There are Abundant Antimicrobial Peptides in Brains of Two Kinds of

ARTICLE pubs.acs.org/jpr

There are Abundant Antimicrobial Peptides in Brains of Two Kinds of Bombina Toads Rui Liu,†,‡ Huan Liu,†,§ Yufang Ma,§ Jing Wu,§ Hailong Yang,§ Huahu Ye,|| and Ren Lai*,‡,§ ‡

Life Sciences College of Nanjing Agricultural University, Nanjing 210095, Jiangsu, China Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, Yunnan, China Beijing Institute of Microbiology and Epidemiology, the Academy of Military Medical Sciences, China

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§

bS Supporting Information ABSTRACT: It is well-known that there is a large amount of antimicrobial peptides in amphibian skins but few antimicrobial peptides are found in amphibian brains. Twenty-two and four antimicrobial peptides were purified and characterized from the brain homogenate of Bombina maxima and B. microdeladigitora, respectively. One hundred fifty-eight cDNA clones encoding 79 antimicrobial peptides were isolated from brain cDNA libraries of B. maxima and B. microdeladigitora. These antimicrobial peptides belong to two peptide groups (maximin and maximin-H). Twenty of them are identical to previously reported antimicrobial peptides (maximin 18, 10, 11, maximin H1, 35, 7, 9, 10, 12, 15, 16) from B. maxima skin secretions. Fiftynine of them are novel antimicrobial peptides. Some of these antimicrobial peptides showed strong antimicrobial activities against tested microorganism strains including Gram-positive and -negative bacteria and fungi. The current diversity in peptide coding cDNA sequences is, to our knowledge, the most extreme yet described for any animal brains. The extreme diversity may give rise to interest to prospect the actual functions of antimicrobial peptides in amphibian brains. KEYWORDS: antimicrobial peptides, brain, amphibian

’ INTRODUCTION Antimicrobial peptides act as important roles in innate immunity. Most of them are 1050 residues in length. They can provide an effective and fast acting defense against harmful microorganisms.1 Amphibian skins are the first line to interact with environments. They depend on their naked skins to prevent from microorganism infection. It has been found that Granular glands in the skin of anuran amphibians synthesize and secrete a remarkably diverse array of antimicrobial peptides.2,3 There is little information about antimicrobial peptides from amphibian brains. Extensive studies have been conducted on amphibian antimicrobial peptides of frogs belonging to the genus Rana and Bombina. About 300 antimicrobial peptides have been identified from more than 20 ranid amphibians.410 The ranid frogs synthesize and secrete multiple active components. The representative case is Odorrana grahami: 107 antimicrobial peptides belonging to 30 different families were found in its skin secretions.7 Bombina is belonging to Bombinatoridae. Presently, the genus Bombina includes 8 species,11,12 including B. bombina, B. fortinuptialis, B. lichuanensis, B. maxima, B. microdeladigitora, B. r 2011 American Chemical Society

pachypus, B. orientalis, and B. variegata. More than 30 antimicrobial peptides belonging to two groups have been found from skin secretions of B. bombina, B. maxima, B. orientalis, and B. variegate.1316 B. maxima and B. microdeladigitora are distributed in southern China and adjacent northern Vietnam. Our previous works have also identified 27 antimicrobial peptides including 11 maximins and 16 maximins H from B. maxima skin.6,17 No antimicrobial peptide has been reported from B. microdeladigitora. In this work, peptidomics and genomics analysis were used to study antimicrobial peptides diversity in brains of B. maxima and B. microdeladigitora.

’ MATERIALS AND METHODS Collection of Toad Brain Homogenates

All of the experimental protocols using animals were approved by the Animal Care and Use Committee at Kunming Institute of Zoology, Chinese Academy of Sciences. Adult specimens of B. Received: October 25, 2010 Published: February 21, 2011 1806

dx.doi.org/10.1021/pr101285n | J. Proteome Res. 2011, 10, 1806–1815

Journal of Proteome Research

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maixma and B. microdeladigitora of both sexes (n = 10; weight range 3050 g) were collected in Chuxiong and Puer, Yunnan Province of China, respectively. According our previous method to avoid contamination from toad skin secretions, we extracted the toad brains.18 Briefly, toads were put into a glass beaker (1000 mL). A piece of absorbent cloth was moistened with anhydrous ether (∼1 mL) and put on top of the container with animals. The container was covered with a lid and we waited for 12 min. Some stress-like behavior may be observed (e.g., amphibians start jumping), but they will become anaesthetized after 12 min treatment and their skins will exude copious secretions. During this process, their glands can completely release their products onto the external surface of the skin in response to the ether stress. The skins were washed with the solution of protease inhibitors in 0.1 M NaCl and 510 sequential washes with 100200 mL of salt water per wash. The anaesthetized amphibianswere used for dissection including getting rid of skins and extracting brains. Extensive washing was carried out again after skinning. The brains were immediately frozen in liquid nitrogen and homogenized by a glass homogenizer in 30 mL of 0.1 M phosphate buffer solution, pH 6.0 (PBS) containing protease inhibitor cocktail (Sigma). The collected solutions were quickly centrifuged and the supernatants were lyophilized. Peptide Purification

The lyophilized sample of B. maixma or B. microdeladigitora brain was dissolved in 10 mL of 0.1 M phosphate buffer solution, pH 6.0 (PBS) and then was applied to a Sephadex G-50 (Superfine, Amersham Biosciences, 2.6 100 cm) gel filtration column equilibrated with 0.1 M PBS. Elution was performed with the same buffer, collecting fractions of 3.0 mL. The absorbance of the eluate was monitored at 280 nm. Every fraction was subjected to an antimicrobial test as described below. Fractions containing antimicrobial activities were collected and pooled. They were subjected to further purification by a reverse-phase liquid chromatography (RP-HPLC) (Hypersil BDS C18, 25 0.46 cm2) column. The elution was performed at a flow rate of 0.7 mL/min with the indicated gradients of acetonitrile in 0.1% (v/v) trifluoroacetic acid (TFA) in water as illustrated in Figure 1. Structural Analysis by Edman Degradation and Mass Spectrometry

Complete amino acid sequences were determined by the automated Edman degradation on an Applied Biosystems pulsed liquid-phase sequencer, model 491. Mass fingerprints (MFPs) were obtained using a Waters ZQ 2000 LCMS spectrometer (Waters, Milford, MA). Chromatography was performed on a Waters C_(18) column (150 mm 4.6 mm, 5 μm). A methanolwater (V:V = 50:50) as the mobile phase was used. A Waters Micromass ZQ detector equipped with an electrospray ionization (ESI) interface was used and operated in the positive ion mode. cDNA Synthesis and cDNA Cloning

Total RNA was extracted using TRIzol (Life Technologies, Ltd.) from the brain of B. maixma or B. microdeladigitora. A SMART PCR cDNA synthesis kit (Clontech, Palo Alto, CA) was used to synthesize cDNA according to the manufacture instruction. Two primers in the kit (30 -SMART CDS Primer IIA, 50 AAGCAGTGGTATCAACGCAGAGTACT(30)N-1N-30 (N = A, C, G or T; N-1 = A, G or C), and SMART II oligonucleotide, 50 -AAGCAGTGGTATCAACGCAGAGTACGCGGG-30 )

Figure 1. Purification of antimicrobial peptides from brain homogenates of B. maxima. (A) Sephadex G-50 gel filtration of brain homogenate of B. maxima. The homogenate was applied on a Sephadex G-50 column equilibrated with 0.1 M phosphate buffer, pH 6.0. Elution was performed with the same buffer, collecting fractions of 3.0 mL. (B and C) Peaks (Indicated as II and III) with antimicrobial activity from Sephadex G-50 were further purified on a Hypersil BDS C18 RP-HPLC column equilibrated with 0.1% (v/v) trifluoroacetic acid in water. The elution was performed with the gradients of acetonitrile in 0.1% (v/v) trifluoroacetic acid in water shown in (B) and (C) at a flow rate of 0.7 mL/min, and fractions were tested for antimicrobial activity. The purified antimicrobial peptides are listed in Table 1. Their mass fingerprint (MFP) results are provided as a Supporting Information (Figure S1).

were used for the first strand synthesis. The second strand was amplified using Advantage polymerase by primer IIA (50 -AAGCAGTGGTATCAACGCAGAGT-30 ) provided by the kit.19 A directional cDNA library was constructed with a plasmid cloning kit (SuperScriptTM Plasmid System, GIBCO/BRL) following the instructions of manufacturer, producing a library of about 5.2 105 and 3.7 105 independent colonies, respectively. A PCR-based method for high stringency screening of DNA libraries was used for screening and isolating the clones. The oligonucleotide primer (M1 (50 -AGATGAATTTTAAGTACATA-30 , in the sense direction) is designed according to the signal peptide sequences of maximin antimicrobial peptides as our previous works.6,17 Primer II A was used as the antisense 1807

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Figure 2. Purification of antimicrobial peptides from brain homogenates of B. microdeladigitora. (A) Sephadex G-50 gel filtration of brain homogenates of B. microdeladigitora. The homogenate was applied on a Sephadex G-50 column equilibrated with 0.1 M phosphate buffer, pH 6.0. Elution was performed with the same buffer, collecting fractions of 3.0 mL. (B) Peak (indicated as II) with antimicrobial activity from Sephadex G-50 was further purified on a Hypersil BDS C18 RP-HPLC column equilibrated with 0.1% (v/v) trifluoroacetic acid in water. The elution was performed with the gradients of acetonitrile in 0.1% (v/v) trifluoroacetic acid in water shown in (B) at a flow rate of 0.7 mL/min, and fractions were tested for antimicrobial activity. The purified antimicrobial peptides are listed in Table 1. Their mass fingerprint (MFP) results are provided as a Supporting Information (Figure S1).

direction primer. The DNA polymerase was Advantage polymerase from Clontech (Palo Alto, CA). The template was the cDNA from the library. DNA sequencing was performed on an Applied Biosystems DNA sequencer, model ABI PRISM 377. Antimicrobial Assay

Antimicrobial activities against Gram-positive bacteria Staphylococcus aureus (ATCC 25923), Bacillus subtilis (ATCC 6633), Gram-negative bacteria Escherichia coli (ATCC 25922), and fungus Candida albicans (ATCC 20032) were assayed according to our previous methods.6,7 Hemolysis Assays

Hemolysis assay was tested with rabbit or human red cells in liquid medium as reported.6,7 Rabbit or human red blood cells in

Alsever’s solution (in g/L: NaCl, 4.2; citric Acid 3 3Na 3 2H2O, 8.0; citric Acid 3 H2O, 0.55; D-glucose, 20.5) were used for the test. Serial dilutions of the peptides were used, and after incubation at 37 °C for 30 min, the cells were centrifuged and the absorbance in the supernatant was measured at 540 nm. Maximum hemolysis was determined by adding 1% Triton X-100 to a sample of cells. Alignment and Phylogenetic Comparison of Peptides

To resolve evolutionary relationships among the obtained genes, phylogenetic analysis were performed using the software package MEGA 4 after multiple alignment of data by CLUSTAL_X. Distances and clustering were based on neighbor-joining method and bootstrap analysis (1000 replications) were used to evaluate the topology of the neighbor-joining tree. 1808



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Table 1. Antimicrobial Peptides Purified from Brain Homogenates of B. maxima and B. microdeladigitoraa name

a

sequence

position in RP-HPLC

origin

Maximin 3

GIGGKILSGLKTALKGAAKELASTYLH-NH2

M1

BM

Maximin 15

GIGTKILGGVKAALKGALKELASTYVN-NH2

M2

BM

Maximin 28

GIGTKFLGGVKTALKGALKELASTYVN-NH2

M3

BM

Maximin 31

GIGGALLSAGKSALKGLAKGLAEHF-NH2

M4

BM

Maximin 32

GIGGKILGGLKTALKGAAKELASTYLH-NH2

M5

BM

Maximin 39

GIGTKFLGGVKTALKGALKELAFTYVN-NH2

M6

BM

Maximin 41

GIGGALLSVGKSALKGLTKGLAEHF-NH2

M7

BM

Maximin 45 Maximin 49

GIGGKILGGLRTALKGAAKELAATYLH-NH2 GIGGVLLSAGKAALKGLTKVLAEKYAN-NH2

M8 M9

BM BM

Maximin 63

GIGGVLLGAGKATLKGLAKVLAEKYAN-NH2

M10

BM

Maximin 68

GIGGALLSAGKAALKGLAKVLV-NH2

M11

BM

Maximin 42

SIGAKILGGVKTFFKGALKELAFTYLQ-NH2

M12

BM

Maximin 77

GIGGALLSAGKSALKGLAKGLAEHL-NH2

M13

BM

Maximin 78

GIGGALLSVGKLALKGLANVLADKFAN-NH2

M14

BMI

Maximin H5

ILGPVLGLVSDTLDDVLGIL-NH2

H1

BM

Maximin H7 Maximin H23

ILGPVIKTIGGVIGGLLKNL-NH2 ILGPVISTIGNVLGGLLKNL-NH2

H2 H3

BM BM

Maximin H24

ILGPVLGLVGDTLGDLL-NH2

H4

BM

Maximin H27

ILGPVLGLVSNALGGLL-NH2

H5

BM

Maximin H37

ILGPVLGLVSNTLDDVLGIL-NH2

H6

BM

Maximin H39

ILGPVLGLVGNALGGLIKKL-NH2

H7

BM

Maximin H46

ISGPVLGLVGNALGGLIKKI-NH2

H8

BM

Maximin H48

ILGPVLGLVGSALGGLI-NH2

H9

BM


ILGLVISTIGNVLGGLLKNL-NH2 ILGPVLGLDGNALGGLIKKI-NH2

H10 H11

BMI BMI

Maximin H55

ILGPVISTIGNALGGLLKNL-NH2

H12

BMI

BM: B. maxima; BMI: B. microdeladigitora.

Synthetic Peptides

Structure Characterization

All of the peptides used in this work were synthesized by GL Biochem (Shanghai) Ltd. (Shanghai, China) and analyzed by HPLC and mass spectrometry to confirmed purity higher than 98%. All peptides were dissolved in water.

Twenty-six purified antimicrobial peptides were sequenced by Edman degradation. Their complete amino acid sequences were obtained as listed in Table 1. Twenty-six mass spectra in Supplemental Figure S1 (Supporting Information) reveal mass fingerprints of these 26 purified peptides. They contain observed molecular weights that match well with the calculated molecular weights. By BLAST search, the 26 peptides are antimicrobial peptides belonging to two different families (Maximin and maximin H, Table 1). Treating these peptides with carboxypeptidase Y did not lead to the release of free amino acids under conditions that free amino acids were released from a peptide with free C-terminal COOH group. The result indicated that the C-terminal ends of these peptides are amidated, which are further confirmed by mass spectrometry analysis.

’ RESULTS Purification of Antimicrobial Peptides

Three fractions were obtaind from the sample of B. maxima brain following Sephadex G-50 gel filtration. Fractions II and III were been found to contain antimicrobial activities and were further separated using C18 RP-HPLC purification as illustrated in Figure 1. The eluted peaks were analyzed by MALDI-TOF-MS and tested for antimicrobial activities. Twenty-two eluted peaks containing antimicrobial activities (indicated M113 and H19 in Figure 1) were found to be pure (Figure S1, Supporting Information). Different from B. maxima, the homogenate sample of B. microdeladigitora brain was only divided into two fractions by the Sephadex G-50 gel filtration. Only fraction II showed antimicrobial abilities. It was subjected to C18 RP-HPLC purification as illustrated in Figure 2. The eluted peaks were analyzed by MALDI-TOF-MS and tested for antimicrobial activities. Four eluted peaks containing antimicrobial activities (indicated M14 and H1012 in Figure 2) were found to be pure (Figure S1, Supporting Information).

Sequence Diversity

We report 158 sequences (GenBank accession numbers EU369581EU369601; EU289759EU289696; EU137911 EU137983) of antimicrobial peptides deduced from cDNA sequences. The peptides could be assembled into two divergent groups containing 79 antimicrobial peptides (Tables 2 and 3). Twenty of them are identical to our previously reported antimicrobial peptide (maximin 18, 10, 11, maximin H1, 35, 7, 9, 10, 12, 15, 16), which are found in skin secretions of B. maxima. Another 59 are novel. In total, 52 and 27 antimicrobial peptides are identified from brain of B. maxima and B. microdeladigitora, respectively (Tables 2 and 3). 1809



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Table 2. Antimicrobial Peptides from Brain of B. maxima name

sequence

name

sequence

Maximin 2

GIGTKILGGVKTALKGALKELASTYVN-NH2

Maximin 56

GIGTKIIGGLKTAVKGALKELAFTYVN-NH2

Maximin 3

GIGGKILSGLKTALKGAAKELASTYLH-NH2

Maximin 63

GIGGVLLGAGKATLKGLAKVLAEKYAN-NH2

Maximin 4

GIGGVLLSAGKAALKGLAKVLAEKYAN-NH2

Maximin 62

GIGGKILSGFKTALKGAAKELAATYLH-NH2

Maximin 5

SIGAKILGGVKTFFKGALKELASTYLQ-NH2

Maximin 64

GIGTKIIGGLKTAVKGALKESAFTYVN-NH2

Maximin 7

GIGAKILGGVKTALKGALKELASTYVN-NH2

Maximin 65

GIGGKILFGLKTALKGAAKELAATYLH-NH2

Maximin 8

GIGTKILGGLKTAVKGALKELASTYVN-NH2

Maximin 66

GIGGKILSGLKTALKGAAKELAATYLH-NH2

Maximin 11

GIGTKIIGGLKTAVKGALKELASTYVN-NH2

Maximin 67

GIGGALLSAGKAALKGLAKVLAEKYAN-NH2


GIGGKILSGFKTALKGAAKELAFTYLH-NH2 GIGTKILGGVKAALKGALKELASTYVN-NH2


GIGGALLSAGKAALKGLAKVLV-NH2 GIGGKILGGVKTALKGALKELASTYAN-NH2

Maximin 18

GIGAKILGGVKTALKGALKELAFTYVN-NH2

Maximin 70

GIGGKILPGFKTALKGAAKELAATYLH-NH2

Maximin 19

GIGTKILGGVKTALKGALKELAFTYAN-NH2

Maximin 71

GIGGVLLSAGKAALKGLARVLAEKYAN-NH2

Maximin 26

GIGGKILGGLKTALKGAAKELAATYLH-NH2

Maximin 72

GIGTKIIGGFKTAVKGALKELAFTYVN-NH2

Maximin 28


Maximin 73

GIGTKILGGVKTALKGALKELAPTYVN-NH2

Maximin 31


Maximin 74

GIGTRIIGGLKTAVKGALKELASTYVN-NH2

Maximin 32

GIGGKILGGLKTALKGAAKELASTYLH-NH2

Maximin 79

SIGAKILGGVKTFFKGALKELAFTYLQ-NH2


ILGPVISTIGGVLGGLLKNL-NH2 ILGPVLGLVGNALGGLIKKI-NH2


ILGPVLGLVGNALGGYLKIL-NH2 ILGPVLGLVGNALGGLIKKL-NH2

Maximin H5

ILGPVLGLVSDTLDDVLGIL-NH2

Maximin H40

ILGPVLGLIGNALGGLIKKI-NH2

Maximin H4

ILGPVISKIGGVLGGLLKNL-NH2

Maximin H41

ILGPVLGLVSGTLDDVLGIL-NH2

Maximin H7

ILGPVIKTIGGVIGGLLKNL-NH2

Maximin H42

VLGPVLGLVSNALGGLL-NH2

Maximin H12

LLGPVLGLVSNALGGLLKNI-NH2

Maximin H43

ILGPVLGLVDNALGGLIKKI-NH2

Maximin H15

ILGPVLGLVGNALGGLLKNL-NH2

Maximin H44

ILGPVLGLVGNALGGLIKEI-NH2

Maximin H16

ILGPVLSLVGNALGGLIKKI-NH2

Maximin H45

ILGPVLGLVGNDLEVYLKI-NH2


ILGPVISTIGNVLGGLLKNL-NH2 ILGPVLGLVGDTLGDLL-NH2


ISGPVLGLVGNALGGLIKKI-NH2 LLGPVLGLVSNDLEVYLKIL-NH2

Maximin H37

ILGPVLGLVSNTLDDVLGIL-NH2

Maximin H48

ILGPVLGLVGSALGGLI-NH2

Table 3. Antimicrobial peptides from brain of B. microdeladigitora Name

Sequence

Name

Sequence

Maximin 1

GIGTKILGGVKTALKGALKELASTYAN-NH2

Maximin 31


Maximin 2

GIGTKILGGVKTALKGALKELASTYVN-NH2

Maximin 33

GIGGKILGGLKTALKGAAKELAATYLQ-NH2

Maximin 4 Maximin 6

GIGGVLLSAGKAALKGLAKVLAEKYAN-NH2 GIGGALLSAGKSALKGLAKGLAEHFAN-NH2


GIGTKFLGGLKTAVKGALKELASTYVN-NH2 GIGGALLSAGKAALKGLAKVLV-NH2

Maximin 7

GIGAKILGGVKTALKGALKELASTYVN-NH2

Maximin 75

GIGTKILGGVKTALKGALKGLASTYAN-NH2

Maximin 10

GIGGALLSAGKSALKGLAKGLAEHFAS-NH2

Maximin 76

GIGGALLSAGKSALKGLAKVLADKFAN-NH2

Maximin 26

GIGGKILGGLKTALKGAAKELAATYLH-NH2

Maximin 77

GIGGALLSAGKSALKGLAKGLAEHL-NH2

Maximin 28


Maximin 78

GIGGALLSVGKLALKGLANVLADKFAN-NH2

Maximin H1

ILGPVISTIGGVLGGLLKNL-NH2

Maximin H23

ILGPVISTIGNVLGGLLKNL-NH2

Maximin H3

ILGPVLGLVGNALGGLIKKI-NH2

Maximin H27

ILGPVLGLVSNALGGLL-NH2


ILGPVISKIGGVLGGLLKNL-NH2 ILGPVLGLVSNALGGLIKKI-NH2


ILGLVISTIGNVLGGLLKNL-NH2 ILGPVLGLDGNALGGLIKKI-NH2

Maximin H10

ILGPVLGLVSNALGGLLKNL-NH2

Maximin H55

ILGPVISTIGNALGGLLKNL-NH2

Maximin H15

ILGPVLGLVGNALGGLLKNL-NH2

All maximin antimicrobial peptides reported here are cationic peptides with positive net charges but some maximin Hs (maximin H5, 24, 27, 29, 30, 32, 37, 41, 42, 44, 45, 47, 48 and 51) are anionic peptides with negative net charges. Especially, maximin 5, 24, 37, 41, and 51 have an isoelectric point less than 4. In these anionic peptides, they have no basic amino acid residue. Our previous work identified a gene-encoded anionic antimicrobial peptide, maximin H5 from B. maxima. It is the first report of the presence of

anionic antimicrobial peptide. The current 14 anionic antimicrobial peptides further confirm this conclusion. Sequence Diversification

We purified and sequenced only 26 of these 79 peptides represented by the cDNAs that we characterized. However, to our knowledge, mRNA sequences isolated from frog universally are found to be translated into peptides. We assumed (but have 1810



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Figure 3. (A) Precursor diversity containing maximin antimicrobial peptides. (B) Three representatives of precursor diverfication of maximins. Mature antimicrobial peptides are boxed. The bar () indicates stop codon.

not proven) this to be the case for our findings in the brains of these Bombina toads. Most of maximin and maximin H are composed of 27 and 20 amino acid residues (aa), respectively but some of them are less than 27 and 20 aa. Maximin 57 has 26 aa; maximin 30, 31, 36, 41, 61, and 77 are composed of 25 aa; maximin 68 has only 22 aa. All of them are shortened from the original length of 27 aa due to the creation of a premature termination codon because the codon is terminated early. The same situation is also found in maximin H27, 42 and 45, foreshortened by a premature stop codon to a length of 17 and 19 aa. Their original length should be 20 aa. For most of maximin peptides, their sequences are started by Gly but maximin 5, 57, and 42 are exceptions, which are started by Ser or Ile. Most of maximin H peptides are started by Ile or leu. Only maximin H42 is started by Val. All of this diversification resulted from point mutations within the “original” peptides. Most of the cDNAs encode one maximin and one maximin H but some cDNAs only encode one maximin, no maximin H. As illustrated in Figure 3, several truncated sequences contain only maximin; they are devoid of maximin H. Antimicrobial Activities

We have observed obvious diversification in primary structures, among the antimicrobial peptides from these two species of Bombina toads, as well as potential genetic mechanisms underlying

their diversification. Perhaps the most intriguing question provoked by this study is why this species exhibits such diversity in its antimicrobial peptide arsenal. We tested antimicrobial activities of these antimicrobial peptides purified the brain homogenates of Bombina toads as listed in Table 4. They exhibited diverse antimicrobial activities. Most of maximins showed strong antimicrobial activities against all tested microorganisms including Gram-positive and Gram-negative bacteria and fungi. The antimicrobial activities of maximin 41, 49, 63, 42, and 77 appear to be narrow, with only antimicrobial abilities against S. aureus. Most of maximins H show weak antimicrobial potency against tested microorganisms with MICs > 100 μg/mL. Only maximin H39 shows strong antimicrobial potency against S. aureus, B. subtilis, and fungus C. albicans. Hemolysis

As listed in Table 5, the hemolytic activities of 26 antimicrobial peptides with three different concentrations (0.0625, 0.125, and 0.25 mg/mL) against human and rabbit red cells were tested. They show stronger hemolytic activities against human red cells than rabbit red cells. The only exception is maximin H52. It is hemolytic ability to rabbit red cells is stronger than human red cells. But at a low concentration (0.0625 mg/mL) of maximin H52, it induced 36.5 and 5% hemolysis of human and rabbit red cells, respectively. Maximin 31, 39, 41, 42, and 77 and maximin H5, H23, H24, H27, H37, H46 and H54 have little hemolytic activity against rabbit red cells while only maximin H5, H23, 1811



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Table 4. Antimicrobial Activities of Maximins or Maximins H (MIC: μg/mL)a C. albicans

S. aureus

E. coli

B. subtilis

Maximin 3

75

4.7

9.4

75


>100 75

9.4 4.7

18.8 9.4

75 75

Maximin 31

>100

37.5

75

>100

Maximin 32

>100

9.4

18.8

>100

Maximin 39

>100

18.8

37.5

>100

Maximin 41

>100

75

>100

>100

Maximin 45

>100

4.7

9.4

75

Maximin 49

>100

18.8

>100


>100 18.8

18.8 9.4

Maximin 42

>100

Maximin 77

Table 5. Hemolytic Activities of Antimicrobial Peptides Maximins or Maximins Ha hemolysis (%) human red cells

rabbit red cells

concentration (μg/mL) 250

125

62.5

250

125

62.5

Maximin 3

43.3

10.9

2.4

8.4

3.5

1.6

Maximin 15

32.8

8.3

0.9

7.9

2

1.1

>100


42.3 29.8

27.7 3.7

15.5 0.4

12.4 5

12.1 0.6

2.2 0.5

>100 37.5

>100 9.4

Maximin 32

10.1

2.3

0.5

3.7

0.4

0.3

Maximin 39

8.2

4.3

1.6

0.5

0.3

0.2

37.5

>100

>100

Maximin 41

17.7

11.8

0.8

3

0.5

0.4

>100

18.8

>100

>100

Maximin 45

66.7

48.9

17.7

54.6

3.2

2.3

Maximin 78

37.5

4.7

>100

37.5

Maximin 49

11.2

9

2.6

11.3

0.7

0.5

Maximin H5

>100

>100

>100

>100

Maximin 63

42.5

23.8

3.4

30.6

11.1

0.5

Maximin H7

18.8

9.4

>100

18.8

Maximin H23

>100

>100

>100

>100


76.1 12.2

44.8 11.2

27.3 6.1

63 2.5

25.9 0.8

15.4 0.8


>100 >100

>100 >100

>100 >100

>100 >100

Maximin 77

10.2

1.5

0.5

0.4

0.4

0.3

Maximin 78

71.7

40.6

17.6

65.4

28.1

17

Maximin H37

>100

>100

>100

>100

Maximin H5

0.5

0.5

0.4

0.4

0.3

0.3

Maximin H39

9.4

9.4

>100

18.8

Maximin H7

79.8

33.4

9.8

35.1

3.2

1.7

Maximin H46

>100

>100

>100

>100

Maximin H23

4.8

1.3

0.9

0.4

0.3

0.3

Maximin H48

>100

>100

>100

>100

Maximin H24

0.6

0.6

0.5

0.4

0.4

0.3

Maximin H52

>100

>100

>100

>100

Maximin H54

>100

>100

>100

>100


0.3 5.3

0.3 4.7

0.3 4.4

0.3 2.5

0.3 0.4

0.3 0.4

Maximin H55 CS

>100 1.2

>100 0.3

>100 0.02

>100 0.6

Maximin H39

91.4

62.5

15.8

43.9

12.3

5.9

Maximin H46

18.3

4.3

1.1

1.6

0.4

0.3 0.5

LH

75

0.3

>100

9.4

Maximin H48

74.4

65.1

60.3

49.2

37.4

LevH

0.02

0.3

0.05

0.1

Maximin H52

50.9

49.8

36.5

63.8

61

5

TS

0.3

4.7

0.6

1.2

Maximin H54

2.6

1.1

1.4

0.3

0.3

0.2

Tin

37.5

>100

37.5

37.5

Maximin H55

91

90.2

78.4

90.2

72.2

50

a

MIC, minimal peptide concentration required for total inhibition of cell growth in liquid medium. These concentrations represent mean values of three independent experiments performed in duplicates. CS, cefoperazone sodium for injection; LH, lincomycin hydrochloride for injection; LevH, levofloxacin hydrochloride for injection; TS, tobramycin sulfate for injection; Tin, tinidazole for injection.

H24, H27 and H54 have little hemolytic activity against human red cells. Maximin 3, 15, 28, and 49 have moderate hemolytic activity against rabbit red cells while maximin 32, 39, 49, 42, and 77 have moderate hemolytic activity against human red cells. Many of these peptides including maximin 45, 63, 68 and 78, and maximin H7, H39, H48, H52 and H55 induce strong hemolysis on rabbit red cells. Many of them (maximin 3, 15, 28, 31, 32, 41, 45, 49, 63, 68, 42, 77 and 78, and maximin H7, H39, H46, H48, H52 and H55) show hemolytic activity against human red cells. Among them, maximin H55 has the strongest hemolytic abilities against both human and rabbit red cells although it has weak antimicrobial activities (Table 4). At the concentration of 250 μg/mL, it leads >90% red cell hemolysis. Phylogenetic Analysis

We constructed nerghbour-joining gene trees for maximin and maximin H listed in this work or published before, respectively (Figure 4). The results showed that there are two main branches

a

These data represent mean values of three independent experiments performed in duplicates.

in the two trees, maximin 63, 4, 71, 49, 78, 67, 41, 76, 10, 6, 31, 77, Hv, Hw, Hu and Ht and maximin H40, H55, H6, H23, H52, H4, H1, H7 and H13 respectively position at very different branches, suggesting that these peptides have evolved independently.

’ DISCUSSION Although antimicrobial peptides from amphibian skins have been extensively studied, little work is related with amphibian brain antimicrobial peptides. Amiche et al. have reported dermatoxin in the brain of Phyllomedusa bicolor.20 Northern blot had revealed low but significant expression of bombinin (maximin) mRNA in B. orientalis brain.15 Recently, preprobrevinin-1, preprotemporin, and preproranatuerin-2 gene transcripts were detected at higher levels in brain of the Oki Tago’s brown frog, Rana tagoi okiensis.21 The present study focused on the analysis of antimicrobial peptides in brain of two species of Bombina toads and successfully characterized a large amount of anitmicrobial peptides that belongs to two groups of maximin and maximin H. B. maxima and B. microdeladigitora have different living environments. B. maxima lives in still ponds connecting with 1812



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Figure 4. Phylogenetic dendrogram obtained by neighbor-joining analysis based on the proportion difference (p-distance) of aligned amio acid sites of the peptide sequences. (A) Maximin peptides; (B) maximin H peptides. Only bootstrap values >50% (expressed as percentages of 1000 replications) are shown at branching points.

brook where the altitude is 25003600 m; B. microdeladigitora lives in tree holes of Secondary Broad-leaf Forests where the altitude is 19002100 m.12 Different living environments may encounter different microorganisms. The diversification of antimicrobial peptide loci potentially might have evolved in response to selective pressure exerted by different microbial pathogens. As listed in Table 4, these antimicrobial peptides have different antimicrobial spectrum and potency. Interestingly, brains of B. maxima and B. microdeladigitora share 12 identical antimicrobial peptides (maximin 2, 4, 7, 26, 28, 31, and 68 and maximin H1, 3, 4, 15 and 23) as listed in Table 2 and 3. Recently, considering the low genetic differences between B. maxima and B. microdeladigitora, and previous morphological and karyological evidence, B. microdeladigitora was reassigned as a subspecies of B. maxima.12 Multiple identical antimicrobial peptides are found in their brains

may provide support for reassigning B. microdeladigitora as a subspecies, not an independent species. These peptides are purified from brain homogenates or deduced from brain cDNAs of Bombina toads. The contamination from amphibian skin secretions is a problem to study components from their tissues other than skins. On the basis of at least five points, we presume that our method can avoid the contamination from the skin secretions although we can not completely prove it: (1) Amphibian skin glands can completely release their products onto the external surface of the skin in response to the ether stress; (2) Extensive washing before removing the skin and again of the carcass after skinning was performed; (3) The anaesthetized amphibians are used for dissection including getting rid of skins and extracting brains. The anaesthetized amphibians have no responses to stress and 1813


Journal of Proteome Research tissue damage (including dissection); (4) Skulls are good barriers to avoid contamination from skin secretions; (5) By peptidomics analysis, we do not find skin marker peptides, such as BSTI-like trypsin inhbitors and BV-8 like peptides2224 in the brain extracts (data not shown). Some of these peptides identified from the toad brains were synthesized and tested for their antimicrobial activities. Most of them contain antimicrobial abilities. Some of them showed potential antimicrobial activities against all the tested microorganisms (Table 4). These results indicated that these peptides identified from toad brains can exert antimicrobial activities as amphibian skin antimicrobial peptides do. There are blood brain barriers (BBB) to defend against microorganisms in brains, but some bacterial species have abilities to translocate across the BBB and enter the subarachnoid space, leading to meningitis.25 These antimicrobial peptides in amphibian brains may act as defensive roles against brain pathogens. It has been observed that many of these peptides have very weak antimicrobial activities, such as maximin H5, H23, H24, H27, H37, H46, H48, H52, H54 and H55. Maximin H5, H24, H27, H37 and H48 are anionic peptides with predicted isoelectric points less than 7.0 whereas others are cationic peptides. It appears that the antimicrobial abilities of antimicrobial peptides are not identical to their net charges. The biological significance of these “structural antimicrobial peptides” with weak or no antimicrobial ability in the brain is unclear. The expression of bombinin (maximin) mRNA in B. orientalis brain and stomach implies that antimicrobial peptides have other physiological functions in the central nervous system and gastrointestinal tract system.15 Our previous work has indicated that the mice injected intraperitoneally with maximin 1 and 3 exhibited typical poisoned symptoms, such as gasp, jerk, and tension, which are possible neurotoxic symptoms.6 Recently, we have found that some anionic maximin H peptides have antinoceptive functions (data not shown), implying that these peptides act as neuropeptides. In addition, many maximin or maximin H antimicrobial peptides showed strong hemolytic activities against human or rabbit red cells (Table 5). It needs much more work to understand how Bombina amphibians conduct self-protection to prevent the toxicities of their antimicrobial peptides in brains. Some antimicrobial peptides show significant different antimicrobial activities although they share high sequence similarity. For example, maximin 15 (GIGTKILGGVKAALKGALKELA STYVN-NH2) and maximin 75 (GIGTKILGGVKTALK GALKGLASTYAN-NH2) have only two amino acid differences (A12/T12 and E20/G20) but they have obviously different functions. Maximin 15 shows moderate antimicrobial abilities against E. coli, S. aureus, and B. subtilis. Maximin 75 only shows antimicrobial potency against S. aureus although it has more positive net charge than maximin 15. Maximin 31 (GIGGALLSAGKSALKGLAKGLAEHF-NH2) and maximin 77 (GIGGALLSAGKSALKGLAKGLAEHL-NH2) have the same situation. They just have one amino acid difference (F25/L25) but their antimicrobial potency and spectrum are different. They also exert significant difference to hydrolyze red cells. Hemolytic activities of maximin 15 and 31 are strong to human red cells while those of maximin 15 and 77 are moderate. All of these results suggest that there are key amino acid residues in these peptides for their functions. For example, the Phe25 in maximin 31 possibly acts as a key role for its antimicrobial or hemolytic activity. Previous works have also suggested the importance of aromatic amino acids in

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antimicrobial peptides.2628 Phenylalanine residues in antimicrobial peptides are required for efficient membrane interaction and antibacterial activity.29 The current work provides a large amount of templates to identify key amino acid residues and to study the structurefunction relationship of antimicrobial peptides. It has been found that several precursor sequences contain only maximin, no maximin H (Figure 3). Their sequence lengths are from 71 to 144 aa. Active evolution appears to be produced in these antimicrobial precursors. It is hypothesized that the sequence with 71 aa is the “original” sequence. The “original” sequence is gradually extended to form corresponding longer sequences by moving the stop codon to a later position. Finally, the original sequence containing only maximin is evolved into the sequence containing both maximin and maximin H. Much more work is necessary to demonstrate this hypothesis.

’ CONCLUSIONS In this study, we identified 79 anitmicrobial peptides belonging to two groups (maximin and maximin H) from brains of two species of Bombina toads (Table 2 and 3). Fifty-two of them are from B. maxima and 27 of them are from B. microdeladigitora. Fifty-nine of them are novel peptides. The current work further reveals the extreme diversity of antimicrobial peptides in single amphibian species. Different from the diversity found in the skin of O. grahami, the diversity reported here occurs in the brain of Bombina toads. ’ ASSOCIATED CONTENT

bS

Supporting Information Supplemental Figure S1. Detailed mass fingerprint (MFP) results of purified antimicrobial peptides. Supplemental Table S1. Summary of the antimicrobial peptides identified from Amphibians. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Dr Ren Lai, Life Sciences College of Nanjing Agricultural University, Nanjing 210095, Jiangsu, China. Tel: þ86-2584396849. Fax: þ86-871-5199086. E-mail: [email protected]. Author Contributions †

These authors contributed equally to this paper.

’ ACKNOWLEDGMENT This work was supported by Chinese National Natural Science Foundation (30830021, 30800185, 31025025) and the Ministry of Science and Technology (2010CB529800). ’ REFERENCES (1) Zasloff, M. Antibiotic peptides as mediators of innate immunity. Curr. Opin. Immunol. 1992, 4, 3–7. (2) Barra, D.; Simmaco, M. Amphibian skin: a promising resource for antimicrobial peptides. Trends Biotechnol. 1995, 13, 205–209. (3) Simmaco, M.; Mignogna, G.; Barra, D. Antimicrobial peptides from amphibian skin: what do they tell us?. Biopolymers 1999, 47, 435–450. 1814


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There are Abundant Antimicrobial Peptides in Brains of Two Kinds of

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