Biochimica et Biophysica Acta, 1132 (1992) 203-206 203 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00

BBAEXP 90396 Short Sequence-Paper

Isolation and nucleotide sequence of cecropin B cDNA clones from the silkworm, Bombyx mori

Kiyoko Taniai a, Yusuke Kato a, Hirohiko Hirochika b and Minoru Yamakawa a

a Laboratory of Biological Defense, National Institute of Sericultural and Entomological Science, Tsukuba (Japan) and b Department of Molecular Biology, National Institute of Agrobiological Resources, Tsukuba (Japan)

(Received 18 June 1992)

Key words: Cecropin B; cDNA cloning; Nucleotide sequence; (Bombyx mori)

Two cDNA clones encoding cecropin B, an antibacterial protein, were isolated from a fat body cDNA library of the silkworm, Bombyx mori. Amino acid sequences of these clones, deduced from nucleotide sequences, were identical, including signal peptide regions. However, the nucleotide sequences were different at 30 positions. Deduced amino acid sequences of Bombyx mori cecropin B showed higher homology with cecropins from Lepidoptera than with those from Diptera.

As a self-defense response against bacterial infec- tion, insects produce several antibacterial proteins [1]. Of these, cecropins are known to be highly potent antibacterial proteins which consist of 31-39 amino acid residues, devoid of cystein, and which have a strongly basic N-terminal region and a long hydropho- bic stretch in the C-terminal half [2]. Cecropins have been isolated from various lepidopteran [3-6] and dipteran insects [7,8]. Recently, the occurrence of a cecropin has been reported from pig intestine, suggest- ing that cecropins are distributed not only in insects but also in vertebrates [9]. In Bombyx mori, cecropin A, B [10-12] and CMIV [1] have been isolated and their amino acid sequences determined. However, molecular cloning of cecropin cDNAs have not yet been performed. In the course of our study on the induction mechanism of cecropin gene activity, we iso- lated cDNAs to prepare probes for Northern blot analysis. In the present study we analyze the cDNA sequences of Bombyx mori (Bin) cecropin B. The pri- mary structure of prececropins is also compared with that of other insects.

Five fifth instar larvae (Tokai × Asahi) were injected with Escherichia coli (4.106 cells/larva) and 9 h later

Correspondence to: M. Yamakawa, Laboratory of Biological De- fense, National Institute of Sericultural and Entomological Science, Tsukuba, Ibaraki 305, Japan.

The sequence data reported in this paper have been submitted to the EMBL/Genbank Data Libraries under the accession numbers Dl1113 and Dlll14.

fat bodies were excised by forceps. A mRNA prepara- tion was extracted using a Quick Prep mRNA Purifica- tion Kit (Pharmacia LKB) from fresh fat bodies (0.56 g). cDNA was prepared from 8 /zg of mRNA using a cDNA Synthesis Kit (Pharmacia LKB). The cDNA was ligated to hgtl0 arms [13] and packaged with a Giga- packII Gold Packaging Extract (Stratagene). The titer was 3.27.106 PFU//~g cDNA. To prepare a probe for plaque hybridization, 40 ng cDNA was used as a tem- plate for polymerase chain reaction (PCR) [14] with Taq DNA polymerase (Promega), using Program Temp Control System PC-700 (Astec). Primers were made based on the amino acid sequences of BM cecropin [12]. Nucleotide sequences for 5' primer (18 mer) were 5 ' -TGGAA(A/G)AT(A/C/T)TT(C/T)AA(A/G)AA- (A/G)-3 ' and for 3' primer (14 mer) 5 '-AC(T/C)TC ( A / G / T)AT(A/ G / T / C ) G C ( A / G / T / C)GG-3'. These were deduced from amino acid residues from Trp-2 to Lys-7 of mature cecropin B, and from Pro-24 to Val-28 for 5' and 3' primer, respectively. 30 cycles of PCR were performed at 40°C for the annealing temperature. Products obtained by PCR were analyzed by 4% low temperature melting agarose (Seakem) gel electrophoresis and the size was confirmed to be as predicted. DNA fragments synthesized by PCR were ligated with M13mp18 phage vector [15] and sequenced by the chain termination method [16]. This confirmed that the cDNA codes for amino acid residues from Ile-8 to Gly-23 of Bm cecropin B. E. coli NM 514 was infected with recombinant phages and 1 • 105 plaques were screened with cecropin B cDNA fragment (80 mer) synthesized by PCR. Radiolabelling of a probe,

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hybridization and washing conditions were as described previously [17]. The single-positive plaques were con- firmed by repeating hybridization three times. Phage DNA was extracted according to published methods [18]. M13mpl8 phage cloning vector [15] and E. coli strain XLl-blue were used for subcloning and transfor- mation. Nucleotide sequences for both strands were determined by the chain termination method [16] with Sequenase (United States Biochemical Corp.). Of 105 plaques, 1000 distinctly positive plaques were obtained. The result indicates that cecropin-related mRNA species occupy about 1% of total mRNAs. Two clones, which possessed the average size of cDNA insert (ca. 400 base pairs), were chosen and their nucleotide se- quences determined.

The complete nucleotide and deduced amino acid sequences of the clones designated as BmcecB3 and BmcecB24 are presented in Fig. 1. These two clones contained an open reading frame and the deduced amino acid sequence showed the characteristics of a cecropin B precursor protein. Deduced amino acid sequences of the mature form of BmcecB3 and Bm- cecB24 were perfectly consistent with those of the purified cecropin B [12]. However, nucleotide se-

quences were different at 30 positions. The results suggest that the differences between the nucleotide sequences may reflect the presence of two different cecropin B genes, or polymorphism at an individual level in B. mori. These possibilities could be distin- guished by the analysis of cecropin genes. The polyadenylation signal, AATAAA, was not seen in these clones. Nucleotide sequences at 3' noncoding region in two clones were AT rich (78.8% and 79.1% for BmcecB3 and BmcecB24, respectively), therefore, the oligo(dT) used for 3' primer to synthesize cDNA might hybridize to this region instead to the poly(A) site. Fig. 2 shows the comparison of amino acid se- quences deduced from nucleotide sequences of ce- cropin cDNAs. The structure of the precursor proteins was divided into two regions containing signal or ma- ture peptides. The homology in amino acid sequences of both regions from Bm cecropin B is higher with cecropins from other Lepidoptera than with those from Diptera (Table I). Results suggest that the score of homology in the deduced amino acid sequences be- tween Bm cecropin B and other cecropins exhibited good correlations to the order of phylogenetic evolu- tion. High homology of cecropin amino acid sequences

BmcecB 3 GT 2 Bm~ae/~ 4 -- 2

BmcecB 3 ~Iumd%(3GT(X3GOdrrAtIAATITACAT~ACAATGAATFrC 62 IMIcecB24 ............................ C ............................... 62

M e ~ 3

Bm~cB 3 GCAAAGATCCTA "rA-I~AGCA 122 IMlcec_ _R24 ............................................................ 122

AlaLysI l~PheVa iPheAlaLeuVa~MetTnrSerAlaAla 23

BmcecB 3 TL-A-Ata%AGAAAATIVgd%AAAA~CATIL~IuAC 182 Bmo~/~24 -G -C 182

ProGl~Ar~ysI lePheLysLysI leGluLysMetGlyArgAsnI leArgAsp 43

BmoecB 3 GGCA AAAGCTATAGGAAAA 242 BmcecB24 .................................. -C ............. -C ......... 242

GlyI leValLysAlaGlyProAla I leGluVa iLeuGlySerAlaLysAlaI leGlyLys 63

BmcecB 3 TGA~L'YI'rAAAA~TACTAGTA~TITAATCT~'I"n~A'I"ATA~AT 302 BmoecB24 .............. TAC ........................................... 302

BmcecB 3 ATGTAATITAATTACAGCTr TAAGACI~AATAATAA C~ATI~AA'I-I"I-n~ 352 13moecB24 ........ A ....... AT-~L~fCGTA ...... -GC-- -TIX2C-TrAT- -T ........... 362

BmoecB 3 AATATAA~AA~ATA'ACITAGTC~T~ATU'A'AU'rAAA 412 BmoecB24 ............. T .......................... 402

BmcecB 3 AATr 416

Fig. 1. Nucleotide and deduced amino acid sequences of two cecropin B cDNAs from the silkworm, Bombyx mori. Dashed lines in the sequence of BmcecB24 indicate that the nucleotides are identical to the sequence of BmcecB3. The termination codon is indicated by asterisks. An arrow

head shows the start position of mature peptides.

B in - B

H c - B

H c - A

| l c - D

M s - D

D r r i - A

D m - B

H p - I A

- - P S i g n a l p e p t i d e s

20 -10 -I

I

~ M a t u r e p e p t J d e s

+10 ~'20 +30 +40

I-- FKKIEKMGRNIRDGI VKAGPAIIEIVL~[ KA~ .... B m - B 6'

, , o - B . . . .

L _ l

Sp--IA W I(RIq~RV~QHT~TIQGLGIAQ--QA NVAATA~

Fig. 2. Comparison of amino acid sequences of cecropins from lepidopteran and dipteran insects. Gaps are introduced to maximize the homologies. Identical amino acid residues in one letter with those of Bm cecropin B are boxed. The C-terminal Gly residues are believed to be the donors of an amide group to the preceding residue. The amino acid residues conserved by cecropins are shown by reversed triangles. Sequence data of cecropins are from the following references: Bombyx mori (Bm) (present data), Hyalophora cecropia (Hc) [20,21], Manduca sexta (Ms) [22], Drosophila

melanogaster (Dm) [8] and Sarcophaga peregrina (Sp) [23].

suggests that they have arisen from a common ances- tral gene through gene duplications. 13 amino acid residues were commonly shared by eight cecropins. Of these, Lys-8, Glu-ll and Arg-18 in the mature peptides were conserved by 24 cecropins including a mammalian cecropin (data not shown). These amino acid residues cluster in a N-terminal half of the mature peptides. It

TABLE I

Comparison of the homology in amino acid sequences deduced from nucleotide sequences of cecropin cDNAs between Bombyx mori cecropin B and a cecropin from other insects

Percentage of the homology in amino acid sequences was calculated from data of Fig. 2. Designation of cecropins is the same as in Fig. 2.

Signal peptide Mature peptide

(Lepidoptera) (%) (%) Hc-B 76.9 80.6 Hc-A 61.5 63.2 Hc-D 57.7 38.5 Ms-D 53.9 31.6

(Diptera) Dm-A 38.5 23.8 Dm-B 46.2 23.8 Sp-IA 38.5 23.8

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has been suggested that cecropins can form nearly perfect amphiphatic a-helices with charged groups on one longitudial side. Hydrophobic helices are often associated with membranes and this secondary struc- ture may be of importance for the membrane-disrupt- ing activity of cecropins [1]. These three common amino acid residues might have an important role from this view point. Recently, Andreu et al. reported artificial hybrids of shortened cecropin A from the Cecropia moth and melittin, which is the main component of bee venom [19]. They synthesized 15-residue hybrids, whose N-terminal half is derived from cecropin A and 'C- terminal half from melittin, with antibacterial activity and spectra similar or better than cecropin A and a 60% reduction in size. Their reduced size and highly a-helical structure require an alternative mechanism for their interaction with bacterial membranes. Inter- estingly, these hybrids did not contain Gly-ll and Arg-18 but Lys-8 of cecropin A from Hayalophora cecropia. The presence of Lys-8 might be indispensable for the full expression of the antibacterial activity•

This work was supported in part by a Grant-in-Aid (Bio Media Program) of the Ministry of Agriculture, Forestry and Fisheries, Japan (BMP 92-IV-2-6) and by a special coordination fund for promoting science and technology (SCF) in the basic research core system by the Science and Technology Agency (STA) Japan.

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