Volume ~SO, number 2. I~t?~.~91 '

FI~I~S t19:~14

March 199i

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Characterization of eDNA clones encoding guinea pig neutrophil cationic peptides Isao N a g a o k a , A k i m a s a S o m e y a . Kazuhisa l w a b u c h i and Tutsuhisa Yamashita Depm'tment o f Bim'hemistry. Juntendo Unh't'rstty. Sdmrd a f Mediehu', Hemga, Bm~kya.ku,

T.kyo 113, lapmt

Received 21 December 1990: revi~e~.lversion received 23 January t991 gDNA elone~ ¢n~:odinl~ antin;i~:rohial ttuinen pi~ neutrophil eatloni¢ pcptides (]NCP.I and GNCP,2 ,,,,'ere i~ohn~d rron'J a bone m~rraw cell eDNA library, Analysi~t of the~e clones indicated tha! both GNCP~ were produced a:~ pre~ttr~or proleifl~ ¢ompri~iOll 9) amino acid residue,~, which wer~ ¢ompo~ed ol'silma| ~equ~nees (N.termina119 residues), pro.peptide sequenee~ 0,3 re~tidue~)and n~atare GNCP ,seqtl~nee~ OI residues), The deduced ~tmino acid ~¢quen¢~:~ ~howed Ih~ll there were only two amino acid dilTerenee,~ between GNCP-I and @NCP-2. one in the pr¢~.peptide re~ion and one in Ihe mature peplide relgion, ltllere~tilliJly. Northern blot anal>'sis and tran~ription rua.ofl'n~say revealed lhat |11¢expression o f G N C P mRNA and the transcription of G N C P g,ene was observed in bone marrow ¢¢11~bat not in mature neatrophiht, The~e obser'~:~tion~ su~!e~t that mature ncutrophil~, despi~e their abundant conlent of GNCPs. lose the capaeit~ to synthesize ¢/)NCPs, Neut rophil eationle peplide; e D N A cloning: Amino acid .,~quen¢~; Gene expression: Tr~tn~¢riptit~n run-off': ¢] uine~, pi~

1. INTRODUCTION Neutrophils play an important role in protecting t~osts against microbial infections. Both oxidative and non-oxidative mechanisms are used by neutrophils to restrict infections [1-3]. Molecules such as H.,O.,, 0.7 and HOCI are examples of microbicidal agcntsthat are derived from oxidative metabolism [1;3]. On the other hand, neutrophil granules contain antimicrobial proteins and peptides that contribute to the oxygenindependent host defense mechanism [2,3]. The most abundant of ~hese antimicrobial components.are low molecular-weight cationic peptides with potent rnicrobicidal activity against bacteria, fungi and viruses [2-8]. In addition, the cationic peptides have been shown to display cytotoxic activity [9] and histaminereleasing activity [10]. Six rabbit, one guinea pig and four human cationic peptides have been purified and characterized [11-14]. Recently, we have purified two guinea p~g neutrophil cationic peptides, GNCP-1 and GNCP-2, and found that GNCP-1 and GNCP-2 are single-chain peptides comprising 31 amino acid residues, which differ only by the substitution of an isoleucine (GNCP-1) for a leucine (GNCP-2) at position 21 [10], and that GNCP-I is the same peptide as that previously purified [12]. However, Correspondence acldress: 1. Nagaoka, Departrneat of Biochernistry, Juntendo University, School of Medicine, Hongo, Tokyo I13, Japan

Bunkyo-ku,

Abbreviations: GNCP, guinea pig neutropbil cationic peptide; bp, base pairs

Published by Elsevier Science Publishers B, V.

tlae precursors of GNCPs are not clear at this stage. In this study, therefore, we have isolated cDNA clones for GNCPs, and analyzed the nucleotide sequences to ¢leduce the amino acid sequences. Furthermore, the expression of the GNCP gene was studied in bone marrow cells and mature neutrophils using G N C P eDNA as a probe. 2, MATERIALS AND METHODS 2. I. PreIJarution Of cells

L3onemarrow cells were obtained from guinea pig femoral bones Gttinea pig perit0neal exudate neutroplfils a~td peripheral blood neutr~l~hils were prer)ared as ~reviously described [15]; tl~e purity of tl~e cells was >95°;0, 2,2, ¢'DNA clonitlg attd sequenchtg Total cellular RNA was isolated from bone marrow cells by the guanidiniunl thiocyanate extraction methoct [16], and poly(A)* RNA was selected byOligo-dT cellulose eolu~n chro~natograpl~y, Double. stranded ¢DNAs were synthesized by a eDNA synthesis kit, and were ligated with EcoRl adaptors, according to tl~e nlanufacturer's it~struc. ions (Pharmacia LKB Bioteehnology), EcoRl-ter,uinated cDNAs were inserted into phage vector Agtl0 and packaged in vitro (Pro1legal0 and then file phages were grown on Esct~erichiacolt C600H fl. Aboul 20000 recombinant phages were screened with a synthetic oligonucleotide probe (5' .GTTCTGGA AGATGC AGGTGCCGAGG C G G C G G T A T G G G A A G C G G C A G G T - 3 '. 48-merJ corresponding 1o line amino acid sequences Thr-9 to Asn.24 of mature GNCP.I ; this sequence was 90% identical to that of tile GNCP eDNA clones isolated, The probe was labeled with ['~"P]dCTP (3000 Ci/mmol. Amerslmm) using random hexanueleotide priaaers (Promega) [17], Tile nucleotide seq tlences of the eDNA inserts were deternlined from both directions using a sequencing ki t (United States Bioehemicall by Lhe dideoxy chain.termination method [18] after st:bcloning the eDNA inserts into a plasmid vector pBluescril0t SK(-) (Stratagene).

287

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Fig. I, Nt, cl¢otld¢ sequences of GNCP cDNA clones and the deduced amino acid sequences of prepro-GNCPs, The nucleotide sequence shown is that of the four overlapping GNCP-I clones, and is numbered starting at the initiation codon ATG, Negative numbers show the upstream region, The deduced amino acid sequence for the precursor of GNCP-I, shown under the nucleotide sequence, is numbered beginning at the N-terminal residue of mature GNCP- I, Negative numbers indicate pre. and pro-sequences, The GNCP-2 eDNA sequence is identical to that of GNCP.I except where Indicated over the given seqLicnce, The amino acid differences between GNCP.I and GNCP-2 are also indicated below the amino acid sequence, The consensus sequence for translation initiation [22] is dot,hie.underlined, The arrows [qdicate the most probable cleavage sites in posttranslational processing [23-25], The triple stars indicate the termination codon, The polyadenylation signal [36] is underlined, The structure of the predicted protein product is shown beneath the sequence data,

2,3 Evaluation of GNCP mRNA transcript Expression of GNCP mRNA was evaluated by Northern blot a,alysis. Total cellular RNA was isolated from bone marrow cells and neutrophils as described above, and eleetrophoresed on a formaldehyde.containing agarose gel, Then, RNA was blotted onto a nylon membrane (Schleicher and Schuell), and hybridized with GNCP-I eDNA probe or 7-actin eDNA probe (pHFTA-I [19]; graciously provided by P. Gunning and L, Kedes, Stanford Univermty) labeled with [nPIdCTP by the random hexanucleotide primer method [171.

2,4 Evaluation of GNCP gene transcription The transcription of the GNCP gene was evaluated bya transcription 'run-off' assay, as described previously 120,21]. Nuclei were isolated from bone marrow cells and neutrophils, and incubated with [nP]UTP (800 Ci/mmol, Amersham) to label nuclear RNA, Transcription of the GNCP gene and, as a control, the 7-actin gene, was determined by hybridization (40 h, 42°C) of the riP-labeled nuclear RNA (10 7 cpm/ml) to the filter.bound (nylon membrane, Schleicher and Schuclh dot-blot apparatus, Bio-Rad laboratories), unlabeled eDNA targets (GNCP-I eDNA and ~,.aetin eDNA) in the presence of 50% formamide and 10o70 dextran sulfate. When the GNCP.2 eDNA was used for Northern blot analysis and transcription run-off assay as a labeled eDNA probe and an unlabeled eDNA target, respectively, the same results as those with the GNCP-I eDNA were obtained (not shown), 288

3. R E S U L T S A N D D I S C U S S I O N

3.1. Isolation o f c D N A clones f o r G N C P s and its sequence analysis T o i so l at e G N C P e D N A s , a e D N A l i b r a r y w a s p r e p a r e d f r o m b o n e m a r r o w cells, a n d a b o u t 2 × 10+ ind e p e n d e n t p l a q u e s w e r e ..creened w i t h a s y n t h e t i c o l i g o n u c l e o t i d e p r o b e b a s e d o:~ the a m i n o acid s e q u e n c e s o f G N C P - 1 . F i f t e e n elo~:es were s e l e c t e d r a n d o m l y f r o m 40 r e c o m b i n a n t c l o n e s h y b r i d i z e d w i t h t h e p r o b e , a n d t h e i r e D N A inserts w e r e a n a l y z e d b y a g a r o s e gel e l e e t r o p h o r e s i s . T h e sizes o f t h e e D N A inserts w e r e 4 5 0 - 5 0 0 b p . A m o n g these inserts, 6 inserts w e r e seq u e n c e d , a n d it was f o u n d t h a t 4 inserts e n c o d e d GNCP-1 and 2 inserts encoded GNCP-2, An open r e a d i n g f r a m e o f 282 n u e l e o t i d e s w as in the v a r i o u s c l o n e s preceded b y a 5 ' - u n t r a n s l a t e d r e g i o n o f 5 5 - 7 8 nucieotides and followed by a 3 ' -untranslated region o f 104-1 17 n u e l e o t i d e s (Fig. 1). T h e a m i n o acid s e q u e n c e s d e d u c e d f r o m the e D N A s e q u e n c e s c o n t a i n e d s e q u e n c e s i d e n t i c a l with the p r e v i o u s l y p u b l i s h e d m a t u r e G N C P - 1

Volume 280. number 2

Mar¢h 1991

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