Discovery of immune-related genes expressed in hemocytes of the tarantula spider Acanthoscurria gomesiana

Developmental and Comparative Immunology 30 (2006) 545–556 www.elsevier.com/locate/devcompimm

Discovery of immune-related genes expressed in hemocytes of the tarantula spider Acanthoscurria gomesiana Daniel M. Lorenzini a, Pedro I. da Silva Jr b, Marcelo B. Soares c, Paulo Arruda d,1, Joa˜o Setubal e,2, Sirlei Daffre a,* a

Departamento de Parasitologia, Instituto de Cieˆncias Biome´dicas, Universidade de Sa˜o Paulo, Avenida Prof. Lineu Prestes, 1374, CEP 05508-900 Sa˜o Paulo, SP, Brazil b Laborato´rio de Artro´podes, Instituto Butantan, CEP 05503-900 Sa˜o Paulo, SP, Brazil c Department of Pediatrics and Biochemistry, University of Iowa, Iowa City, IA 52242, USA d Centro de Biologia Molecular e Engenharia Gene´tica, Universidade Estadual de Campinas, CEP 13083-970 Campinas, SP, Brazil e Laborato´rio de Bioinforma´tica, Instituto da Computac¸a˜o, Universidade Estadual de Campinas, CEP 13083-970 Campinas, SP, Brazil Received 5 July 2005; revised 28 August 2005; accepted 2 September 2005 Available online 7 October 2005

Abstract The present study reports the identification of immune related transcripts from hemocytes of the spider Acanthoscurria gomesiana by high throughput sequencing of expressed sequence tags (ESTs). To generate ESTs from hemocytes, two cDNA libraries were prepared: one by directional cloning (primary) and the other by the normalization of the first (normalized). A total of 7584 clones were sequenced and the identical ESTs were clustered, resulting in 3723 assembled sequences (AS). At least 20% of these sequences are putative novel genes. The automatic functional annotation of AS based on Gene Ontology revealed several abundant transcripts related to the following functional classes: hemocyanin, lectin, and structural constituents of ribosome and cytoskeleton. From this annotation, 73 transcripts possibly involved in immune response were also identified, suggesting the existence of several molecular processes not previously described for spiders, such as: pathogen recognition, coagulation, complement activation, cell adhesion and intracellular signaling pathway for the activation of cellular defenses. q 2005 Elsevier Ltd. All rights reserved. Keywords: Hemocytes; Innate immunity; Expressed sequence tags (ESTs); Hemocyanin; Coagulation; Lectins; Serine-proteases; Antimicrobial peptides

1. Introduction

* Corresponding author. Tel.: C55 11 309 17272; fax: C55 11 309 17417. E-mail address: [email protected] (S. Daffre). 1 Alellyx Applied Genomics, CEP 13067-850, Campinas, SP, Brazil. 2 Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Bioinformatics 1, Box 0477, Blacksburg, VA 24060, USA.

0145-305X/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2005.09.001

Invertebrates developed an efficient immune system to control infections. The hemolymph circulating cells, named hemocytes, are essential components of this system that play two important roles. One is the cellular activity that causes the phagocytosis and/or encapsulation of the pathogens [1]. The second, but not less important, is the production of peptides and proteins that participate in the immune response, as seen in the following examples. The hemocytes of horseshoe crabs

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store clotting factors, proteinase inhibitors and lectins [2,3]. The insects and crustaceans’ hemocytes produce phenoloxidase [4]. Antimicrobial peptides have been isolated from hemocytes of animals that belong to different phyla of invertebrates [5]. The constitutive production and storage of these peptides and proteins in the hemocytes keeps invertebrates ready to fight invading pathogens. To investigate the immune response of spiders, initially, we worked in the characterization of antimicrobial molecules. Two peptides (gomesin and acanthoscurrin) and one acylpolyamine (mygalin) with antimicrobial activity have been isolated from the hemocytes of mygalomorph spider A. gomesiana. The use of mygalomorph spiders as an experimental model is very useful, because they are one of the oldest species in the order Araneae. Gomesin is a cationic peptide of 18 amino acids and two disulfide bridges, produced from a precursor containing a signal peptide and an anionic segment on the C-terminus, which is constitutively synthesized in the hemocytes and stored in their granules [6,7]. Acanthoscurrin is a glycine-rich peptide, which is post-translationally processed by the removal of the signal peptide and C-terminal amidation. Acanthoscurrin is released into the cell free hemolymph following immune challenge [8]. Mygalin is a bis-acylpolyamine N1,N8-bis(2,5-dihydroxylbenzoil)spermidine active against E. coli, and its activity is inhibited by catalase [9]. The discovery of novel invertebrate genes related to the immune response has been accelerated by high throughput sequencing techniques combined with searches for homologous sequences on public databases. The sequencing of expressed sequence tags (ESTs) is specially useful, since it allows simultaneously the novel gene discovery and gene expression analysis. The sequencing of ESTs has been done with hemocytes of shrimps [10,11], mollusks [12,13] and insects [14], resulting in the identification of several immune related genes. In the mosquito Anopheles gambiae, the EST clones were used to prepare a cDNA microarray, and the modulation of immune genes’ expression was analyzed under different immune stimuli [15]. In this context, exploring novel genes on invertebrates such as spiders, which are phylogenetically distant from the organisms mentioned above, would produce meaningful information that can support the search for immune features that are conserved throughout the invertebrates. This study reports the production of ESTs from the hemocytes of the spider A. gomesiana obtained through two cDNA libraries, one prepared by directional

cloning (primary) and the other by the normalization of the first (normalized). The random selection of clones from the primary cDNA library resulted in the identification of transcripts abundantly expressed in the hemocytes, while the normalized library produced a large number of unique sequences. In addition, several of these sequences are related to invertebrate immune response. 2. Material and methods 2.1. Spiders A. gomesiana is a tarantula spider of the Theraphosidae family that is distributed in southeast Brazil. Adults from this species are medium-sized (approximately 5 cm in length) and can live over 23 years. The animals used in the experiments were adults of both sexes and in the intermolt stage. These spiders were not reared in the laboratory, but donated to the Arthropods Laboratory of the Butantan Institute (Sa˜o Paulo, Brazil) by citizens of Sa˜o Paulo and neighbor towns, where they were kept. 2.2. Hemocyte isolation and RNA extraction Hemolymph was collected from 15 spiders (approximately 0.5 ml/animal) as previously described [6]. Hemocytes were separated from cell free hemolymph by centrifugation at 800!g for 10 min at 4 8C before RNA extraction. Total RNA was isolated from hemocytes using Trizol reagent (Gibco/BRL). 2.3. Construction of cDNA libraries 2.3.1. Primary One directionally cloned cDNA library was prepared as described previously [16]. The mRNA (1 mg), purified from total RNA (300 mg) with an oligo-(dT) column, was annealed with 2-fold mass excess of a NotI-(dT)18 primer and reverse transcribed with Superscript Reverse Transcriptase (Life Sciences). Following the second strand synthesis, the doublestranded cDNAs longer than 350 bp were size selected by gel filtration on a Bio-Gel A-50M column (BioRad), joined to a 500- to 1000-fold molar excess of EcoRI adapter, digested with Not I, and size selected over a second Bio-Gel column to remove the excess of EcoRI adapter. The selected cDNAs were cloned into the EcoRI and NotI sites of the pT7T3-Pac phagemid vector and electroporated into E. coli DH10B host cells (Invitrogen). The primary library plasmid DNA was

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purified using Qiagen-tip 100 (Qiagen) and stored at K70 8C.

and not identified as contaminants were considered as high quality sequences.

2.3.2. Normalized The normalized library was prepared following the method 4 described in [16]. The primary library plasmid DNA was electroporated into E. coli DH5aF 0 , infected with the helper phage M13K07 (Pharmacia) and harvested to prepare the single-stranded plasmids. An aliquot of the single-stranded plasmids was amplified by PCR to produce the cDNA inserts, which were hybridized in a 20-fold excess with the single-stranded plasmids. Following hybridization at a relatively low Cot (y5), the remaining single-stranded circles (normalized library) were purified on a hydroxyapatite (HAP) column, converted to double-stranded circles by primer extension and electroporated into E. coli DH10B host cells (Invitrogen). The normalized library plasmid DNA was purified and stored as described above.

2.5.2. Sequence assembly High quality sequences from both libraries were assembled by sequence similarity using CAP3 set to minimum of 40 overlap and 95% identity (non-default parameters: -o 40 -p 95). The longest ORF size of each assemble sequence was accessed by Flip (http:// megasun.bch.umontreal.ca/ogmp/aboutflip.html).

2.4. Template preparation and DNA sequencing E. coli DH10B host cells (Invitrogen) were electroporated with plasmid DNA from the cDNA libraries and spread on LB agar plates containing 60 mg/ml of ampicillin. Random selected colonies were grown on Circle Grow medium (Qbiogene) containing ampicillin at 100 mg/ml for 22 h at 37 8C. Plasmid DNA from the transformed bacteria was prepared in 96-well plates using a modified alkaline lysis method (http:// sucest.lbi.ic.unicamp.br/public/protocols.html). The 5 0 end of cDNA inserts was sequenced on an automatic DNA sequencer ‘ABI 3100’ (Applied Biosystems) using T3 primer and ABIe Big Dye terminator kit (Applied Biosystems). 2.5. Sequence analysis 2.5.1. Sequence trimming and contaminant discarding The chromatograms from sequenced clones were automatically processed for base calling and low quality trimming using Phred set to minimum quality 10 (non-default parameters: -trim_alt -trim_cutoff 0.09). Vector sequence trimming was done by Crossmatch with the pT7T3-Pac sequence (non-default parameters: -minmatch 10 -minscore 20) and contaminant sequences were identified by BlastN set to e-value cutoff 1!10K30 (non-default parameters: Ke 1!10K30), using a database of possible contaminants (ribosomal RNA, E. coli genome, mitocondrial and plasmid sequences, all from Genbank). Sequences containing more than 200 bp after trimming (quality and vector)

2.5.3. Functional annotation The assembled sequences (AS) were submitted to similarity searches (BlastX—e-value cutoff 1!10K6, InterProScan) against public databases (nr-NCBI, SwissprotCTREmbl, and Interpro). The GeneOntology (www.geneontology.org) terms associated with Interpro, Swissprot or TREmbl sequences found in the similarity searches were automatically annotated to the corresponding AS. Two additional protein databases with GeneOntology associations were prepared with immune related sequences from horseshoe crabs [17] and Drosophila melanogaster [18], and these databases were used for automatic functional annotation as described above. The AS containing at least one EST from the primary library were manually annotated, when possible, with one GeneOntology entry for molecular function ontology. The manual annotation was based on the results of automatic annotations and similarity searches on public databases. 2.5.4. Increasing cDNA coverage The longest clones of AS related to immune system were sequenced from 5 0 and 3 0 ends (using T3 and T7 primers, respectively) and manually assembled together with corresponding ESTs, using the software Seqman (Lasergene package, DNAStar, USA) 3. Results For the discovery of genes expressed in the spider hemocytes, 7584 ESTs were sequenced from primary and normalized cDNA libraries (Table 1). After sequence trimming and contaminant rejection, almost 90% of these ESTs (High Quality ESTs) had enough information for sequence analysis. The low frequency of ESTs rejected for the presence of contaminants indicated the high quality of the cDNA libraries. The elevated efficiency of the DNA sequencing is also attested by the low frequency of sequences discarded due to quality and the high average length of the sequences (Table 1). The high quality ESTs were

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Table 1 Statistics of expressed sequence tags (EST) from cDNA libraries of Acanthoscurria gomesiana hemocytes

Sequenced ESTs High quality ESTs Discarded ESTs Average length of sequencesa Discarded reason Low qualityb Sizec No insert Contaminants E. coli Mitocondrial Ribossomal Vector

Normalized

Primary

Total

5760 5156 604 524.7

1824 1634 190 651.0

7584 6790 794

540 6 13

100 4 1

640 10 14

7 1 14 23

1 1 1 82

8 2 15 105

a

Sequences length after quality trimming (bp). Sequences shorter than 200 bp with Phred quality above 10. c Sequences shorter than 200 bp after vector and low quality trimming. b

deposited in the GenBank (accession codes DR442119–DR448908). The sequence assembly of the high quality ESTs revealed 3723 assembled sequences (AS), which correspond to the estimated number of transcripts identified. From this total, 814 AS contain at least one EST from the primary library (Primary Assembled Sequences—PAS). When the ESTs of each library were assembled separately, the 20 most abundant AS from the primary library corresponded to 34% of the ESTs sequenced from this library, while in the normalized library only 6% of the ESTs sequenced from this library were found in the 20 most abundant AS. This demonstrates that the abundance of transcripts found in the primary library was significantly reduced by the normalization process. Consequently, the discovery of new transcripts was more efficient in the normalized library, as verified by the higher number of unique sequences obtained from this library (819 primary, 1308 normalized) when equivalent number of ESTs from each library were used on separate identical sequence assemblies. In order to assign function to all putative transcripts obtained, the assembled sequences were submitted to similarity searches with several public sequence databases (Table 2). The number of AS with matching sequences on NR and SwisprotCTREmbl was very similar, and much higher than on Interpro. However, 213 AS had matches only with Interpro search. Using the Gene Ontology (GO) information available for entries of SwissprotCTREmbl and Interpro

Table 2 Automatic functional annotation statistics of assembled sequences (AS) Database

AS with match on Database

AS annotated with GO

NRa Swisprot C TREmbl Interpro Imune Drosophila Imune Limulus Total

2192

–

2171 873 106 91 2411

1937 378 106 91 1971

a

Protein sequence database from NCBI.

databases, the assembled sequences were automatically associated with GO terms. Most of the AS with matches on these databases were associated with GO terms (Table 2). Several AS were associated with more than one term, making it difficult to analyze the distribution of AS on GO terms. On the other hand, this annotation was very useful for the manual annotation of PAS and identification of immune related transcripts. Each PAS was manually annotated with only one term of the molecular function ontology (Gene Ontology). In this way, 459 of the 814 PAS were annotated. The number of primary library ESTs in each PAS was used to evaluate the abundance of the corresponding transcripts in the spider hemocytes, and the GO annotation grouped these ESTs by function (Fig. 1). The functional class related to hemocyanins has the highest number of ESTs. Components of ribosome and cytoskeleton were also very abundant. In the sugar binding (lectin) class, it was found one PAS containing 27 ESTs from the primary library (AGCCAR1041B12, Table 4). There was also an elevated number of ESTs related to transposable elements. For the identification of immune related AS, a list of functional classes was prepared from literature dedicated to this issue (Table 3). The AS annotated with GO terms that correspond to these functional classes were individually analyzed. Special attention was given to AS annotated through the immune related sequences from horseshoe crabs and D. melanogaster. This analysis led to the identification of 123 AS, which was reduced to 73 after sequencing both ends and manually assembling each AS. These AS represent several functional classes involved in the innate immune response (Table 4). 4. Discussion The present study presents the identification of transcripts related to immune system by

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549

1000

504

Primary library ESTs

356

92

100

55

48

47 35

35

34

34

30

26

24

23

n tio

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ct

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nc

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A

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sti

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10

Fig. 1. Gene expression profile of Acanthoscurria gomesiana hemocytes using Gene Ontology. Values indicate the number of sequenced clones from the primary library grouped in categories of Molecular Function ontology. Only the fourteen most abundant categories are presented.

high-throughput sequencing of hemocyte ESTs of the tarantula spider A. gomesiana. The sequencing of clones from both a primary and a normalized cDNA library yielded a total of 3723 transcripts that included at least 20 abundantly expressed. The number of transcripts is considered over estimated, because the sequence assembly software (CAP3) often separates identical ESTs into different AS [19]. This separation may be due to errors or polymorphisms in the sequences, or to regions of the transcripts with low coverage of ESTs [19]. The number of transcripts without match in the NR database was high (1531, 41%), suggesting the finding of a great number of novel genes. However, the percentage of novel genes is much higher on transcripts with ORFs shorter than 400 bp than on transcripts of longer ORFs (Fig. 2). Not all short ORFs without matches in public databases should be considered as novel genes, since it is difficult to find significant matches (e-value !1!10K6) on database searches with short protein sequences. As reported before, transcripts with low protein coding capacity (short ORFs) may correspond to ESTs of either very short sequences or sequences with long 5 0 UTR (Untranslated Region) [20]. Therefore, the estimated percentage of

novel genes should be closer to that found with ORFs longer than 900 bp (20%). The gene expression profile of spider hemocytes was characterized by the abundance of primary library ESTs (Fig. 1). Among the sequences with functional attribution, the AS related to hemocyanin subunits were remarkably abundant (356 ESTs), indicating the involvement of hemocytes in the production of this oxygen transport protein. In the tarantula spider Eurypelma californicum, the hemocyanins are Table 3 GeneOntology (GO) terms related to the immune system GO:0004252 GO:0003810 GO:0003796 GO:0006961 GO:0008329 GO:0003823 GO:0005530 GO:0017114 GO:0004867 GO:0004888 GO:0003700 GO:0006952 GO:0004503 GO:0003793

Serine-type endopeptidase Protein-glutamine gamma-glutamyltransferase Lysozyme Antibacterial humoral response (sensu Invertebrata) Pattern recognition receptor Antigen binding Lectin Wide-spectrum protease inhibitor Serine protease inhibitor Transmembrane receptor Transcription factor Defense response Monophenol monooxygenase Defense/immunity protein

550

Table 4 Immune related Assembled Sequences (AS) identified in the cDNA libraries of A. gomesiana hemocytes AS code

# of ESTsa Norm.

Gi

Description

Organism

59 35 21 71 40 39 4 103

122792 20138395 20138398 17376946 70624 20138397 20138397 20138396

Hemocyanin A chain Hemocyanin B chain Hemocyanin C chain Hemocyanin D chain Hemocyanin E chain Hemocyanin F chain Hemocyanin F chain Hemocyanin G chain

Eurypelma Eurypelma Eurypelma Eurypelma Eurypelma Eurypelma Eurypelma Eurypelma

2 1 1 0 0

52782738 52782737 28445738 28445738 1085148

Acanthoscurrin 1 precursor Acanthoscurrin 2 precursor Gomesin precursor Gomesin precursor lysozyme S

A. gomesiana A. gomesiana A. gomesiana A. gomesiana Drosophila melanogaster

2!10K30 2!10K30 3!10K41 6!10K38 2!10K16

0 0 1 2 0 1 1 0 0 0 0 0 1 0 0

542517 542517 542517 129688 18542425 913964 7387836 3928787 1817554 25989209 23266416 28194028 28194028 847761 26332511

coagulation factor B precursor coagulation factor B precursor coagulation factor B precursor Proclotting enzyme precursor factor C precursor factor C Carcinoscorpius Limulus clotting factor C precursor factor B SpBf limulus factor D coagulation factor-like protein 3 serine protease PC5-A prothrombin precursor prothrombin precursor SPC3 unnamed protein product

Tachypleus tridentatus Tachypleus tridentatus Tachypleus tridentatus Tachypleus tridentatus Tachypleus tridentatus rotundicauda Carcinoscorpius rotundicauda Strongylocentrotus purpuratus Tachypleus tridentatus Hyphantria cunea Rana esculenta Takifugu rubripes Takifugu rubripes Branchiostoma californiense Mus musculus

5!10K50 1!10K58 2!10K54 1!10K81 3!10K75 1!10K100 1!10K129 8!10K50 1!10K117 5!10K60 8!10K44 2!10K50 1!10K09 3!10K56 2!10K11

1 2

17223666 1078956

serine proteinase inhibitor serpin-3 intracellular coagulation inhibitor type 2 (LICI 2) intracellular coagulation inhibitor type 2 (LICI 2) similar to serine (or cysteine) proteinase inhibitor alpha-2-macroglobulin complement component C3

Rhipicephalus appendiculatus Tachypleus tridentatus

9!10K30 5!10K65

Tachypleus tridentatus

2!10K65

Rattus norvegicus

1!10K47

Limulus sp. Branchiostoma belcheri

1!10K139 8!10K49

AGCCAR1003H02

7

3

1078956

AGCCAR1005F08

2

0

34881479

AGCCAR1002C10 AGCCAR1003A01

3 5

4 0

7521905 13928544

E-value californicum californicum californicum californicum californicum californicum californicum californicum

0 0 0 0 0 0 1!10K180 0

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Phenoloxidases AGCCAR1001B07 9 AGCCAR1019B12 8 AGCCAR1008E07 5 AGCCAR1004D09 9 AGCCAR1013F04 4 AGCCAR1001H09 34 AGCCAR1009A03 5 AGCCAR1003A06 13 Antimicrobial peptides and proteins AGCCAR1001C06 7 AGCCAR1006D12 2 AGCCAR1034B01 1 AGCCAR1010G04 1 AGCCAR1035C06 2 Serine proteases AGCCAR1044G07 1 AGCCAR1057G02 1 AGCCAR2017E09 0 AGCCAR1026C07 5 AGCCAR1003H07 1 AGCCAR1006G12 1 AGCCAR1011F11 1 AGCCAR1012C07 2 AGCCAR1013H11 6 AGCCAR1006A04 3 AGCCAR1006C09 4 AGCCAR1019E09 2 AGCCAR2001D06 0 AGCCAR1020D11 3 AGCCAR1013F12 3 Serine protease inhibitors AGCCAR2016C11 0 AGCCAR1001D03 10

Prim.

AGCCAR1013G08 AGCCAR1013A03 AGCCAR1056G07 AGCCAR1037F11 AGCCAR2006A03

1 1 3 1 0

AGCCAR1031C10 AGCCAR1009B05 AGCCAR1049A04

1 3 1

20302747 25149822 25149822 13346812 22901764

Branchiostoma floridae Caenorhabditis elegans Caenorhabditis elegans Haemonchus contortus Ancylostoma caninum

1!10K56 6!10K11 4!10K11 2!10K35 1!10K31

2133556

unknown thrombospondin thrombospondin thrombospondin Kunitz-like protease inhibitor precursor cystatin precursor

0

Tachypleus tridentatus

2!10K14

27 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 1 0 0 0 0 0 1 2 0 0

1346296 6630613 21666693 1346296 1346296 17942826 5851893 17942826 5851893 17942826 5851897 17942826 17942826 5851893 5851893 31217088 27808640 4878035 4505245 4505245 4505245 12738842 84651 6981152 9857647 2833353

Hemocytin precursor Hemolectin hemolectin-like protein Hemocytin precursor Hemocytin precursor Tachylectin 5a Tachylectin 5a Tachylectin 5a Tachylectin 5a Tachylectin 5a Tachylectin 5b Tachylectin 5a Tachylectin 5a Tachylectin 5a Tachylectin 5a ENSANGP00000012978 peptidoglycan recognition protein neurocan core protein precursor mannose receptor C type 1 precursor mannose receptor C type 1 precursor mannose receptor C type 1 precursor polydomain protein C-reactive protein chain 3.3 lectin, galactose binding galectin LEC-4 Galectin-4 (Lactose-binding lectin 4)

Bombyx mori Drosophila melanogaster Penaeus monodon Bombyx mori Bombyx mori Tachypleus tridentatus Tachypleus tridentatus Tachypleus tridentatus Tachypleus tridentatus Tachypleus tridentatus Tachypleus tridentatus Tachypleus tridentatus Tachypleus tridentatus Tachypleus tridentatus Tachypleus tridentatus Anopheles gambiae Bos Taurus Gallus gallus Homo sapiens Homo sapiens Homo sapiens Mus musculus Limulus sp. Rattus norvegicus Caenorhabditis elegans Sus scrofa

1!10K104 1!10K21 2!10K10 5!10K31 1!10K25 4!10K68 7!10K75 2!10K79 6!10K71 6!10K61 7!10K76 1!10K73 5!10K72 1!10K70 3!10K80 2!10K44 6!10K36 6!10K18 1!10K28 2!10K23 1!10K25 4!10K49 2!10K15 3!10K18 5!10K08 4!10K16

0 1 1 1

22651842 22651842 22651842 9965396

Aedes aegypti Aedes aegypti Aedes aegypti Xenopus laevis

5!10K17 9!10K29 1!10K40 7!10K34

0 1 0

24650493 15718457 345423

Toll-related protein; AeTehao Toll-related protein; AeTehao Toll-related protein; AeTehao Toll/IL-1 receptor binding protein MyD88 spatzle CG6134-PI Peroxinectin protein-glutamine gamma-glutamyltransferase (EC 2.3.2.13)

Drosophila melanogaster Penaeus monodon Tachypleus tridentatus

2!10K10 1!10K89 2!10K58

551

The BLASTX hits on the protein sequence database from NCBI (NR) with the lowest E values (implying the most significant similarities) are indicated in the table. a Number of ESTs in each assembled sequence obtained from Normalized (Norm.) and Primary (Pri.) cDNA libraries.

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AGCCAR1013B05 1 Lectins AGCCAR1041B12 2 AGCCAR1010G07 1 AGCCAR1012D08 2 AGCCAR1053A05 1 AGCCAR1036E09 3 AGCCAR1045A09 1 AGCCAR1046E07 1 AGCCAR1005B06 1 AGCCAR1006D08 2 AGCCAR1009B07 1 AGCCAR1017A07 4 AGCCAR1017E06 1 AGCCAR1018D08 1 AGCCAR1052G07 1 AGCCAR1018F02 6 AGCCAR1008C06 6 AGCCAR2018F05 0 AGCCAR1044F01 1 AGCCAR1014G02 3 AGCCAR1015G05 1 AGCCAR1027E01 2 AGCCAR1025B08 4 AGCCAR2010G02 0 AGCCAR1036D05 3 AGCCAR1043F05 1 AGCCAR1055H06 1 Other immune related molecular functions AGCCAR1010C07 1 AGCCAR1028H03 1 AGCCAR2016F04 0 AGCCAR1015B09 2

0 0 0 3 1

552

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600

80 70

500

60

Number of AS

50 300

40 30

200

20

Novel Genes (%)

400

100 10 0

0 300

400

500

600

700

800

900

1000

1500

2000

Mais

ORF Length (bp) Fig. 2. Distribution of Assembled Sequences (AS) according to longest ORF length and to percentage of novel genes. The AS considered as novel genes had no matches on NR database with e-value cut-off 1!10K6.

synthesized in hemocytes attached to the inner heart wall, and the hematopoiesis is induced subsequent to bleeding the animal [21]. Hemocyanins in circulating hemocytes have never been described, suggesting that the ESTs identified in A. gomesiana may be originated from hemocytes detached from heart wall during bleeding. The frequency of hemocyanin ESTs also demonstrates the efficiency of the cDNA library normalization. In the primary library hemocyanin represented 228 of every 1000 sequenced ESTs, followed by 17 in the normalized library. Therefore, the normalization reduced 13-fold the abundance of hemocyanins ESTs, and a similar result is expected for other abundant transcripts. Other abundant transcripts were identified for functional classes related to protein biosynthesis, cytoscheleton organization, energy metabolism, regulation of gene expression and DNA transposition (Fig. 1). Interestingly, one single transcript related to a lectin, AGCCAR1041B12, contained 27 ESTs from the primary library and other four single transcripts, each containing more than 10 ESTs, were assigned to unknown function. The functional annotation of spider transcripts aided the identification of several sequences related to immune response. These sequences represent various functional classes and may be involved in diverse processes of the immune response. The transcripts with similarity to hemocyanin subunits, besides the participation on oxygen transport, may participate in the immune response as

phenoloxidase (Table 4). In insects and crustaceans, phenoloxidases are enzymes responsible for melanin formation on wounds or invading organisms. This enzyme is synthesized as an inactive precursor, prophenoloxidase, and activated by the removal of a fragment by serine-proteases [4]. The phenoloxidases and hemocyanins from arthropods are similar in both amino acid sequences and physico-chemical properties of the active site [22]. Differing from other arthropods, the chelicerates do not have phenoloxidases, and some reports have demonstrated phenoloxidase activity by hemocyanins [23–25]. The hemocyanin of the tarantula spider E. californicum acquires phenoloxidase activity after limited proteolysis with trypsin or chymotrypsin [23], while in horseshoe crabs this conversion is observed with non-enzymatic interaction of hemocyanin with clotting factors or antimicrobial peptides [24,25]. A. gomesiana hemocyanin presents phenoloxidase activity when incubated with the detergent sodium dodecylsulfate (SDS), but no activity is found after incubation with trypsin or chymotrypsin (Daffre, personal communication). The similarity between the antimicrobial peptides gomesin from A. gomesiana and tachyplesin from horseshoe crabs [6] suggests that gomesin may induce the phenoloxidase activity in the spider hemocyanins as observed for its analog in horseshoe crabs [25]. The alignment of hemocyanin subunits’ sequences from A. gomesiana and E. californicum suggests the finding of an eighth hemocyanin sequence (AGCCAR1009A03, data not shown). The hemocyanin of E.

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californicum contains only seven subunits, which have been completely sequenced [21]. For each A. gomesiana hemocianin AS it was found one matching E. californicum subunit with sequence identity O96%, except for AGCCAR1009A03 that showed less than 50% sequence identity to any of the E. californicum subunits. We are investigating if product of this cDNA is a component of the A. gomesiana hemocyanin complex. Among the sequences related to antimicrobial proteins and peptides (Table 4) we found the sequences of the previously isolated antimicrobial peptides, gomesin, acanthoscurrin 1 and 2 [6–8]. A novel isoform of gomesin (AGCCAR1010G04) was found, presenting 89% amino acid sequence identity to the gomesin precursor, as well as the transcript of the original gomesin (AGCCAR1034B01). The alignment of these sequences shows that the novel gomesin differs from the original by five substitutions within the mature peptide region and other four substitutions in the precursor regions (Fig. 3). Two substitutions in the mature gomesin region conserved the hydrophobic nature (residues 30(Y to F) and 35(V to L), probably maintaining the hydrophobic patch of the gomesin structure [26]. In addition, two conservative substitutions were observed in the mature gomesin region (residues 31(K to R) and 32(Q to N)). The only nonconservative substitution in the mature gomesin region (residue 39(R to S)) is in the C-terminal of the mature peptide. In a structure-activity relationship study of gomesin, a similar substitution (R to A) did not affect the antimicrobial activity [27]. Therefore, the substitutions in the sequence of the novel gomesin indicate that this peptide presents antimicrobial activities similar to the original gomesin. The transcript identified with similarity to lysozyme may participate in bacterial killing through hydrolysis of its cell wall. Lysozyme gene expression in hemocytes was observed in shrimps [28] and ticks [29]. In mammals, the lysozymes are stored in granules of macrophages and neutrophils, and are involved in killing of Gram-positive bacteria [30].

Fig. 3. Sequence alignment of gomesin precursor isoforms. The spider Assembled Sequences (AGCC.) are compared with the previously described gomesin precursor [7]. Positions containing substitutions are marked in gray for conservative and in black for non-conservative.

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Several spider transcripts were found with similarity to serine proteases or serine protease inhibitors (Table 4). The proteases involved in invertebrate immune response are members of the chymotrypsin family that are produced as zymogens and activated by limited proteolysis [31]. These proteases are organized in cascades and controlled by specific inhibitors. Four different spider transcripts (AS) have similarity to proteases (Factor B and Proclotting Enzyme) that contain two conserved domains: CLIP and Chymotrypsin. These domains are observed in proteases of crustaceans and insects that activate phenoloxidase [4] or trigger the Toll signaling pathway through the cleavage of Spa¨etzle [32]. The clotting cascade from horseshoe crabs contains four proteases (Factors C, G, B and Proclotting Enzyme), three inhibitors (LICI 1, 2 and 3) and the clottable coagulogen. This cascade is started with the autoactivation of Factor C and G, induced by their binding to components of bacterial and fungal surfaces, respectively [2]. The finding of several transcripts with similarity to components of the horseshoe crabs clotting cascade (Factors C and B, Proclotting Enzyme, LICI 2) suggests the presence of a homologous cascade in A. gomesiana, that may participate in blood coagulation. Interestingly, no spider sequence with similarity to the clottable coagulogen was found. Besides the clotting cascade, the spider transcripts with similarity to Factor C could also act as hemocyte receptors. In horseshoe crabs, the Factor C present in the cell surface is activated by LPS, which triggers a cell-signaling cascade that leads to the degranulation of hemocytes [33]. The corresponding spider sequences contain the SUSHI conserved domains, which are the LPS binding regions [34]. The sequences of one serine protease and one protease inhibitor from the spider show similarity to components of the vertebrate complement system. The serine protease (AGCCAR1012C07) is similar to Factor B from the alternative pathway for complement activation, and the protease inhibitor (AGCCAR1003A01) is similar to complement factor C3. In vertebrates, these proteins form the C3 convertase of the alternative pathway, which cleave C3 to C3a and C3b. The C3b binds to the surface of pathogens to promote phagocytosis, and released C3a participates on chemotaxis and activation of leucocytes [35]. A complement-like protein from A. gambiae hemocytes also promotes phagocytosis of Gram-negative bacteria [36]. Another spider transcript (AGCCAR1002C10) presented a conserved domain of alpha-2-macroglobulins.

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These wide-spectrum protease inhibitors are present at high concentration in the plasma of horseshoe crabs, and assist the hemocytes in the clearance of circulating proteases [37]. Several transcripts with similarity to lectins were identified (Table 4). Lectins are proteins with sugar binding activity that, in animals, participate in the immune response as recognition molecules. Different glycoconjugates present on the surface of pathogens are recognized by specific lectins, and the absence of these glycoconjucates on host cells serves as a marker to distinguish between self and non-self [38]. The most abundant lectin transcript has 27 ESTs from the primary library (AGCCAR1041B12), and presents very significant similarity (1!10K104) to hemocytin from Bombyx mori hemocytes [39]. This protein presents sugar binding and hemocyte aggregating activities, and its synthesis is induced upon infection [39]. Ten spider transcripts present similarity to tachylectin 5 from horseshoe crab (Table 4). In this organism, this protein is found in the cell free hemolymph at high concentration (O10 mg/ml) and agglutinates bacteria. The tachylectin 5 presents a fibrinogen conserved domain, also found in vertebrate ficolins (involved in the lectin pathway of complement system activation) and fibrinogen (involved in blood clotting) [40]. In mollusks, fibrinogen-like proteins are present as different isoforms in the mucous glands of Limax flavus [41] and in the hemolymph of Biomphalaria glabrata, where they are produced after infection with a trematode [42]. The variety of transcripts containing fibrinogen domain found in the spider was also observed in the genomes of A. gambiae and D. melanogaster [18]. Some transcripts are related to other lectins (Table 4), such as: peptidoglycan recognition protein (PGRP), C type lectins, pentraxins and galectins. PGRPs show binding activity to bacterial cell wall, and are involved in the phagocytosis of Gram-negative bacteria in insects [43], activate the phenoloxidase cascade and trigger the Toll [44] and Imd signaling pathways [45]. The C-type lectin conserved domain was found in a protein present in the prophenoloxidase activating complex of Manduca sexta [46] and in two vertebrate proteins: colectins (involved in lectin pathway of complement activation) and selectins (involved in leukocyte traffic to infected tissues) [38]. Pentraxins are found in the serum of vertebrates, and in horseshoe crabs it is one of the most abundant proteins of the free cell hemolymph [38]. Some galectins are secreted from mammal macrophages and are involved in activation and recruiting of leukocytes [47].

Five spider transcripts were found with similarity to components of the Toll signaling pathway (Table 4), including the Toll receptor, the MyD88 Toll adaptor and Spa¨tzle. This pathway activates the synthesis of antimicrobial peptides in the fat body of D. melanogaster and the production of inflammatory mediators (citokines, chemokines) on vertebrate macrophages [48,49]. In vertebrates, the Toll receptors bind to microbial surface molecules directly, while in D. melanogaster the activated Spa¨tzle is the Toll target [48]. After the binding, the Toll receptors interact with MyD88 and start the intracellular signaling pathway that regulates the transcription of immune related genes. The spider sequence with similarity to Spa¨tzle suggests a Toll activation mechanism similar to the one observed in D. melanogaster. Finally, transcripts with similarity to peroxinectins and transglutaminases were found (Table 4). Peroxinectins are adhesion molecules stored in hemocyte granules of crustaceans. Following the degranulation induced by infection, the released peroxinectins are activated by serine-proteases to stimulate cell adhesion, phagocytosis and encapsulation. The sequence of this protein contains a peroxidase domain followed by a C-terminal domain, which is involved in adhesion [50]. Transglutaminases are enzymes that catalyze the covalent binding of glutamine residues to lysine residues or other primary amines. In crustaceans, this protein is released from hemocytes and polymerizes the clot protein, forming a stable clot around the wound [51]. The horseshoe crab’s transglutaminase promotes the attachment of the hemocytes over the clot through the covalent binding of a hemocyte surface protein, proxins, to coagulin [52]. The sequences from the spider A. gomesiana produced for this work increased immensely the diversity of araneae genes deposited in public databases, specially the immune related genes. These sequences will be an useful material for comparative and evolutionary studies. In addition, this material will support the characterization of the spider hemocyte proteome by a mass spectrometry approach (in progress). Acknowledgements This work was supported by grants from Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) (Brazil) and Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) (Brazil). We are thankful to Dr Ana Teresa R. de Vasconcelos (LNCC/MCT) for processing the Interpro searches,

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Almir Samuel Zanca (CBMEG/UNICAMP) for DNA sequencing, Renato Vicentini dos Santos (CBMEG/UNICAMP) and Apua˜ Ce´sar de Miranda Paquola (ICB/USP) for valuable discussions.

[14]

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