RESEARCH REPORT

JOURNAL OF INTERFERON & CYTOKINE RESEARCH Volume 29, Number 8, 2009 © Mary Ann Liebert, Inc. DOI: 10.1089/jir.2008.0078

Identification of a Putative Invertebrate Helical Cytokine Similar to the Ciliary Neurotrophic Factor/Leukemia Inhibitory Factor Family by PSI-BLAST-Based Approach Gong Cheng,1,* Xin Zhao,1,* Zuofeng Li,1,2 Xinyi Liu,3 Weiyao Yan,1 Xiaoyan Zhang,3 Yang Zhong,1,2 and Zhaoxin Zheng1

Most of our knowledge of helical cytokine-like molecules in invertebrates relies on functional assays and similarities at the physicochemical level. It is hard to predict helical cytokines in invertebrates based on sequences from mammals and vertebrates, because of their long evolutionary divergence. In this article, we collected 12 kinds of fish cytokines and constructed their respective consensus sequences using hidden Markov models; then, the conserved domains region of each consensus sequence were further extracted by the SMART tool, and used as the query sequence for PSI-BLAST analysis in Drosophila melanogaster. After two filtering processes based on the properties of helical cytokines, we obtained one protein named CG14629, which shares 25% identities/46% positives to fish M17 cytokine in the half length of the N-terminus. Considering the homology between M17 and LIF/CNTF (leukemia inhibitory factor/ciliary neurotrophic factor), and the close relationship between Dome, the putative cytokine receptor in Drosophila cells, and LIFR/CNTFR (LIF receptor/CNTF receptor), the results suggest that CG14629 is a good candidate for the helical cytokine ortholog in D. melanogaster.

Introduction

H

elical cytokines are secreted mediators involved in extracellular signal mechanisms, playing significant roles in balancing host immune responses. All helical cytokines fold into a bundle of four to six α-helices, and their primary amino acid sequences share little to no sequence similarity (Bazan 1990b; Thoreau and others 1991). In recent years, the cytokines, which have been well characterized within mammals, have begun to be cloned and sequenced within nonmammalian vertebrates, especially in fish (Altmann and others 2003; Fujiki and others 2003; Lutfalla and others 2003; Kaiser and others 2004; Bird and others 2005; Huising and others 2005; Krause and Pestka 2005; Gunimaladevi and others 2006; Huising and others 2006; Igawa and others 2006). In the invertebrate chordate Ciona intestinalis, the genome analysis showed the existence of orthologs of JAK/STAT signaling proteins (Hino and others 2003), but ascidian (sea squirts, a class in the Tunicata subphylum) helical cytokine or cytokine receptor genes have not been described to date. These findings imply that the origin of helical cytokines predates vertebrates.

Previous works have described some experimental traces suggesting that cytokine-like molecules exist in invertebrate animals (Hughes and others 1992; Paemen and others 1992; Ottaviani and others 1995; Wajant and others 1998). However, no further molecular data are provided in those works to identify which cytokines exert their biological activity in immunocytochemical analysis. In Drosophila melanogaster, the comparative genomic analysis did not reveal the existence of any homologs to vertebrate cytokines (Kaiser and others 2004). However, compelling evidence has emerged for the involvement of cytokine signaling in various physiological processes. A Drosophila JAK/STAT pathway was first identified through its role in embryonic segmentation. The Drosophila JAK is named hopscotch (hop), and shares 27% overall amino acid identity with human JAK2 (Binari and Perrimon 1994). The single Drosophila STAT identified to date is named STAT92E, and shares 37% amino acid identity with human STAT5 (Hou and others 1996; Yan and others 1996). BLAST searches showed that two loci (CG14225 and CG14226) were predicted to encode probable type I cytokine receptor chains (Boulay and others 2003).

1

School of Life Science, Fudan University, Shanghai, People’s Republic of China. Shanghai Center for Bioinformation Technology, Shanghai, People’s Republic of China. 3 School of Life Science and Technology, Tongji University, Shanghai, People’s Republic of China. *These authors contributed equally to this work. 2

461

462 In D. melanogaster, the unpaired (upd) gene has been identified as a ligand that is capable of activating JAK/STAT signaling (Gergen and Wieschaus 1986; Harrison and others 1998), and then the same phenotype was observed in the absence of the gene activity of dome, hop, and upd. In light of this, the protein was considered the ligand of the Dome receptor (Hombria and Brown 2002). Based on the homology of the JAK/STAT signaling proteins (JAKs, STATs, and cytokine receptors) between vertebrate and Drosophila, we speculated that the ligand of the Dome receptor should have sequence homology to one vertebrate cytokine. However, the Upd characteristics are considered to be unlike those any vertebrate helical cytokine, and no direct data proved that Upd could bind to the Dome receptor (Harrison and others 1998). Could Upd be the genuine ligand of Drosophila JAK/STAT signaling, or only an intermediate to induce the expression of the real Drosophila helical cytokine? Ottaviani and colleagues have reported a gene encoding for a putative helical cytokine in D. melanogaster, which is significantly increased after immune stimulation (Malagoli and others 2007). In this work, we collected the fish cytokines and constructed the consensus sequences, then reexamined the Drosophila genome by PSI-BLAST. The result showed that one locus in Drosophila genome has significant similarity to fish M17, which is a member of LIF/CNTF (leukemia inhibitory factor/ciliary neurotrophic factor) family.

Materials and Methods Identifi cation of cytokine-like molecules from the Drosophila RefSeq protein database The starting point for the search was the collection of sequences of 12 helical cytokine families (EPO, GH, IFN, IFN-γ, IL-6, IL-10, IL-11, IL-12, PRL, SL, Leptin, and M17) in different fishes. These sequences are deposited in GenBank database (http://www.ncbi.nlm.nih.gov) (GenBank accession no. shown in Appendix) by various authors (Kaiser and others 2004; Krause and Pestka 2005; Huising and others 2006). The sequences of each cytokine were aligned and a FASTA format was using ClustalW, then a consensus sequence derived from multiple sequence alignments was constructed by hidden Markov models (HMMER, Version 2.3.2) (Churchill 1989; Eddy 1995). The conserved domains of each consensus sequence were further confirmed by the SMART tool and, finally, we extracted all the conserved cytokine domain sequences to a single FASTA file. The conserved domain sequences were PSI-BLAST with BLOSUM45 matrix against the Drosophila RefSeq protein database, respectively (http://www.ncbi.nlm.nih.gov and http://www.flybase. org/). All blast results were screened by the threshold 0.005, and the proteins matching the properties of vertebrate helical cytokines were identified for further analysis.

Sequence analysis The Mw (molecular weight) and pI (isoelectric point) were computed online (http://www.expasy.ch/tools/pi_ tool.html). The signal sequences were predicted using the SignalP program (www.cbs.dtu.dk/services/SignalP). The protein sequence alignment and percentage identities/positives were calculated using the DNAStar MegAlign software (DNASTAR, Inc., Madison, WI). The secondary structure

CHENG ET AL. was predicted and analyzed using the Jpred (http://www. compbio.dundee.ac.uk/~www-jpred/advanced.html) and PROF (http://www.aber.ac.uk/~phiwww/prof/) Web services. The conserved domain was predicted by Simple Modular Architecture Research Tool (SMART: http://smart. embl-heidelberg.de/) (Schultz and others 1998; Letunic and others 2006).

Results and Discussion Why were only fi sh cytokines chosen to identify invertebrate cytokines? From Porifera to Coelenterate, the metazoan diverges to Protostomia and Deuterostomia. The Protostomia branch evolves to mollusca and arthropoda; Deuterostomia is the other branch, which produces protochordata and vertebrates (Fig. 1). Under the plausible assumption that helical cytokines constitute a monophyletic group, the cytokine ancestral gene produces an enormous helical cytokine family in the Deuterostomia branch (Bazan 1990a). It is thus natural to ask how the ancestral gene evolves into the Protostomia branch? Helical cytokine was never identified from Protochordata and Echinodermata, so the vertebrate helical cytokine may be formed between the Protochordata stage and the vertebrate stage and appears first in fish, which, in phylogenetic time, is coincident with the period of formation of arthropoda (450–550 million years) (Fig. 1). As a hypothesis, all helical cytokines originate from an ancestral cytokine gene. If the ancestral genes kept the same phylogenetic steps in Deuterostomia and Protostomia, the time of formation of helical cytokines could be consistent within these two branches. If the helical cytokines exist in invertebrate, we consider that the fish cytokines would have the most homology to them. However, it is difficult to identify helical cytokines in invertebrate based on the known sequences of vertebrate cytokines, due to the different evolutionary rates on different branches and sites. The high divergence of the helical cytokines also limits the applicability of similarity-based gene finding methods, such as GenomeScan (Yeh and others 2001), which use homologous protein sequences to guide the gene finding process (Conklin and others 2005). However, we could predict those invertebrate cytokine genes using ancestral mimic genes. To recover the ancestral helical cytokine gene maximally, we chose the helical cytokine sequences in fish, which are the lowest form of vertebrates. In this work, we only choose fish cytokines, but not mammal or all vertebrate ones, as templates to identify the helical cytokines in Drosophila.

Strategy and results of identifi cation The process that we developed has the objective to discover new candidates for helical cytokine proteins within a comprehensive Drosophila protein database. Considering the rapid genetic evolution resulting in the large genomic discrepancy between vertebrates and invertebrates, it is hard to identify invertebrate helical cytokine by means of largescale comparative genomic analysis. Therefore, instead, we chose the sequence search by PSI-BLAST analysis. The complete process of identification is schematically shown in Figure 2.

463

PUTATIVE INVERTEBRATE HELICAL CYTOKINE 0

Mammalia

Aves 250

Reptilia

Time to Present (million years)

Amphibia

Pisces Agnatha

450

VERTEBRATES Protochordata (Urochordata, Cephalochordata) Hemichordata

Arthropoda

FIG. 1. Simplified phylogenetic tree of animals, based on previous work.

Mollusca

Annelida Echinodermata

Nematoda

700

Platyhelminthes

DEUTEROSTOMIA

PROTOSTOMIA

Coelenterata Porifera 900

In Module 1 (sequence collection and consensus sequence construction), the protein sequences of 12 fish helical cytokines families were collected from the Genbank database based on published articles (Kaiser and others 2004; Krause and Pestka 2005; Huising and others 2006). Multiple sequence alignment files of each cytokine were created to produce the consensus sequences by hidden Markov models, which takes the relative frequency of every amino acid at each position into account and thus provides a better representation of an alignment.

MultiFasta Files of Fish Cytokines Sequences

HMMER

In Module 2 (conserved domain prediction and exploring PSI-BLAST), based on the 12 consensus sequences, the conserved domains of each cytokine were predicted by SMART tool. The conserved domain sequences were used as the templates of the PSI-BLAST analysis in the D. melanogaster protein database (Table 1). In Module 3 (the blast results filtration), we obtained 1,082 Drosophila protein sequences using PSI-BLAST analysis in the upper work. Considering the sequence properties of vertebrate helical cytokine, the invertebrate cytokine should

Consensus Sequences of Each Cytokines

SMART

Domain Sequences

Putative Cytokine-like Domain in Invertebrate

Filtration

PSI-BLASTs in Invertebrate Protein Database

FIG. 2. Schematic representation of the validating PSI-BLAST process for the identification of new helical cytokine proteins.

464

CHENG ET AL. A The Secondary Structure of CG14629 and M17

B PSI-BLAST Result Between CG14629 and M17 Consensus Sequence

M17 Consensus Sequencene CG14629 M17 Consensus Sequencene CG14629 Identities = 25% Positives = 46%

C Putative Conserved Domain of M17 Consensus Sequence 50

100

150

M17 Consensus Sequence CNTF Conserved Domain

200

216

CNTF

Chicken CNTF Identities = 28% Positives = 50%

M17

Fish LIF Identities = 80% Positives = 88%

D

LIFR Dome tities

Iden

CNTFR

s=

%

21

tie

ti en

Id

s ive

=

% = 26

es =

iv Posit

41%

% 39

sit

Po

FIG. 3. (A) The secondary structure of CG14629 and M17. (B) PSI-BLAST result of identifying a novel helical cytokine in Drosophila melanogaster. (C) The relationship among M17, chicken CNTF, and fish LIF. (D) Domain structure of Drosophila Dome and vertebrate IL-6 receptor family members LIFR/CNTFR. The numbers represent the percentages of amino acid identities or positives between cytokine-binding modules (CBM) of the Dome receptor and LIFR/CNTFR. As vertebrate helical cytokine receptors, Dome has two cytokine-binding modules. The first CBM of the Dome receptor has two pairs of conserved cysteines, and the second CBM has a partially conserved WSxWS motif that has been shown in vertebrates to be essential for the signaling process.

465

PUTATIVE INVERTEBRATE HELICAL CYTOKINE meet two qualifications: a peptide length below 450 amino acids (Upd is considered as a ligand to Dome receptor; the length of Upd is 413AA) and an α-helix structure. After the first filtration by the qualification of peptide length, only 312 sequences remained. Then, the secondary structure was predicted in all remaining sequences, where 311 of these could be predicted by Jpred Web server, and the one without a Jpred result was analyzed by PROF instead. Finally, we got one D. melanogaster protein, CG14629 (NP_569881), which matched the upper two qualifications and had 25% identities/46% positives to M17 in 101 amino acids of the N-terminus (the M17 consensus sequence is 216 AA in total) (Fig. 3A).

The relationship among CG14629, M17, LIF, and CNTF M17 is a class-I helical cytokine common to most teleost fish, and is a member of the LIF/OSM/CNTF family (Fujiki and others 2003; Huising and others 2006). The alignment showed that the M17 consensus sequence has 80% identities/88% positives to zebra fish LIF and 28% identities/50% positives to chicken CNTF, and the SMART analysis showed that the M17 consensus sequence belongs to the putative CNTF conserved domain (Fig. 3B). Based on the upper analysis, we considered that D. melanogaster CG14629 protein has a close relationship to the M17/LIF/ CNTF family. On the Drosophila predicted protein genome database, BLAST searches showed that two loci (CG14225 and CG14226) were predicted to encode probable type I cytokine receptor chains (Boulay and others 2003). CG14226,

Table 1.

called Dome receptor, has five extracellular fibronectin type III (FNIII) modules, of which the first and second show homology to the cytokine-binding modules (CBM) of LIFR and CNTFR (Fig. 3C). The first CBM of the Dome receptor has two pairs of conserved cysteines, and the second CBM has a partially conserved WSxWS motif that has been shown in vertebrates to be essential for the signaling process (Hombria and Brown 2002) (Fig. 3D). From the homological analysis, we have a well-founded belief that the ligand of the Dome receptor should have a relationship to LIF or CNTF, which were the members of IL-6 family. The upper relationship for CG14629 and M17/LIF/CNTF could verify that the CG14629 is a good candidate for the ligand of the Dome receptor.

The characterization of CG142629 CG14629 codes a 319 amino acid precursor protein, which contains a signal peptide of 20 amino acids, and the mature protein possesses a calculated molecular mass of 32.9 kDa and a predicted theoretical isoelectric point (pI) of 5.13, with three potential N-linked glycosylation sites. The investigation of its secondary structure indicates that CG14629 has strong putative α-helical content: the helical percentage is 59.1% and the sheet percentage is only 1.7%. The structural and biochemical parameters showed that CG14629 has a close relationship to vertebrate helical cytokines (Table 2). The SMART and BLAST analysis suggested that the cytokine-like protein CG14629 belongs to a conserved domain DUF1397 family. The proteins of this family were found to exist in insects, and are annotated as being a

Comparison of Structural and Biochemical Parameters

Drosophila melanogaster

Primary structure Precursor (AA) Signal peptide (AA) Mature peptide (AA) Mature peptide Molecular mass (KDa) Cysteine (AA) Prolinea (%) Leucinea (%) Positive chargeda (%) Isoelectric point Secondary structure Helical percenta,b (%) Sheet percenta,c (%) Loop percenta (%) a

M17

LIF

CNTF

CG14629

CG11378

CG9917

Upd

GF

PF

CP

ZF

Mu

Hu

CH

Mu

Hu

319 20

312 24

301 25

413 27

223 36

NA NA

215 29

215 32

203 23

202 22

195 198 200 None None None

299

288

276

386

187

175

186

183

180

180

195

198

200

32.9

31.6

31.0

44.1

21.0

19.1

21.0 20.8

19.9

19.7

21.3

22.6

22.9

12 4.7 14.1 11.0

12 5.2 8.7 11.5

12 2.5 9.1 10.1

2 3.4 7.5 21.5

6 6.4 11.2 14.4

4 10.3 8.6 14.3

6 6 5.9 6.6 10.8 10.9 15.6 15.8

6 6.7 11.1 12.8

6 6.1 13.9 13.3

1 5.1 16.4 13.3

1 2.5 12.6 15.7

1 3.5 13.0 15.5

5.13

5.53

4.93

11.49

8.92

9.59

9.22 9.41

9.20

9.28

5.3

6.05

6.35

59.1

54.5

60.1

47.4

61.5

50.3

59.7 65.0

58.3

55.6

59.5

61.0

58.0

1.7 39.2

5.2 40.3

3.3 36.6

4.4 48.2

0 38.5

2.9 46.8

0 0 40.3 35.0

0 41.7

2.8 41.6

0 40.5

0 31.9

1.5 40.5

Percentage of amino acid in the mature peptide. Helical percent: The amino acid percentage of α-helix in the mature peptide. c Sheet percent: The amino acid percentage of β-sheet in the mature peptide. Abbreviations: GF, goldfish; PF, puffer fish; CP, common carp; ZF, zebra fish; Mu, mouse; Hu, human; CH, chicken; NA, not available. b

466

CHENG ET AL. Table 2.

Cytokine

SMART Results of Helical Cytokines Start position

End position

SMART E value

SMART domain

21 6 1 88 — 56 38 45 6 16 10 18

213 180 200 300 — 207 177 191 205 166 213 227

5.80e-03 1.30e-24 1.60e-53 2.40e-04 — 8.63e-03 3.62e-25 9.47e-01 5.40e-05 4.60e-04 3.40e-76 1.50e-68

CNTF EPO-TPO Hormone-1 Interferon — IL6 IL10 IL6 IL12 Leptin Hormone-1 Hormone-1

M17 EPO GH IFN IFN-γ IL-6 IL-10 IL-11 IL-12 Leptin PRL SL

hemolymph glycoprotein precursor and synthesized in the fat body (Samaraweera and Law 1995; Conklin and others 2005). As we know, the fat body is an important immune organ in insects. Moreover, we evaluated CG14629 as a cytokine using the CytoPred Web site (http://www.imtech.res.in/raghava/ cytopred/index.html). The result indicates that CG14629 belongs to the cytokine family based on the SVM (support vector machine) method (data not shown).

In the D. melanogaster protein database, we found other two proteins (CG11378 and CG9917) that also included the DUF1397 domain. These three DUF1397 proteins are all located on chromosome X (Fig. 4A) and share ~30% identities in protein sequences. CG14629 and CG9917 are intronless genes, but CG11378 consists of two exons (Fig. 4B).

Conclusion In this work, we identified a putative cytokine-like protein CG14629 by PSI-BLAST analysis. Our evidence to support this protein as a good candidate for an ortholog of helical cytokines in invertebrate is as follows: First, CG14629 matches the properties of helical cytokine sequences. It has a suitable length of protein sequence and consists of α-helices. Second, CG14629 has high relative similarity to the IL-6 family cytokines M17/LIF/CNTF. As the known cytokine receptor, Dome also shows partial homology to LIFR and CNTFR. CG14629 could be a good candidate for the ligand of Dome receptor. Furthermore, CG14629 contains the DUF1397 domain. The proteins of this family are annotated as being hemolymph glycoprotein precursors, and synthesized in the fat body, an important insect immune organ. This work is the first report on an invertebrate cytokinelike sequence sharing relative similarity to vertebrate helical cytokines, and should supply new evidence for the existence of helical cytokines in invertebrates.

A Mbp 0 1 2 3 4 5

CG14629 Mbp 14 CG11378 15 16 17 18

CG9917

Drosophila melanogaster Chromosome X

B

The Intron/Exon Structure of the Proteins Including DUF 1397 Domain

FIG. 4. (A) The genomic locations of proteins, including the DUF1397 domain in Drosophila melanogaster. (B) The intron/exon structure and the protein sequences identity percentages of the proteins, including the DUF1397 domain.

CG14629 CG11378 CG9917 The Coding Region The Non-Coding Region

Percent Identities CG14629

CG11378

CG9917

34.6

31.2

CG11378

34.6

CG14629

CG9917

CAH33828

CAA31060

Leptin

PRL

M17

IFN-γ

AAM52337

CAE45012

IL-12 p35

SL

CAG14936

IL-11

CAE82301

BAD94444

BAC81422

CAD62446

BAC76885

CAM82751 CAM82750

IL-10

AAY56128

IFN

AAC60105

CAD67609

JE0144

GH

AAQ72467

Fugu rubripes (Takifugu rubripes)

IL-6

CAH39855 CAH39856

EPO

Carp (Cyprinus carpio)

NP_001073302

NP_998029

AAH92358

CAJ33891

BAD26596

CAI61347 CAI61346

AAW78362

NP_997523

CAI79040

ABB77436

Zebrafish (Danio rerio)

GenBank Accession Number of Helical Cytokines

Appendix

CAF99247

AAR25695

AAR25696

AAR25701

AAR25699

AAP57415

CAD88198

CAD67779

AAR25694

AAR25698

Puffer fish (Tetraodon nigroviridis)

P21993

CAJ14971

CAI29480

BAD20648

ABI48359

CAE45642

AAA49555

ABB89952

Rainbow trout (Oncorhynchus mykiss)

ABI17540

P79697

P87495

AAR20886

Goldfish (Carassius auratus)

AAW21707

AAP51036

Salmon (Salmo salar)

AAA49445

BAF80790

ABB90401

BAC07253

Halibut (Hippoglossus hippoglossus)

ABD46706

CAK29522

CAM32185

Sea bass (Dicentrarchus labrax)

AAS98173

BAD94448

Medaka (Oryzias latipes)

PUTATIVE INVERTEBRATE HELICAL CYTOKINE 467

468

Acknowledgments This work was supported by a Fudan University grant to G.C. (CQH1322025) and a grant from the National Programs for Science and Technology Development to W.Y. (2004BA519A38). This work was also partly supported by a grant from the National High Technology Research and Development Program of China (863 Program) to Z.L. (2006AA02A414) and by the Shanghai Leading Academic Discipline Project (B111).

Author Disclosure Statement No competing financial interests exist.

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Address correspondence to: Dr. Zhaoxin Zheng State Key Laboratory of Genetic Engineering Fudan University 220 Handan Road Shanghai 200433 People’s Republic of China Tel: 86-21-65642504 Fax: 86-21-65642504 E-mail: [email protected] and Dr. Zuofeng Li Shanghai Center for Bioinformation Technology Shanghai 200235 People’s of Republic of China E-mail: [email protected] Received 30 September 2008/Accepted 4 February 2009