine-rich domain, known to enhance the inhibition of collagen-induced platelet aggregation caused by PIII metalloproteinases through interaction with

453

the platelet integrin a2b1 (Kamiguti et al., 2003; Jia et al., 2000). Thus, the presence of the cysteine-rich domain may function to synergistically increase the hemorragic effect of these toxins. The predominant expression of NIII metalloproteinases in B. insularis venom gland over other classes of metalloproteinases may contribute to its highly toxic effect, known as being much more potent than the venom of B. jararaca, with a faster lethal effect (Mosmann, 2001; Melgarejo, 2003). Therefore, we can suggest that the extremely competitive habitat to which B. insularis has been submitted for thousands of years at Queimada Grande island, including high snake density and little food availability, leads to the selection of lineages expressing preferentially more potent toxins as PIII metalloproteinases. More recently, a new gene structure of the disintegrin family was identified in Agkistrodon contortrix contortrix (acostatin) and Agkistrodon piscivourus piscivourus (piscivostatin) venoms. It consists of a signal peptide, a pro-domain and a disintegrin domain, lacking the metalloproteinase domain (Okuda et al., 2002). In Bitis gabonica EST library, two clusters presented homology with this kind of structure, named gabonin 1 and 2, described as new members of the short coding region family of disintegrins (Franscischetti et al., 2004). On the other hand, in B. jararaca transcriptome, we did not find any disintegrin belonging to this group, as observed for B. insularis and B. jararaca transcriptomes. Therefore, we may suggest that the absence of such molecules is a shared character among members of Bothrops genus. 3.3.2. Serine-proteinases Snake venom serine-proteinases are enzymes that affect hemostasis and thrombosis upon envenomation, acting on a variety of components of the coagulation cascade in order to cause an imbalance of the haemostatic system of the prey (Matsui et al., 2000). They can act as procoagulants causing in vivo activation of the coagulation system, but because of the excessive consumption of coagulation factors they frequently lead to clinical anticoagulation (Markland, 1998). These enzymes are defined by a common catalytic mechanism that includes a highly reactive serine residue that forms an active site within a histidine and aspartic acid residues. A high number of isoforms have been described for venom serine-proteinases, in which small changes in sequence or structure have a great impact on activity (Saguchi et al., 2005; Serrano and

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Maroun, 2005). Thus, venom gland serine-proteinases are known to form a multigene family (Deshimaru et al., 1996). The identification of most of the serine-proteinase clusters was given by the 30 UTR region, which is particularly long, as noted in the B. insularis transcriptome (Junqueira-de-Azevedo and Ho, 2002). This class of toxin follows the metalloproteinases in abundancy of toxins present in B. jararaca transcriptome (Fig. 1). Among them, the most representative molecules (62 clusters/173 reads) are homologs of the serine a-fibrinogenase isolated from Macrovipera lebetina, known to present anticoagulant activity (NCBI accession number (acc) AF528193) (Table 2). The second most abundant group (38 clusters/107 reads) of serine-proteinases

Fig. 1. Graphics showing the representation of clones from each toxin category over the total of toxin transcripts from B. jararaca (A) and B. insularis (B) venom glands transcriptome (B was reproduced with authorization from Junqueira-de-Azevedo and Ho, 2002). BPP—bradykinin-potentiating peptide; LAO— L-amino acid oxidase; svVEGF—snake venom vascular endothelium growth factor; PLA2—phospholipase A2; NGF—nerve growth factor.

presents homology to KN-BJ2 (acc AB004067), which is a serine-proteinase isolated from B. jararaca venom that displays kinin-releasing and fibrinogen-clotting activity, being fully or largely responsible for bradykinin release (Serrano et al., 1998). The high amount of cDNAs coding for serine-proteinases found in B. jararaca venom gland is in accordance with the clinical effects observed upon accidents caused by this species, as the major effect of these toxins in the envenomation is the excessive consumption of coagulation factors and formation of abnormal fibrin clots resulting in the absence of blood coagulation (Matsui et al., 2000). 3.3.3. C-type lectins There are two major C-type lectin classes of proteins in snake venoms which belong to the C-type lectin superfamily: the ‘‘true’’ lectins, generally homodimeric and presenting saccharide-binding activity in a calcium-dependent manner (Xu et al., 1999) and the C-type lectin-like proteins, generally heterodimeric and that bind to protein ligands rather than saccharides (Wei et al., 2002). The main reported activity of the first class is hemagglutination (Ozeki et al., 1994; Xu et al., 1999; de Carvalho et al., 2002) while members of the second class interfere in the coagulation pathways in various ways and exhibit anticoagulant or proaggregating effects, which makes them promising candidates for the development of new drugs (Andrews and Berndt, 2000; Andrews et al., 2001). The C-type lectin-like proteins can be divided into four main groups, according to their binding characteristics: (1) inhibition of conversion of prothrombin into thrombin through binding to coagulation factors IX and X (Matsuzaki et al., 1996; Lee et al., 2003); (2) direct inhibition of thrombin action, such as caused by bothrojaracin (Arocas et al., 1997); (3) inhibition of platelet aggregation through binding to GPIb present on platelet surfaces (Shin et al., 2000) and (4) stimulation of platelet aggregation: as an example, the twochain bothrocetin binds to von Willebrand factor, changing its conformation and activating its binding to platelet glicoprotein (GP) Ib (Sen et al., 2001). In our transcriptome analysis, we could identify clusters presenting homology with all C-type lectin-like protein classes described before (Table 2). The most abundant C-type lectin-like proteins are homologues of the glycoprotein Ibbinding protein, previously isolated from B. jararaca venom (Kawasaki et al., 1996), followed by

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bothrojaracin homologues (Arocas et al., 1997), factor IX/X binding proteins (Matsuzaki et al., 1996) and botrocetin homologues (Sen et al., 2001). In the B. jararaca venom gland transcriptome, there was only one C-type lectin cluster, which codes for a putative homodimeric C-type ‘‘true’’ galactose-binding lectin. It was composed of 25 reads, and corresponded to 7.4% of toxin-related sequences (Kashima et al., 2004). The presence of true lectins in B. jararaca venom has already been described, and associated with strong hemagglutination (de Carvalho et al., 2002). In fact, gum bleeding has been observed in only 6.9% of Brazilian B. jararaca-caused accidents, as opposed to 24% of B. jararaca-caused accidents (Franc- a and Malaque, 2003). This feature may be, in part, due to the predominance of hemagglutinating true-lectins in B. jararaca venom, which are less abundant in B. jararaca. In the Bitis gabonica venom gland transcriptome, 4 clusters of C-type lectins were found, one of them being similar to galactosebinding lectin and the others similar to C-type lectin-like proteins (Franscischetti et al., 2004). The cloning, characterization and structural analysis of a true-lectin (BiL) from B. insularis was recently reported (Guimara˜es-Gomes et al., 2004). BiL displayed hemagglutinating activity that was inhibited by galactose, lactose and EDTA, evidencing that it is a typical calcium-dependent galactosebinding lectin. In B. insularis venom gland transcriptome, among the 16 C-type lectin clusters, 2 showed best similarities with galactose-binding truelectins (Junqueira-de-Azevedo and Ho, 2002). It seems that true lectins have a minor participation in the B. insularis envenomation pathology, since they corresponded to only 14% of the C-type lectinrelated transcripts. No true lectin was found in our transcriptome analysis, even when the library was screened by PCR using specific primers, in spite of it having been previously described in B. jararaca venom (Ozeki et al., 1994). This is probably due to individual variation in venom composition. 3.3.4. Bradykinin-potentiating peptides (BPPs) The BPPs are proline-rich oligopeptides of 5-14 amino acid residues. They usually present a pyroglutamyl residue at the N-terminus and an Ile-Pro-Pro (IPP) sequence at the C-terminus. They are known to inhibit the endothelial metalloproteinase angiotensin-converting enzyme (ACE), which produces angiotensin II, a potent vasoconstrictor, through hydrolysis of angiotensin I (Hayashi and

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Camargo, 2005). ACE is responsible for the maintenance of normal blood pressure levels, and is also able to inactivate the hypotensive peptide bradykinin, which is released in the blood of envenomed victims through the action of venom serine-proteinases. Therefore, BPPs are involved with the hypotension observed after envenomation by Bothrops species, and have been used as models for the development of new antihypertensive drugs (Fernandez et al., 2004). In our analysis, we found 25 clusters (80 reads) corresponding to BPP precursor transcripts, accounting for 6.2% of the toxin transcripts (Tables 2 and 4). Most of them present only the 30 UTR, known to be particularly long in BPP precursors (Murayama et al., 1997; Higuchi et al., 1999; Soares et al., 2005). These transcripts are known to contain several BPP-coding regions tandemly arranged at the N-region and a C-type natriuretic peptide at the C-region of the precursor (Hayashi and Camargo, 2005). Cluster JARO001047D04 consists of a partial sequence that presented 100% identity with the 7 BPPs located at the N-terminal region of a BPP precursor previously described in B. jararaca (Murayama et al., 1997). In B. insularis transcriptome, BPP-related transcripts accounted for 19.7% of toxin transcripts. In addition, a higher redundancy of BPPs was observed, since 61 reads could be grouped in a same cluster (Junqueira-de-Azevedo and Ho, 2002). When we compare these two pit vipers, it seems that B. insularis became specialized in the production of high amounts of extremely conserved BPPs while B. jararaca presents more variability among BPP precursors. This high frequency of BPPs in B. insularis transcriptome is in accordance with the activity of its venom. Therefore, we could speculate that the action of B. insularis venom, which is able to kill the prey immediately, is fully or largely related to a BPPcaused hypotensive shock. 3.3.5. Phospholipases A2 and their inhibitors Bothrops venoms contain numerous components that could potentially affect neuromuscular responses, including the phospholipases A2 (PLA2) myotoxins. They are the major components responsible for the intense local damage observed after some Bothropic accidents, being capable of inducing inflammatory events through the enhancement of vascular permeability and the recruitment of neutrophils (Zuliani et al., 2005). Some Bothrops venoms (B. jararaca, B. moojeni and B. neuwiedi)

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are known to cause high levels of muscle damage when compared to B. jararaca and B. erythromelas venoms (Zamuner et al., 2004). These findings are in accordance with our results, in which PLA2 seems to have a minor participation in the envenomation process since it accounts for only 0.7% of the toxin transcripts, indicating that the local damage is less representative than the vascular disorders induced by metalloproteinases, serine-proteinases and C-type lectins (Fig. 1A). The same pattern was observed in the analysis of B. insularis transcriptome, in which the PLA2 accounted for 6.7% of the toxin transcripts (Junqueira-de-Azevedo and Ho, 2002). On the other hand, the high frequency of PLA2 transcripts in B. jararacussu transcriptome (58% of the toxin transcripts) evidences the clear predominance of the local action of this venom (Kashima et al., 2004). In Bitis gabonica transcriptome, a high number of transcripts coding for PLA2 was also found (32% of the toxin transcripts), PLA2 being the most expressed toxin (Franscischetti et al., 2004). This result indicates a predominance of local action over systemic injury in both B. jararacussu and Bitis gabonica envenoming accidents. Interestingly, we found some clusters coding for PLA2 inhibitors, which are mostly present in snake sera. The most frequent ones, comprising only of the 30 UTR, are homologous to a Trimeresurus flavoviridis phospholipase A2 class A inhibitor (Nobuhisa et al., 1997) (Table 2). PLA2 inhibitors have been purified from the sera of many venomous snakes such as Notechis ater (Hains et al., 2000), Crotalus durissus terrificus (Faure et al., 2000) and Trimeresurus flavoviridis (Nobuhisa et al., 1997) and also from nonvenomous snakes such as Elaphe quadrivirgata (Okumura et al., 2002) and Pyton reticulatus (Thwin et al., 2002). Several studies showed that the resistance of some animals to snake venoms can be explained by the presence of protein factors in their blood which inhibit the activity of important toxic compounds. These proteins are either metalloproteinase inhibitors (Perales et al., 2005) or PLA2 inhibitors (Lizano et al., 2003). To our knowledge, PLA2 inhibitors have never been described as venom components, although it is well known that some venoms contain competitive components. B. jararaca venom, for instance, contains not only botrocetin but also GPIb-BP which inhibits the botrocetin-induced platelet agglutination (Kawasaki et al., 1996). If these kinds of PLA2 inhibitors are present in the venom and are playing some specific role in the

envenomation caused by B. jararaca is a question to be further elucidated. Noteworthy, in our laboratory we have previously detected the activity of PLA2 inhibitors in Bothrops jararaca venom, although a molecular characterization of its structure was not performed (Sousa et al., 2001). There are very few reports describing the presence of low molecular weight PLA2 inhibitors in the venom, in particular from Bothrops snakes (Vidal and Stoppani, 1971 and 1980). These molecules are much smaller than the PLA2 inhibitors isolated from sera. The absence of a complete characterization of these peptides, especially regarding its amino acid sequence, makes it difficult to fully appreciate the significance of these molecules. 3.3.6. Other toxins present in B. jararaca venom gland transcriptome In the EST database we found some venom cysteine-rich secretory proteins (CRISPs) (Table 2). These proteins are widely distributed in snake venoms, but little information is available about their action in the envenomation. Until now, two types of activities were found: the inhibition of smooth muscle contraction and the blocking of cyclic-nucleotide-gated ion channels (Yamazaki and Morita, 2004; Oispov et al., 2005). We also found some toxin classes that seem to have minor participation in B. jararaca envenomation process, since they are present in small amounts in the database. The L-amino acid oxidases, which are able to induce apoptosis, affect platelets and have hemorragic effects (Aird, 2002), were found at a frequency of 0.5% of toxin-related reads (Table 4). In addition, the snake venom vascular endothelium growth factor (svVEGF) accounts for 0.2% of the toxin-related reads in B. jararaca transcriptome, which is less represented than in B. insularis transcriptome (4.3%). B. insularis svVEGF was cloned, expressed and shown to be capable of increasing vascular permeability thus acting as a toxin dispersion agent and probably contributing to the hypotensive action observed upon envenomation (Junqueira-de-Azevedo et al., 2001). 3.4. Analysis of transcripts related to cellular functions Proteins related to cellular functions, including housekeeping proteins, comprise 22.4% of the matching transcripts. These transcripts were divided

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Table 5 Representation of the putative cellular protein transcripts from B. jararaca venom glands according to their cellular functions, as well as their redundancy (clones/clusters) Cellular proteins

Clusters (%)

Clones (%)

Redundancy

General metabolism Transcription and translation Processing and sorting Polypeptide degradation Structural functions Cell regulation and other functions

42 68 38 09 15 56

54 (14.5) 105 (28.3) 103 (27.8) 14 (3.8) 19 (5.1) 76 (20.5)

1.3 1.5 2.7 1.5 1.3 1.4

(18.4) (29.8) (16.7) (3.9) (6.6) (24.6)

into 6 categories, according to their functional types (Table 5). The most abundant transcripts are related to DNA transcription and mRNA translation (28.7%), closely followed by the transcripts related to post-translational processing and sorting of molecules (27.8%). Among the former group we could identify mostly ribosomal proteins, besides factors involved in translation initiation and elongation (Table 3). In the latter group the most frequent transcripts are difulfide-isomerases, enzymes involved in disulfide-bridge formation (Freedman et al., 1994). These bridges are frequently found in toxins and are largely responsible for the maintenance of the three-dimensional structure of these molecules. Another protein found in the EST database and included in the post-translational processing and sorting group is calreticulin, a Ca2+-binding protein that has already been characterized as a molecular chaperone, among other functions (Coppolino and Dedhar, 1998). Almost all of the calreticulin-related transcripts (except for one) could only be identified through homology with the UTRs of B. insularis calreticulin (BlastN against EST database), suggesting that these regions are conserved among the snakes. The pattern of cellular protein transcripts described here is similar to that observed in B. insularis transcriptome (Junqueira-de-Azevedo and Ho, 2002). These results are in accordance with what is expected for this highly specialized secretory tissue, as its main function is to express, process and secrete toxins to the lumen of the venom gland. 3.5. Evolutionary considerations A comparison of the transcriptomes of the phylogenetically related Bothrops species reveals beyond an apparent similarity that some differences

can be outlined, such as the higher amount of BPPrelated transcripts in B. insularis database, which indicates a higher hypotensive action of its venom. These BPPs also seem to be more conserved in B. insularis, as 61 reads could be grouped in the same cluster. In B. jararaca, an apparent higher variability among BPPs was observed. The predominant expression of PIII-class of metalloproteinases by B. insularis is a contrast to the similar levels of expression of PII and PIII-classes by B. jararaca, which also indicate a more specialized expression pattern of B. insularis and may explain, in part, an increased potency of its venom. This feature may be a consequence of the adverse situations to which this endemic snake has been submitted for thousands of years including long starvation periods and high competition for feeding because of the high snake density in the island, in which 12,000–20,000 snakes are distributed over 430,000 m2 (Mosmann, 2001). Moreover, diet has already been described as an influencing factor in venom evolution, concerning the differences in susceptibility of the preys to different venoms and the natural selection for feeding on local prey (Daltry et al., 1996). The more homogeneous diet to which B. insularis has been submitted for a long time, composed basically of birds and some invertebrates available in the island (Duarte et al., 1995), may be an important factor in its present venom composition, more effective against birds than mammalians (Cogo et al., 1993; da Cruz Hofling et al., 2001). In contrast, B. jararaca can be found from Bahia (Northeast) to Rio Grande do Sul (South) States at Brazil, and the wide geographical occurrence can enhance the variability of its venom composition (Jayanthi and Gowda, 1988; Rodrigues et al., 1998). B. jararaca feeding-habits are more variable and these snakes present high adaptability, being able to survive at agrarian, silvian and urban areas

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(Mosmann, 2001; Melgarejo, 2003). In spite of the apparent differences, we have to consider these conclusions carefully, since the number of generated ESTs for both transcriptomes is different (610 for B. insularis and 2318 for B. jararaca) and this can lead to some bias in the analysis. Moreover, individual variation may have contributed for some of the observed differences. 3.6. Perspectives Snake venom is a natural library for screening valuable bioactive substances for hemostasis and thrombosis, among others (Marsh and Williams, 2005). This work provided a catalog of the transcripts contained in B. jararaca venom gland, which has been used for supporting proteomic studies of this species (unpublished). Moreover, the sequences can be used to directly probe the genetic material from other snake species or to investigate differences in gene expression pattern in response to factors such as diet, aging and geographic localization. Attempts for the cloning and expression of toxins from the database are in course, aiming to elucidate some structure-activity relationships. Acknowledgments This work was supported by grants from the Brazilian agencies Fundac- a˜o de Amparo a` Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Cientı´ fico e Tecnolo´gico (CNPq), Rede Proteoˆmica do Rio de Janeiro and Riogene.

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