Sequences of two cDNA clones from the medfly Ceratitis capitata encoding antibacterial peptides of the cecropin family

Gene, 134 (1993) 241-243 0 1993 Elsevier Science Publishers

B.V. All rights reserved.

241

0378-t 119/93/$06.00

GENE 07348

Sequences of two cDNA clones from the medfly Ceratitis capitata encoding antibacterial peptides of the cecropin family* (Insect immunity; ceratotoxin; sarcotoxin; homology; medfly cDNA library)

magainin;

Marco Rosettoa, Andrea G.O. Manetti”, Cosima T. Baldari” aDepcwtmrnt

ofEvolutionaryBiology,

Received by G. Bernardi:

18 January

53100 Sienu, Ituly;

recombinant

Daniela

and bl.R.I.S.,

1993; Revised/Accepted:

DNA; nucleotide

Marchini”,

Via Fiorrntina

3 May/l0

sequence;

Roman0

amino

acid sequence

Dallai”, John L. Telfordb and

I. 53100 Siena, Itdy.

May 1993; Received at publishers:

Tel. (39-577)

293470

17 June 1993

SUMMARY

Using a backtranslated oligodeoxyribonucleotide probe, encoding a conserved we have isolated two cDNA clones from the medfly, Ceratitis capituta. Sequence encode two closely related peptides which are members of the cecropin family.

INTRODUCTION

Antimicrobial peptides have been isolated from both invertebrates and vertebrates. They include, among others, the cecropins (Ccc) and the diptericins from insects (reviewed in Boman and Hultmark, 1987; Hoffmann and Hoffmann, 1990), the magainins from amphibians (Zasloff, 1987) and the defensins from mammalian polymorphonuclear lymphocytes (Selsted et al., 1985). Although different in primary structure, many of these peptides can form amphipathic a-helices and kill bacteria by attacking the cell membrane, probably through the formation of ion channels (Okada and Natori, 1985a; Wade et al., 1990). Most antibacterial peptides are produced in insects in response to infection. Among the most potent of the inDr. C.T. Baldari, Department of Evolutionary Via Mattioli 4, 53100 Sierra, Italy. Tel. (39-577) 293470; Fax 298898.

Corrrspondencr

Biology. (39-577)

to:

*On request. the authors will supply detailed experimental the conclusions reached in this Short Communication.

evidence

for

Abbreviations: aa, amino acids(s); bp. base pair(s); cDNA, DNA complementary to RNA; Ccc, cecropin(s); Ctx, ceratotoxin; kb, kilobase or 1000 bp; nt, nucleotide(s); oligo, ohgodeoxyribonucleotide; ORF, open reading frame; SP. signal peptide(

motif in insect antibacterial peptides, determination shows that the cDNAs

ducible antimicrobial peptides are the Ccc (reviewed in Boman and Hultmark, 1987). Interestingly, the Ccc locus in Drosophila melanogaster has been shown to include the gene encoding andropin, a male-specific antimicrobial peptide which is constitutively produced in the ejaculatory duct (Samakovlis et al., 1991). Recently two closely related antibacterial peptides have also been purified from the female reproductive accessory glands secretion in Ceratitis capitata (Marchini et al., 1993). Here we present the sequence of two cDNA clones from the medfly Ceratitis capitata encoding peptides of the Ccc family.

EXPERIMENTAL

AND DISCUSSION

(a) Cloning and sequence determination of two medfly cDNAs We have screened a hgtll cDNA library from adult females of Ceratitis capitata for the genes coding for the female antibacterial peptides ceratotoxins (Ctx) (Marchini et al., 1993). The oligo which was used in the screening was designed by backtranslation of the middle portion of medfly Ctx A and Ctx B (Marchini et al., 1993) using Drosophila codon bias (see Fig. 1). This region con-

242 A

to the backtranslated M

60 1

Q

T

120 21

AAATCGAACGCGTTGGTCAGCATACCA I E R" G Q H T

R

180 41

GGTTCCCGCGTCTTAAACAGTTTAGGTATAcATccT*TTGT~G~TATcTGTc~

TGAATTTCAACARAGTCTTCATCCTCGTTGCCATCGTCATCGC~TTTTCGCAGGGCN F N K V F I L VA I V I A I F CTGAAGCGGGTTGGCTG~ E A G W L

K

K

I

G

K

K

A

GAGATGCCACGATCCAAACCATCGCTGTGGCCC~C~GCTGCT~TGTCGCAGCCACCG I Q T I A "A Q Q AA NV DA T

A

G

A

T

240 A 61

CCCGGGGTTAAAAACTGAAATTTGCTAGCGAGCTTCATTACC~GATACGAGCATTTCAC R G

300 63

ACAATATTTACTGCCTAATTTATTTAATTTATCAGTATGCGCTTATATTATGTTAAGACTTTTAAAATAAATTCATATTCAAAATT

360 389

B 60

ATTCGAACGTCACAGCAAATAGCAGCCTTCGCAACTGCAGTACAC GAATAACACAAAGARATAAACATTCGAAATGAATTTCAAGTATTGGTTCTCCTC FNKVLVLL M N

120 10

GCTGTCATCTTTGCCGTTTTCGCTGGACRAACCGAGGCAGGCAGGTTGGCTG~ATTGGC AVIFAVFAGQTEAGWLKKIG

180 30

BBG&4AATTGAGCGCGTCGGCCAACATACATACACGAGATGCCAC~TCCAGACCATCGGTGTG K K I ERVGQHTRDATIQTIG"

240 50

GCCCAACAGGCTGCCAATGTTGCTGCAACGCTGRGGGATAAACTTGTTGTTTTATTTATATT AQQAANVAATLKG

300 63

AGCTGATAAACTATAAATGTAAATGTAGATTAAGTGATT~TTTATTTATTTCAGCTGTT TGATATTAAAGCAAAACTTTTGCACGAAAATT

360 392

Fig. 1. Nucleotide sequences of two Ccc cDNA clones, Cccl (A) and Cec2 (B), from the medfly Cerafitis capirutu. The deduced aa sequence of the ORFs is shown below the nt sequence. The oligo used for the screening was the 30-mer 5’-CCGGTGGCCAAGAAGATTGGCAAGATTGCC encoding the lo-aa peptide PVAKKIGKIA. The nt sequence was determined using the Sequenase kit from US Biochemical (Cleveland, OH, USA). The underlined regions show the homology with the oligo. The nt sequences will appear in the EMBL and GenBank nt sequence X70029 (B).

databases

with

accession

numbers

X70030

(A)

and

tains the Lys-rich motif KKIGK present as such (KKIGK) or with one substitution in other antibacterial peptides, namely Ccc A (KKIEK; Steiner et al., 1981) and B (KKIEK; von Hofsten et al., 1985) from Hyalophora cecropia, Ccc B from Antherea (KKIEK; Craig et al., 1987), Ccc A and Ccc B from Bombyx mori (KKIEK; Teshima et al., 1986), sarcotoxin IA from Sarcophugu peregrinu (KKIGK; Okada and Natori, 1985b), Ccc A from Drosophila melunoguster (KKIGK; Kylsten et al., 1990) and magainin 2 from Xenopus luevis (KKFGK; Zasloff, 1987). Screening of the cDNA library with this oligo, in addition to Ctx cDNA, revealed two other clones which hybridised at low stringency. The nt sequence of the two cDNAs was determined and is presented in Fig. 1A and 1B. The two cDNAs show a high sequence homology, with 75% identity. Their homology

1. 2. 3. 4. 5. 6.

a short sequence

oligo used as a probe is limited

of 16 bp encoding

served in other antibacterial

the aa sequence

to

con-

peptides.

(b) The aa sequence analysis of the ORFs Each cDNA has a single ORF which codes for 63 aa. The conceptual translation products of these two clones revealed 84% aa identity and 98% similarity over most of the sequence

(Fig. 2). The lowest identity

is found be-

tween the N-terminal

16 aa of the two molecules,

this region

cases resembles

in both

closely

secretion signal peptides (SP), with a putative tidase cleavage site after aa 21 (von Heijne,

however eukaryotic

signal pep1986). The

presumptive mature peptides differ by only 3 aa. The two cDNAs are closely related to the members of the melunoguster gene cluster encoding the antibacterial peptides known as Ccc (Kylsten et al., 1990). As shown in Fig. 2, the conceptual translation products of the two cDNAs show a very high aa sequence homology with Drosophilu Ccc (with over 70% identity and about 90% similarity) (Fig. 2). Extensive aa sequence homology can also be found with another dipteran Ccc, sarcotoxin IA from the flesh fly Surcophuga peregrina (Matsumoto et al., 1986). Comparison of medfly and lepidopteran Ccc shows a limited homology (see the extensive aa sequence comparison between dipteran and lepidopteran Ccc precursors recently carried out by Taniai et al. (1992)). In Drosophila, the Ccc are a family of at least four closely related peptides (Tryselius et al., 1992). They are produced as pre-pro peptides which, by analogy to the well characterised Ccc from Hyulophora cecropiu (Boman et al., 1989) are assumed to be processed to yield the mature forms by removal at the N terminus of a SP followed by removal of a pro-sequence by a dipeptidyl aminopeptidase (Kylsten et al., 1990). In addition, as in Hyulophoru and Surcophugu (von Hofsten et al., 1985; Matsumoto et al., 1986), the C-terminal Gly is probably split resulting in amidation of the second last aa. Although there is little aa identity between the putative SP of the medfly cDNA translation products and other dipteran Ccc, the mature peptides are up to 90% identical in aa sequence, including the presumptive dipeptidase cleavage site and the C-terminal Gly (Fig. 2). Ccc are produced in response to bacterial infection and Drosophila

MNFNKVFILVAIVIAIFAGQTEAGWLKKIGKKIERVGQHTRDATIQTIAVAQQ~~TARG G wLIryI%Y XHL YE u TL S GhGI S VhGL LYE 66L EL.Bzz flk I &GI XIYE Id& % s LE I QM1 F LLH Y SQ

63 LK

Fig. 2. Comparison of the aa sequences of medfly Cccl (1) and Cec2 (2) with Drosophila CecA (3). CecB (4). Ccc C (5) and with sarcotoxin IA (6). Only the aa which differ from medfly Cccl primary structure are shown. Similar aa are underlined. Arrowheads show the potential cleavage site of the SP (left arrowhead) and the potential cleavage site by a dipeptidyl aminopeptidase (right arrowhead).

243 injury

(Boman

and Hultmark,

tion has however in Drosophila (Kylsten

1987). Ccc gene transcrip-

been shown to be completely

repressed

only when the flies were grown

axenically

et al., 1990). Northern

used to construct

blot analysis

the medfly cDNA library

of the RNA revealed

only

a very low amount of Ccc-specific mRNA (data not shown). As in Drosophila, this could be accounted for by the possible presence of bacterial contamination in the food. We conclude,

therefore,

that the two cDNAs

in this paper code for medfly homologues

presented

of other insect

Ccc and as such name them Cccl and Cec2.

antibacterial peptides from the female reproductive of the medfly Ceratiris capitata (Insecta, Diptera).

accessory glands Insect Biochem.

Mol. Biol. (1993) in press. Matsumoto,

N., Okada,

M., Takahashi,

H., Ming, Q.X., Nakajima.

Komano, H. and Natori. S.: Molecular assignment of the C-terminal of sarcotoxin protein of Sarcophuga peregrina. Okada, M. and Natori, S.: lonophore

Y.,

cloning of a cDNA and IA, a potent antibacterial

Biochem. J. 239 (1986) 717-722. activity of sarcotoxin I, a bacteri-

cidal of Sarcophagu peregrina. Biochem. J. 260 (1985a)4533458. Okada, M. and Natori, S.: Primary structure of sarcotoxin I, an antibacterial protein induced in hemolymph of Sarcophuga peregrina (flesh fly) larvae. J. Biol. Chem. 260 (1985b) 717447177. Samakovlis, C., Kylsten, P.. Kimbrell. D.A., EngstrBm, A. and Hultmark. D.: The An&pin gene and its product, a male-specific antibacterial peptide in Drosophilu melnnogasrrr. EMBO J. IO (1991) 1633169. Selsted, M.E., Brown, D.M., DeLange, R.G., Harwig, S.S.L. and Lehrer, RI.: Primary structures of six antimicrobial peptides of rabbit peri-

ACKNOWLEDGEMENTS

toneal neutrophils. Steiner, H., H&mark,

Research supported by the Italian National Research Council (CNR), Special Project RAISA, Subproject No. 2, paper No. 1053.

Sequence and specificity of two antibacterial proteins involved in insect immunity. Nature 292 (1981) 2466248. Taniai, K., Kato. Y., Hirochika, H. and Yamakawa. M.: Isolation and

H.G. and H&mark,

H.G.:

nucleotide sequence of cecropin B cDNA clones from the silkworm, Bombyx mori. Biochim. Biophys. Acta 1132 ( 1992) 2033206. Teshima, T., Ueki, Y., Nakai, T., Shiba, T. and Kikuchi, M.: Structure determination of lepidopteran, self-defense substance produced by silkworm. Tetrahedron 42 (1986) 8299834. Tryselius, Y., Samakovlis, C., Kimbrell, D.A. and Hultmak, D.: CecC,

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