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RECTED PROOF

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,

Joao Setubal e,2, Sirlei Daffre a,*

a Departamento de Parasitologia, Instituto de Ciencias Biomedicas, Universidade de Sao Paulo, Avenue Prof. Lineu Prestes,

1374, CEP 05508-900 Sao Paulo, SP, Brazilb Laboratorio de Artropodes, Instituto Butantan. CEP 05503-900 Sao Paulo, SP, Brazil

c Department of Pediatrics and Biochemistry, University of Iowa, Iowa City, IA 52242, USAd Instituto da Computacao, Centro de Biologia Molecular e Engenharia Genetica, Universidade Estadual de Campinas,

CEP 13083-970 Campinas, SP, Brazile Laboratorio de Bioinformatica, Instituto da Computacao, Universidade Estadual de Campinas, CEP 13083-970 Campinas, SP, Brazil

Received 5 July 2005; revised 28 August 2005; accepted 2 September 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

UNCOR

0145-305X/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.dci.2005.09.001

* Corresponding author. Tel.: C55 11 309 17272; fax: C55 11 309

17417.

E-mail address: sidaffre@icb.usp.br (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.

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1. Introduction

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 encapsula-

tion 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 anti-

microbial 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 on 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 a 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 amida-

tion. Acanthoscurrin is released into the cell free

hemolymph following immune challenge [8]. Mygalin

is a bis-acylpolyamine N1,N8-bis(2,5-dihydroxylben-

zoil)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 data-

bases. The sequencing of expressed sequence tags

(ESTs) is specially useful, since it allows simul-

taneously 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 phylogeneti-

cally 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

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TED PROOF

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 Therapho-

sidae family that is distributed in southeast Brazil.

Adults from this species are medium-sized (approxi-

mately 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 (Sao Paulo, Brazil)

by citizens of Sao Paulo and neighbor towns, where

they were kept.

2.2. Hemocyte isolation and RNA extraction

Hemolymph was collected from 15 spiders (approxi-

mately 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 pre-

pared 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 double-

stranded cDNAs longer than 350 bp were size selected

by gel filtration on a Bio-Gel A-50M column (Bio-

Rad), 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.

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.

2.4. 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!10K

30), using a database of possible contaminants

(ribosomal RNA, E. coli genome, mitocondrial and

plasmid sequences, all from Genbank). Sequences

DCI 848—7/10/2005—02:17—-[-no entity-]-—166836—XML MODEL 3dc+ – pp. 1

containing more than 200 bp after trimming (quality

and vector) and not identified as contaminants were

considered as high quality sequences.

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).

TED PROOF

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 GeneOntol-

ogy (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 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

–12

C

Table 1

Statistics of expressed sequence tags (EST) from cDNA libraries of

Acanthoscurria gomesiana hemocytes

Normalized Primary Total

Sequenced ESTs 5760 1824 7584

High quality ESTs 5156 1634 6790

Discarded ESTs 604 190 794

Average length of

sequencesa

524.7 651.0

Discarded reason

Low qualityb 540 100 640

Sizec 6 4 10

No insert 13 1 14

Contaminants

E. coli 7 1 8

Mitocondrial 1 1 2

Ribossomal 14 1 15

Vector 23 82 105

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

trimming.

Table 2

Automatic functional annotation statistics of assembled sequences

(AS)

Database ASs with match on

Database

ASs annotated with

GO

NRa 2192 –

Swisprot C

TREmbl 2171 1937

Interpro 873 378

Imune Drosophila 106 106

Imune Lımulus 91 91

Total 2411 1971

a Protein sequence database from NCBI.

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UNCORRE

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 avail-

able for entries of SwissprotCTREmbl and Interpro

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TED PROOF

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 (AGC-

CAR1041B12, 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 dedi-

cated 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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PROOF

356

92

5548 47

35 35 34 3430

26 24 23

504

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100

1000

hemoc

yanin

struc

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cons

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cytos

kelet

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some

recep

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DNA bind

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suga

r bind

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RNA bind

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trans

posa

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cell

adhe

sion m

olecu

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pepti

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GTP bind

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trans

lation

regu

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cripti

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unkn

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uncti

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Prim

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Lib

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EST

s

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.

Table 3

GeneOntology (GO) terms related to the immune system

GO:0004252 Serine-type endopeptidase

GO:0003810 Protein-glutamine gamma-glutamyltransferase

GO:0003796 Lysozyme

GO:0006961 Antibacterial humoral response (sensu Inverteb-

rata)

GO:0008329 Pattern recognition receptor

GO:0003823 Antigen binding

GO:0005530 Lectin

GO:0017114 Wide-spectrum protease inhibitor

GO:0004867 Serine protease inhibitor

GO:0004888 Transmembrane receptor

GO:0003700 Transcription factor

GO:0006952 Defense response

GO:0004503 Monophenol monooxygenase

GO:0003793 Defense/immunity protein

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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

DCI 848—7/10/2005—02:17—-[-no entity-]-—166836—XML MODEL 3dc+ – pp. 1

TED 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

–12

UNCORRECTED PROOF

Table 4

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

AS code # of ESTsa

Norm. Prim. Gi Description Organism E-value

Phenoloxidases

AGCCAR1001B07 9 59 122792 Hemocyanin A chain Eurypelma californicum 0

AGCCAR1019B12 8 35 20138395 Hemocyanin B chain Eurypelma californicum 0

AGCCAR1008E07 5 21 20138398 Hemocyanin C chain Eurypelma californicum 0

AGCCAR1004D09 9 71 17376946 Hemocyanin D chain Eurypelma californicum 0

AGCCAR1013F04 4 40 70624 Hemocyanin E chain Eurypelma californicum 0

AGCCAR1001H09 34 39 20138397 Hemocyanin F chain Eurypelma californicum 0

AGCCAR1009A03 5 4 20138397 Hemocyanin F chain Eurypelma californicum 1!10K180

AGCCAR1003A06 13 103 20138396 Hemocyanin G chain Eurypelma californicum 0

Antimicrobial peptides and proteins

AGCCAR1001C06 7 2 52782738 Acanthoscurrin 1 precursor A. gomesiana 2!10K30

AGCCAR1006D12 2 1 52782737 Acanthoscurrin 2 precursor A. gomesiana 2!10K30

AGCCAR1034B01 1 1 28445738 Gomesin precursor A. gomesiana 3!10K41

AGCCAR1010G04 1 0 28445738 Gomesin precursor A. gomesiana 6!10K38

AGCCAR1035C06 2 0 1085148 lysozyme S Drosophila melanogaster 2!10K16

Serine proteases

AGCCAR1044G07 1 0 542517 coagulation factor B precursor Tachypleus tridentatus 5!10K50

AGCCAR1057G02 1 0 542517 coagulation factor B precursor Tachypleus tridentatus 1!10K58

AGCCAR2017E09 0 1 542517 coagulation factor B precursor Tachypleus tridentatus 2!10K54

AGCCAR1026C07 5 2 129688 Proclotting enzyme precursor Tachypleus tridentatus 1!10K81

AGCCAR1003H07 1 0 18542425 factor C precursor Tachypleus tridentatus 3!10K75

AGCCAR1006G12 1 1 913964 factor C Carcinoscorpius rotundicauda 1!10K100

AGCCAR1011F11 1 1 7387836 Limulus clotting factor C precursor Carcinoscorpius rotundicauda 1!10K129

AGCCAR1012C07 2 0 3928787 factor B SpBf Strongylocentrotus purpuratus 8!10K50

AGCCAR1013H11 6 0 1817554 limulus factor D Tachypleus tridentatus 1!10K117

AGCCAR1006A04 3 0 25989209 coagulation factor-like protein 3 Hyphantria cunea 5!10K60

AGCCAR1006C09 4 0 23266416 serine protease PC5-A Rana esculenta 8!10K44

AGCCAR1019E09 2 0 28194028 prothrombin precursor Takifugu rubripes 2!10K50

AGCCAR2001D06 0 1 28194028 prothrombin precursor Takifugu rubripes 1!10K09

AGCCAR1020D11 3 0 847761 SPC3 Branchiostoma californiense 3!10K56

AGCCAR1013F12 3 0 26332511 unnamed protein product Mus musculus 2!10K11

Serine protease inhibitors

AGCCAR2016C11 0 1 17223666 serine proteinase inhibitor serpin-3 Rhipicephalus appendiculatus 9!10K30

AGCCAR1001D03 10 2 1078956 intracellular coagulation inhibitor

type 2 (LICI 2)

Tachypleus tridentatus 5!10K65

AGCCAR1003H02 7 3 1078956 intracellular coagulation inhibitor

type 2 (LICI 2)

Tachypleus tridentatus 2!10K65

AGCCAR1005F08 2 0 34881479 similar to serine (or cysteine) pro-

teinase inhibitor

Rattus norvegicus 1!10K47

AGCCAR1002C10 3 4 7521905 alpha-2-macroglobulin Limulus sp. 1!10K139

AGCCAR1003A01 5 0 13928544 complement component C3 Branchiostoma belcheri 8!10K49

AGCCAR1013G08 1 0 20302747 unknown Branchiostoma floridae 1!10K56

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UNCORRECTED PROOF

AGCCAR1013A03 1 0 25149822 thrombospondin Caenorhabditis elegans 6!10K11

AGCCAR1056G07 3 0 25149822 thrombospondin Caenorhabditis elegans 4!10K11

AGCCAR1037F11 1 3 13346812 thrombospondin Haemonchus contortus 2!10K35

AGCCAR2006A03 0 1 22901764 Kunitz-like protease inhibitor pre-

cursor

Ancylostoma caninum 1!10K31

AGCCAR1013B05 1 0 2133556 cystatin precursor Tachypleus tridentatus 2!10K14

Lectins

AGCCAR1041B12 2 27 1346296 Hemocytin precursor Bombyx mori 1!10K104

AGCCAR1010G07 1 0 6630613 Hemolectin Drosophila melanogaster 1!10K21

AGCCAR1012D08 2 0 21666693 hemolectin-like protein Penaeus monodon 2!10K10

AGCCAR1053A05 1 0 1346296 Hemocytin precursor Bombyx mori 5!10K31

AGCCAR1036E09 3 0 1346296 Hemocytin precursor Bombyx mori 1!10K25

AGCCAR1045A09 1 0 17942826 Tachylectin 5a Tachypleus tridentatus 4!10K68

AGCCAR1046E07 1 0 5851893 Tachylectin 5a Tachypleus tridentatus 7!10K75

AGCCAR1005B06 1 0 17942826 Tachylectin 5a Tachypleus tridentatus 2!10K79

AGCCAR1006D08 2 0 5851893 Tachylectin 5a Tachypleus tridentatus 6!10K71

AGCCAR1009B07 1 0 17942826 Tachylectin 5a Tachypleus tridentatus 6!10K61

AGCCAR1017A07 4 1 5851897 Tachylectin 5b Tachypleus tridentatus 7!10K76

AGCCAR1017E06 1 0 17942826 Tachylectin 5a Tachypleus tridentatus 1!10K73

AGCCAR1018D08 1 0 17942826 Tachylectin 5a Tachypleus tridentatus 5!10K72

AGCCAR1052G07 1 0 5851893 Tachylectin 5a Tachypleus tridentatus 1!10K70

AGCCAR1018F02 6 1 5851893 Tachylectin 5a Tachypleus tridentatus 3!10K80

AGCCAR1008C06 6 0 31217088 ENSANGP00000012978 Anopheles gambiae 2!10K44

AGCCAR2018F05 0 1 27808640 peptidoglycan recognition protein Bos Taurus 6!10K36

AGCCAR1044F01 1 0 4878035 neurocan core protein precursor Gallus gallus 6!10K18

AGCCAR1014G02 3 0 4505245 mannose receptor C type 1 precursor Homo sapiens 1!10K28

AGCCAR1015G05 1 0 4505245 mannose receptor C type 1 precursor Homo sapiens 2!10K23

AGCCAR1027E01 2 0 4505245 mannose receptor C type 1 precursor Homo sapiens 1!10K25

AGCCAR1025B08 4 0 12738842 polydomain protein Mus musculus 4!10K49

AGCCAR2010G02 0 1 84651 C-reactive protein chain 3.3 Limulus sp. 2!10K15

AGCCAR1036D05 3 2 6981152 lectin, galactose binding Rattus norvegicus 3!10K18

AGCCAR1043F05 1 0 9857647 galectin LEC-4 Caenorhabditis elegans 5!10K08

AGCCAR1055H06 1 0 2833353 Galectin-4 (Lactose-binding lectin 4) Sus scrofa 4!10K16

Other immune related molecular functions

AGCCAR1010C07 1 0 22651842 Toll-related protein; AeTehao Aedes aegypti 5!10K17

AGCCAR1028H03 1 1 22651842 Toll-related protein; AeTehao Aedes aegypti 9!10K29

AGCCAR2016F04 0 1 22651842 Toll-related protein; AeTehao Aedes aegypti 1!10K40

AGCCAR1015B09 2 1 9965396 Toll/IL-1 receptor binding protein

MyD88

Xenopus laevis 7!10K34

AGCCAR1031C10 1 0 24650493 spatzle CG6134-PI Drosophila melanogaster 2!10K10

AGCCAR1009B05 3 1 15718457 Peroxinectin Penaeus monodon 1!10K89

AGCCAR1049A04 1 0 345423 protein-glutamine gamma-glutamyl-

transferase (EC 2.3.2.13)

Tachypleus tridentatus 2!10K58

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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ORF Length (bp)

Num

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el G

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)

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.

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UNCORRE

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, regu-

lation 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

CI 848—7/10/2005—02:17—-[-no entity-]-—166836—XML MODEL 3dc+ – pp. 1–

TED PROphenoloxidase (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 hemocya-

nin with clotting factors or antimicrobial peptides

[24,25]. A. gomesiana hemocyanin presents phenolox-

idase 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 (AGC-

CAR1009A03, data not shown). The hemocyanin of E.

12

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921

922

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924

925

926

ORRE

californicum contains only seven subunits, which have

been completely sequenced [21]. For each A. gomesi-

ana 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 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 substi-

tutions were observed in the mature gomesin region

(residues 31(K to R) and 32(Q to N)). The only non-

conservative 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 substi-

tutions 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].

UNCFig. 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.

DCI 848—7/10/2005—02:17—-[-no entity-]-—166836—XML MODEL 3dc+ – pp. 1

927

928

929

930

931

932

933

934

935

936

TED PROOF

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 Chymo-

trypsin. These domains are observed in proteases of

crustaceans and insects that activate phenoloxidase [4]

or trigger the Toll signaling pathway through the

cleavage of Spaetzle [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 (AGC-

CAR1003A01) 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) pre-

sented a conserved domain of alpha-2-macroglobulins.

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1038

1039

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UNCORRE

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 signifi-

cant 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 tachy-

lectin 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 Biompha-

laria 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 path-

way 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].

CI 848—7/10/2005—02:17—-[-no entity-]-—166836—XML MODEL 3dc+ – pp. 1–

TED PROOF

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 Spatzle. This pathway activates the synthesis of

antimicrobial peptides in the fat body of D. melanoga-

ster 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 Spatzle 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 Spatzle

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). Perox-

inectins 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 Fundacao

de Amparo a Pesquisa do Estado de Sao Paulo

(FAPESP) (Brazil), Conselho Nacional de Desenvolvi-

mento Cientıfico e Tecnologico (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/U-

NICAMP) and Apua Cesar de Miranda Paquola

(ICB/USP) for valuable discussions.

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