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peptidespeptides 27 (2006) 30853091available at ls1. IntroductionIn recent years, it has been widely recognized that manyorganisms use antimicrobial peptides (AMPs) as part of theirhost defense systems against the invasion of microorganisms4,28,29. To amphibian species, the skin glands are the richestsources for AMPs. Up to now, on the basis of broad structuralcharacteristics, amphibians have been grouped into variousfamilies including gaegurins (2437 residues), brevinvins-1(1724 residues) and -2 (3034 residues), ranalexin (20residues), ranatuerins-1 (25 residues) and -2 (33 residues),esculentins-1 (46 residues) and -2 (37 residues), palustrin (31residues), japonicin-1 (14 residues) and -2 (21 residues),nigrocin-2 (21 residues), rugosins (3337 residues) and tem-* Corresponding author at: Key Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, College ofLife Sciences, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China. Tel.: +86 258 4396849; fax: +86 258 4396673.E-mail address: (R. Lai).1These authors have the same contribution to this paper.0196-9781/$ see front matter # 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.peptides.2006.08.01722 August 2006Accepted 23 August 2006Published on line 26 September 2006Keywords:AmphibianAntimicrobial peptideDiversityBrevinins-ALTemporins-ALAmolops loloensistemporins-ALa, b, and c. The brevinins-AL family which is structurally related to brevinins-1from skin secretions of the European frog, Ranabrevipoda, is composed of 24 amino acids andhas an intra-disulfide bridge at the C-terminus. The temporins-AL family, composed of 13 or16 amino acid residues, is related with temporins from the skin secretions of R. temporaria.The findings of this study will facilitate the solutions to the taxonomic questions of the ranidgenus Amolops and Staurois. In the work of this paper, both brevinins-ALb and temporin-Mainduced mast cell degranulation and histamine release, and had cytotoxic activity towardsolid tumor cell line HepG2. Brevinins-ALb also exerted strong hemolytic activity whiletemporin-Ma had no such activity.# 2006 Elsevier Inc. All rights reserved.Yi Lua,1, Jianxu Lib,e,1, Haining Yuc, Xueqing Xub,e, Jianguo Lianga,Yongqiang Tiand, Dongying Mab, Guoqing Lina, Guoqiang Huangb, Ren Laia,b,*aKey Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, College of Life Sciences,Nanjing Agricultural University, Nanjing, Jiangsu 210095, ChinabBiotoxin Units of Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology,Chinese Academy of Sciences, Kunming 650223, Yunnan, ChinacCollege of Life Sciences School of Heibei Normal University, Shijiazhuang, Hebei 050016, ChinadSchool of Chemical Engineering, Sichuan University, Chengdu 610051, ChinaeGraduate School of the Chinese Academy of Sciences, Beijing 100009, Chinaarticle infoArticle history:Received 25 July 2006Received in revised formabstractThere are around 27 species of Amolops amphibian distributed in South-east of Asia. Sevenantimicrobial peptides (AMPs) belonging to two different families were purified from skin ofrufous-spotted torrent frog, Amolops loloensis, and designated brevinins-ALa, b, c, and d, andAmolops loloensisTwo families of antimicrobialfunctions from skin of rufous-spottedjournal homepage: www.ewith multipletorrent frog,/locate/peptidescentrifuged and the supernatants were lyophilized.peptides 27 (2006) 3085309130862.2. Peptide purificationLyophilized skin secretion sample of A. loloensisi (1.2 g, totalOD280nmof 300) was dissolved in 10 ml 0.1 M phosphate buffer,pH 6.0, containing 5 mM EDTA. The sample was applied toa Sephadex G-50 (Superfine, Amersham Biosciences,2.6 cm C2 100 cm) gel filtration column equilibrated with0.1 M phosphate buffer, pH 6.0. Elution was performed withthe same buffer, with collecting fractions of 3.0 ml. Theabsorbance of the elute was monitored at 280 nm. Theantimicrobial activity of fractions was determined as indi-cated below. The protein peak containing antimicrobialactivity was pooled, lyophilized, and re-suspended in 2 ml0.1 M phosphate buffer solution, pH 6.0, and purified further byC18reverse phase high performance liquid chromatography(RP-HPLC, Hypersil BDS C18,30cmC2 0.46 cm) column.2.3. Structural analysisporins (1016 residues). Most of amphibian AMPs from ranidfrogs share a conserved disulfide-bridged heptapeptide seg-ment at the C-terminal end 2,5,6,8,11,12,21,24, except thetemporins without disulfide bonds. The ranid AMPs have acommon N-terminal preproregion, which is highly conservedboth intra- and interspecifically, followed by a markedlydifferent C-terminal domain corresponding to the matureAMPs.According to the classical taxonomic analysis, Amolopsamphibians belong to Ranidae, Amolopinae, members ofwhich were not clear with the ranid genus Staurois Cope 18659,16 until Inger differentiated them with marked differencesin morphology and ecology of their larval forms 14. Amolopsloloensisi, distributed in mountainous regions (21003200 mheight) of southwest China, is proved as a member of Amolops.Although many AMPs are identified from Rana amphibians, noAMPs from Amolops amphibians have ever been reported. Inthis study, the isolation, characterization, cDNA cloning, andbiological activities of AMPs derived from skin secretions of A.loloensisi were described.2. Materials and methods2.1. Collection of frog skin secretionsAdult specimens of A. loloensisi of both sexes (n = 30; weightrange 3040 g) were collected in Yunnan Province of China.Skin secretions were collected as follows: frogs were put into acylinder container. A piece of absorbent cotton immersed withanhydrous ether was put on the top of the container. Thecontainer was covered with a lid and permeated withvolatilized anhydrous ether. Being stimulated by anhydrousether for 12 min, frog skin surface was seen to exude copioussecretions. Skin secretions were collected by washing thedorsal region of each frog with 0.1 M NaCl solution containing0.01 M EDTA. The 500 ml of collected solution were quicklyComplete peptide sequencing was undertaken by Edmandegradation on an Applied Biosystems pulsed liquid-phasesequencer, model 491. Fast atom bombardment (FAB) massspectrometry was carried out on an Autospec-3000 spectro-meter, equipped with a high field magnet, using glycerol:3-nitrobenzyl alcohol:dimethyl sulphoxide (1:1:1, v:v:v) as mixedmatrix. The ion gun was operated at 25 kV with a current of1 mA, using Cs+as the bombarding gas.2.4. SMART cDNA synthesisTotal RNA was extracted (Life Technologies, Ltd.) from the skinof single sample of O. grahami using TRIzol reagent. cDNA wassynthesized by using a SMARTTMPCR cDNA synthesis kit(Clontech, Palo Alto, CA). The first strand was synthesized withcDNA 30SMART CDS primer II A, 50-AAGCAGTGGTATCAACG-CAGAGTACT (30) N-1N-30(N = A, C, G or T; N-1 = A, G or C), andSMART II A oligonucleotide, 50-AAGCAGTGGTATCAACGCA-GAGTACGCGGG-30. The second strand was amplified usingAdvantage polymerase by 50PCR primer II A, 50-AAGCAGTGG-TATCAACGCAGAGT-30.2.5. Screening of cDNA encoding antimicrobial peptidesThe cDNA synthesized by SMARTTMtechniques was used astemplate for PCR to screen the cDNAs encoding AMPS. Twooligonucleotide primers, S1(50-ccaaa(G/c)atgttcacc(t/A)tgaa-gaAA(T/C)-30), in the sense direction, a specific primerdesigned according to the signal peptide sequences ofantimicrobial peptides from ranid frogs, and primer II A asmentioned in SMART cDNA synthesis in the antisensedirection were used in PCR reactions. The DNA polymeraseused was Advantage polymerase from Clontech. The PCRconditions were: 2 min at 94 8C, followed by 30 cycles of 10 s at92 8C, 30 s at 50 8C, 40 s at 72 8C. Finally, the PCR products werecloned into pGEM1-T Easy vector (Promega, Madison, WI).DNA sequencing was performed on an Applied BiosystemsDNA sequencer, model ABI PRISM 377.2.6. Peptide synthesisBrevinins-ALb (FLPLAVSLAANFLPKLFCKITKKC) and amidatedTemporins-ALa (FLPIVGKLLSGLSGLL-NH2) were synthesizedby solid phase synthesis on an Applied Biosystems model 433Apeptide synthesizer according to the manufacturers standardprotocols. After the cleavage and deprotection of side-chain,the crude synthetic peptide was purified on a Vydac C18RP-HPLC column (25 cm C2 1 cm), eluting at a flow rate of 1 ml/minby a linear gradient of acetonitrile in 0.1% trifluoroacetic acidin water. Identity of the peptide was confirmed by automatedEdman degradation with a protein sequencer and massspectrometry analysis. Fast atom bombardment mass spec-trometry was carried out on an Autospec-3000 spectrometer,equipped with a high field magnet 15. The synthetic peptideswere next subject for biological activities evaluation.2.7. Antimicrobial assaysStandard bacterial and fungal strains used in antimicrobialassays including Gram-positive bacterium Staphylococcusaureus (ATCC2592), Gram-negative bacteria Escherichia coli(ATCC25922), Bacillus dysenteriae, and fungus Candida albicans2.11. Cytotoxic activitySolid tumor cell line HepG2was a kind gift from Chinese TypeCulture Collection (Kunming Institute of Zoology, CAS). It wasmaintained in RPMI-1640 (GIBCO) medium supplemented with10% (v/v) heat-inactivated fetal calf serum (FCS), 2 mMglutamine (Sigma), 10 mM Hepes (Sigma), 50 mM 2-mercap-toethanol (Bio-Rad), 100 units/ml penicillin and streptomycin.One hundred microliters of HepG2cells (3 C2 105/ml) wereseeded into a microtiter plate. Various concentrations of thesample tested were added and incubated for 48 h. Cytotoxicitywas measured by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method 22.IC50was definedas the concentration of the sample at which the absorbance at490 nm was reduced by 50%.3. Results3.1. Purification of antimicrobial peptidesThe supernatant of A.loloensis skin secretions was divided intoseveral peaks by Sephadex G-50 as illustrated in Fig. 1A. Thepeak with antimicrobial activity was marked with a bar, andfurther applied to an RP-HPLC column. More than 20 peakspeptides 27 (2006) 30853091 3087(ATCC2002) were obtained from Kunming Medical College.Bacteria were first grown in Luria-Bertani (LB) broth to anOD600nmof 0.8. A 10 ml aliquot of the bacteria was then takenand added to 8 ml of fresh LB broth with 0.7% agar and pouredover a 90 mm Petri dish containing 25 ml of 1.5% agar in LBbroth. After the top agar hardened, a 20 ml aliquot of the testsample filtered on a 0.22 mm Millipore filter was dropped ontothe surface of the top agar and completely dried before beingincubated overnight at 37 8C. If the sample examined hadantimicrobial activity, a clear zone would be formed on thesurface of the top agar representing inhibition of bacterialgrowth. Minimal inhibitory concentration (MIC) was deter-mined in liquid LB medium by incubating the bacteria in LBbroth with variable amounts of the sample tested. The MIC atwhich no visible growth occurred was recorded.2.8. Hemolysis assaysHemolysis assays were undertaken using rabbit red blood cellsin liquid medium as reported by Bignami 3. Serial dilutions ofthe peptide were used, and after incubation at 37 8C for 30 min,the cells were centrifuged and the absorbance in the super-natant was measured at 540 nm. Maximum hemolysis wasdetermined by adding 1% Triton X-100 to a sample of cells.2.9. Mast cell degranulation asssysMast cell degranulation was determined by measuring therelease of b-D-glucosaminidase from lysed mast cells. Wistarrats were killed by cervical dislocation and mast cells wereobtained by peritoneal washing of adult Wistar rats withTyrodes solution (137 mM NaCl, 2.7 mM KCl, 1.36 mM CaCl2,0.49 mM MgCl2, 0.36 mM NaH2PO4, 11.9 mM NaH2CO3, and5.04 mM D-glucose). Ten microliter samples were added to90 ml mast cells suspensions and incubated at 37 8C for 15 min.After centrifugation, 50 ml supernatants were added to 50 mlofthe substrate (3 mg of r-nitrophenyl-N-acetyl-b-D-glucosami-nidine dissolved in 10 ml of 200 mM sodium acetate, pH 4.5solution) for b-D-glucosaminidase assay and the mixtureswere incubated at 37 8C for 6 h. The reaction was terminatedby addition of 100 ml 0.1 M Na2CO3(pH 10.0). The absorbance ofthe colored product was assessed at 405 nm and the valueswere expressed as the percentage of total b-D-glucosamini-dase activity from rat mast cell suspensions in the presence of0.1% (v/v) Triton X-100 (used as 100% control).2.10. Histamine release assayWistar rats were killed by cervical dislocation and mast cellswere obtained by peritoneal washing of adult Wistar rats withTyrodes solution. Ten microliter samples were added to 90 mlmast cells suspensions and incubated at 37 8C for 15 min. Thereaction was terminated by adding the ice-cold Tyrodessolution (2.9 ml). The cell suspensions were then centrifugedand both the supernatants and cell pellets were tested forhistamine concentration as described by Evans et al. 10.Histamine-releasing activity was expressed as the percentageof the total cellular content and the values were corrected forspontaneous release without 5% exceeding in the absence ofpeptides.Fig. 1 Fractionation of A. loloensis skin secretion. (A)Sephadex G-50 gel filtration of A. loloensis skin secretion.A. loloensis skin secretion was applied on a Sephadex G-50(Superfine, Amersham Biosciences, 2.6 cm C2 100 cm)column equilibrated with 0.1 M phosphate buffer, pH 6.0.Elution was performed with the same buffer, collectingfractions of 3.0 ml (A). The peak (indicated by an arrow)with antimicrobial activity from Sephadex G-50 wasfurther purified on a Hypersil BDS C18RP-HPLC column(30 cm C2 0.46 cm) equilibrated with 0.1% (v/v)trifluoroacetic acid/water. The elution was performed withthe indicated gradient of acetonitrile in (B) at a flow rate of0.7 ml/min, and fractions were tested for antimicrobialactivity. The purified antimicrobial peptides are indicatedby 1a, 1b, and 2a, respectively (B).peptides 27 (2006) 308530913088encoding antimicrobial peptides. The clones wereprecursors. The family name of antimicrobial peptides wass. Gaps (-) have been introduced to optimize the sequencesequenceswere obtained from this separation (Fig. 1B). The peak withantimicrobial activity (marked by 1a, 1b, and 2a) was collected.3.2. Structural characterizationThree purified AMPs (1a, 1b, and 2a in Fig. 1B) named asbrevinins-ALa, brevinins-ALb, and temporins-ALa were sub-ject to amino acid sequence analysis by automated Edmandegradation. The amino acid sequences for brevinins-ALa andbrevinins-ALb (1a and 1b) were FLPMLAGLAANFLPKLFCK-Fig. 2 The amino acid sequences deduced from the cDNA clonesnumbered as ZDn at the left of amino acid sequences of theindicated in the right of amino acid sequences of the precursorhomology. Dots (.) indicate identical amino acid residues. Thesignal peptide sequences are italicized.ITKKC and FLPLAVSLAANFLPKLFCKITKKC, respectively. Theyare both composed of 24 amino acid residues, with molecularweight of 2665.3 and 2663.7 Da of each analyzed by fast atombombardment mass spectrometry. These results matchedvery well with the theoretical molecular weights (2665.5 and2663.5 Da). Analysis by the ExPASy MW/pI tool (http:/www.expasy.ch/tools/pi_tool.html) showed that they havethe same predicted pI of 9.7. The third AMP of temporins-ALa(2a) has a 16-amino acid sequence of FLPIVGKLLSGLSGLL, andcontains a C-terminally amidated residue.3.3. cDNA cloningFifty different cDNA clones encoding the precursorof brevinins-ALs and temporins-ALs were screened and sequenced from theskin cDNA library of A. loloensis. Thirteen different cDNAsequences encoding 12 different precursor proteins wereobtained (GeneBank Accession Numbers: DQ673109DQ673121). Seven mature AMP sequences belonging to threedifferent families were deduced from the 13 cDNAs since thesynonymous mutation occurred. They were designated brevi-nins-ALa, b, c, and d and temporins-ALa, b, and c (Fig. 2). Thestructural organization of these precursors is quite similar,comprising a signal peptide sequence, an N-terminal spacerpeptide region containing several aspartic and glutamic acidresidues, and the antimicrobial peptide at the C-terminus. All ofthe precursors encoding the three different families of AMPshave signal sequences of highly conserved, although themature peptides are significantly different (Fig. 2). The aminoacid sequences deduced from the cDNA sequences containthree AMPs that matched well with the sequences determinedby Edman degradation. By BLAST search, brevinins-ALa andtemporins-ALa were found to share similarity with brevininsand temporins identified from skin secretions of the Ranabrevipoda 21 and R. temporaria 26,respectively(Fig. 3).of mature peptides are boxed. The predicted3.4. Antimicrobial activityBrevinins-ALb and temporins-ALa exhibited antimicrobialactivity against the tested strains (Table 1). Among the testedFig. 3 The comparison of antimicrobial peptides from A.loloensis skin with other amphibian antimicrobialpeptides. Brevinins-ALa and b, and temporins-ALa arefrom this report; brevinin 1E is from Ref. 21, andtemporin-F is from Ref. 26. Gaps (-) have been introducedto optimize the sequence homology. Stars (*) indicateidentical amino acid residues.peptides 27 (2006) 30853091 3089strains, B. dysenteriae was the most sensitive to these twoAMPs. The MICs of brevinins-ALb and temporins-ALa for B.dysenteriae was 2.20 and 1.50, respectively (Table 1). Theantibiotic activity was proved to be lethal for the sensitivestrains, such as S. aureus, E. coli, B. dysenteriae, and C. albicans.The sensitive strains were not capable of resuming growth onagar plates after a 6-h treatment with concentrations abovethe corresponding MICs.3.5. Hemolytic activitySome AMPs exhibit hemolytic activities 15. Rabbit red bloodcells were used to check for hemolytic capability in ourexperiments.

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