- Email: [email protected]
Purification of a lysozyme from skin secretions of Bufo andrewsi
Comparative Biochemistry and Physiology, Part C 142 (2006) 46 – 52 www.elsevier.com/locate/cbpc
Purification of a lysozyme from skin secretions of Bufo andrewsi Yu Zhao a,b, Yang Jin a, Wen-Hui Lee a, Yun Zhang a,* a
Department of Animal Toxinology, Kunming Institute of Zoology, The Chinese Academy of Sciences, 32 East Jiao Chang Road Kunming, Yunnan 650223, China b Graduate School of the Chinese Academy of Sciences, Beijing 100039, China Received 2 July 2005; received in revised form 2 October 2005; accepted 3 October 2005 Available online 23 November 2005
Abstract A novel toad lysozyme (named BA-lysozyme) was purified from skin secretions of Bufo andrewsi by a three-step chromatography procedure. BA-lysozyme is a single chain protein and the apparent molecular weight is about 15 kDa as judged by SDS-PAGE. The specific lytic activity against Micrococcus lysodeikticus of BA-lysozyme is 2.7 105 units/mg, indicating that it is a potent lysozyme. It displayed potent bactericidal activity against Staphylococcus aureus and Escherichia coli with minimal inhibitory concentrations (MIC) of 1 and 8 AM, respectively. The deduced primary structure of BA-lysozyme from cloned cDNA was confirmed by N-terminal sequencing and peptide mass fingerprinting. Its amino acid sequence shares 56.5% identity with that of chicken egg-white lysozyme. Phylogenetic analysis indicates that B. andrewsi lysozyme is closely related to that of turtle. This is the first report on the isolation and primary structure determination of amphibian lysozyme. D 2005 Elsevier Inc. All rights reserved. Keywords: Lysozyme; cDNA; Bufo andrewsi; Skin secretions
1. Introduction Amphibian skin is a morphologically, biochemically and physiologically complex organ which fulfills a wide range of functions necessary for amphibian survival, including respiration, water regulation, anti-predator, antimicrobial defense, excretion, temperature control, etc. (Clarke, 1997). Skin gland secretions are a primary source of amphibian biochemical, and numerous peptides with diverse biological activities have been isolated from amphibian skin (Erspamer et al., 1985; Bevins and Zasloff, 1990). In particular, numerous antimicrobial peptides have been found in the skin secretions of many amphibian species that are responsible for the innate immune defense, and most of them have a molecular weight less than 5000 (Simmaco et al., 1998; Apponyi et al., 2004; Bevier et al., 2004; Conlon et al., 2004a,b; King et al., 2005). Bufonidae is a major family of Anura and widely distributed in the world. Unlike other families of anura, bufonoid toad skin secretions contain much more organic molecules (e.g. bufadienolides or bufadienolide-like steroids, * Corresponding author. Tel.: +86 871 5198515; fax: +86 871 5191823. E-mail address: [email protected] (Y. Zhang). 1532-0456/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2005.10.001
alkaloids and biogentic amines), but less peptides or proteins (Daly, 1995; Clarke, 1997). Recently, investigations on the proteins of bufonoid toad skin secretions have been conducted in our lab, and some bioactive proteins, such as protease inhibitor have been isolated and characterized (Zhao et al., 2005). Lysozyme (EC.3.2.1.17) is widely distributed among eukaryotes and prokaryotes and takes part in protecting microbial infections or digestion. Lysozyme kills bacteria by hydrolyzing h-1,4-glycosidic linkages between N-acetylglucosamine and N-acetylmuramic acid of the peptidoglycan layer in the bacterial cell wall. Lysozymes are classified into three major types: chicken type (c-type) (Canfield, 1963), goose type (g-type) (Simpson et al., 1980) and invertebrate type (i-type) (Ito et al., 1999). The c-type lysozyme has been found in many organisms including virus, bacteria, plants, insects, reptiles, birds and mammals. Lysozyme was also found in the skin of leopard frog (Rana pipiens) two decades ago (Ostrovsky et al., 1976). Since then, there have had no further investigations on amphibian skin lysozyme. In this study, we firstly report the isolation and complete amino acid sequence of an amphibian lysozyme from the skin secretions of a common bufonoid toad, Bufo andrewsi, in China.
Y. Zhao et al. / Comparative Biochemistry and Physiology, Part C 142 (2006) 46 – 52
2. Materials and methods
47
but without DTT). The gel was stained with Coomassie brilliant blue R-250 (Sigma).
2.1. Materials 2.5. Amino acid sequence determination Adult specimens of B. andrewsi of both sexes were collected in Qujing county, Yunnan province of China. DEAE-Sephadex A-50 and Sephadex G-50 (superfine) were purchased from Pharmacia (Sweden). Trifluoroacetic acid (HPLC grade) was from Perkin Elmer. Acetonitrile (HPLC grade) was from Fisher Chemicals. Zorbax 300 SB-C8 reversephase HPLC (RP-HPLC) column (5 Am; 25 0.46 cm) was from Agilent. Micrococcus lysodeikticus was obtained from Institute of Microbiology, the Chinese Academy of Sciences. Chicken lysozyme (EC 3.2.1.17) was purchased from Fluka and bovine serum albumin (BSA) was from Sigma-Aldrich. RNeasy Midi Kit was purchased from Qiagen and SuperScripti Plasmid System was from Invitrogen. All other reagents used were of the highest purity available. 2.2. Animals and secretions collection Adult specimen of B. andrewsi of both sexes (n = 50; weight range 80– 120 g) were used. The toads were electrically stimulated three times (24 V, 50 Hz) for 5 –10 s each and washed in 0.9% NaCl. The secretions were centrifuged at 10,000g at 4 -C for 30 min. The supernatant was filtered and lyophilized and stored at 80 -C. 2.3. Purification of BA-lysozyme The lyophilized skin secretions were dissolved in 50 mL of 50 mM Tris– HCl, pH 7.8 buffer, dialyzed against the same buffer and then applied on a DEAE-Sephadex A-50 ion exchanger chromatography column (3.5 50 cm), equilibrated with the same buffer. The elution was firstly performed with two column volumes of the same buffer without NaCl gradient. Proteins were monitored at 280 nm. The fractions containing lysozyme activity were collected and concentrated, then applied on a Sephadex G-50 gel filtration column (2.6 100 cm) pre-equilibrated with 50 mM PBS, pH 6.0 buffer, containing 100 mM NaCl. Fractions with lysozyme activity were lyophilized, then dissolved in water and applied to a Zorbax 300 SB-C8 reverse-phase HPLC (RP-HPLC) column (5 Am; 25 0.46 cm) equilibrated with 0.1% (v/v) trifluoroacetic acid/water. Elution was carried out with an acetonitrile linear gradient at a flow rate of 0.7 mL/min. Proteins were detected by monitoring absorbance at 220 nm. 2.4. SDS-polyacrylamide gel electrophoresis Fifteen percentage of non-continuous SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was used to determine the apparent molecular weight of BA-lysozyme according to the method of Laemmli (1970). Proteins were mixed with 6 reducing loading buffer (1% SDS, 30% glycerol, 0.28 M Tris – HCl, pH 6.8, 0.001% bromphenol blue, 0.5 M DTT) or 6 non-reducing loading buffer (same as reducing loading buffer,
The amino acid sequence of N-terminal was determined by automated Edman degradation on an Applied Biosystems sequencer (model 476A). 2.6. Enzyme assays Lysozyme activity was measured with a continuous spectrophotometric assay using M. lysodeikticus bacterial suspension as substrate, as described by Shugar (1952). The initial decrease in 450 nm of the suspensions caused by the lysis of M. lysodeikticus cells was measured at 30 -C for 2 min. A decrease of 0.001 in OD was defined as 1 unit of lysozyme activity. The concentration of the protein was determined by a protein assay kit (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as standard. 2.7. Effects of pH and temperature on enzyme activity Lysozyme activity was determined in 0.1 M PBS buffer at different pHs (5.0 – 8.0). For measuring the effects of temperature on enzyme activity, BA-lysozyme was incubated in 0.1 M PBS buffer (pH 6.0) at a temperature range from 20 -C to 80 -C for 30 min. Enzyme activity was determined at pH 6.0 and 30 -C, using M. lysodeikticus suspension as substrate. 2.8. Antimicrobial assays Standard bacterial and fungal strains used in antimicrobial assays are gram-positive bacterial strain Staphylococcus aureus (ATCC2592), gram-negative bacterial strain Escherichia coli (ATCC25922) and fungal strain Candida albicans (ATCC2002). The purified protein was examined for antimicrobial activity according to the method described before (Lai et al., 2002). Briefly, bacteria were first grown in LB (Luria – Bertani) broth to an OD 600 nm of 0.8. A 10-AL aliquot of the bacteria was then taken and added to 8 mL of fresh LB broth with 0.7% agar and poured over on a 90-mm Petri dish containing 25 mL of 1.5% agar in LB broth. After the top agar hardened, a 20-AL aliquot of the sample (0.5 mg/mL) filtered on a 0.22-Am Millipore filter was dropped onto the surface of the top agar and completely dried before incubated overnight at 37 -C. If the sample contains antimicrobial activity, a clear zone formed on the surface of the top agar representing inhibition of bacterial growth. Minimal inhibitory concentration (MIC) was determined in liquid LB medium by incubating the bacteria in LB broth with various amounts of the sample tested. The MIC at which no visible growth occurred was recorded. 2.9. Construction and screening of a cDNA library mRNAs were prepared from the total RNA extracted from the skin of a single B. andrewsi by oligo(dT) cellulose
48
Y. Zhao et al. / Comparative Biochemistry and Physiology, Part C 142 (2006) 46 – 52
chromatography with a RNA purification kit. A directional cDNA library was constructed with plasmid cloning kit (SuperScripti Plasmid System, Invitrogen) with some modifications as described previously (Zhang et al., 1995). Briefly, linkers containing a NotI restriction site and a SalI site were added to size-selected cDNAs. These cDNAs were cloned into the pSPORT1 vector (Gibco-BRL) by insertion at the NotI and SalI site of pSPORT1 vector arms and used to transform E. coli DH52 competent cells, producing a library of about 4 107 independent colonies. Degenerate primer (5V-GCNAARGCTYTGAARAARGG-3V) was designed according to N-terminal sequence of BAlysozyme (AKALKKG) and a vector SP6 promoter primer (5V-CATACGATTTAGGTGACACTATAG-3V, in the antisense direction) located in 3Vof the cloned insert, were used in PCR screening in the cDNA library of B. andrewsi skin. Positive clones were picked and applied to DNA sequencing. 2.10. Confirmation of the deduced protein sequences by peptide mass fingerprinting Peptide mass fingerprinting analysis was conducted as described previously (Lee et al., 2003). Briefly, purified BAlysozyme (10 Ag) was run on a 15% SDS-PAGE gel and the corresponding band was cut and the rest of the steps were according to the standard procedure provided by the manufacture. Proteomics Solution I system (PSI) (Applied Biosystems) was used to detect the corresponding trypsin-digested peptide masses. Peptide mass detecting range was among 800 –3000 Da. 2.11. Multiple alignment and phylogenetic sequence analyses The amino acid sequences of BA-lysozyme and other reported c-type lysozymes of various organisms from the Protein Information Resource and Swiss-PROT were aligned using Clustal W (Thompson et al., 1994). Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 3.0 with neighbor-joining method (Kumar et al., 2004).
Fig. 1. Purification of BA-lysozyme from Bufo andrewsi skin secretions. (A) G50 gelfiltration chromatography. The NaCl-free fractions of DEAE Sephadex A50 were collected and concentrated, then applied on a Sephadex G-50 gel filtration column (2.6 100 cm) pre-equilibrated with 0.05 M PBS, pH 6.0 buffer, containing 0.1 M NaCl. Elution was achieved with the same buffer at a flow rate of 22 mL/h. Four protein peaks were observed and peak III showed lysozyme activity. (B) The pooled peak III from Sephadex G-50 was concentrated and applied to a Zorbax 300 SB-C8 reverse-phase HPLC (RPHPLC) column (5 Am; 25 0.46 cm), equilibrated with 0.1% (v/v) trifluoroacetic acid/water. Elution was performed with an acetonitrile linear gradient at a flow rate of 0.7 mL/min. Lysozyme activity was found in the peak indicated by the arrow. Insert: SDS-PAGE profile of BA-lysozyme. Lane 1, molecular mass markers. Lane 2, reducing BA-lysozyme. Lane 3, non-reducing BA-lysozyme.
3.2. Lysozyme activity, optimum pH and thermal stability 3. Results 3.1. Purification of BA-lysozyme The secretions dissolved in 50 mM Tris – HCl (pH 7.8) were applied to the DEAE-Sephadex A-50 column pre-equilibrated by the same buffer. The lysozyme activity was detected in the unabsorbed fractions. These fractions were collected, concentrated, and loaded on a gel filtration column (Sephadex G-50). This chromatography step resolved in four peaks and lysozyme activity was concentrated in peak III (Fig. 1A). Then the peak of interest was pooled and applied to a reverse-phase C8 column. Fraction with lysozyme activity was eluted at 35% acetonitrile (Fig. 1B). The final preparation was subjected to SDS-PAGE analysis and a single band corresponding to a molecular mass of ¨ 15 kDa was detected after Coomassie blue staining (Fig. 1B: insert). The N-terminal amino acid sequence of BA-lysozyme was determined to be QKYERXELAKALKKGGL.
The activity of purified BA-lysozyme, as determined by the turbidometric method of Shugar (1952), was determined to be 2.7 105 units/mg protein. The lytic activity of BA-lysozyme against M. lysodeikticus was examined at various pHs (pH 5.0– pH 8.0). As shown in Fig. 2, BA-lysozyme displayed highest lytic activity at pH 6.0. The thermal stability of BAlysozyme lytic activity was measured in the range of 20 -C – 80 -C (Fig. 3). It was stable between 20 and 70 -C, and it was significantly inactivated at 80 -C. 3.3. Antimicrobial assays BA-lysozyme exerted antibacterial activity towards both gram-positive (S. aureus) and gram-negative bacteria (E. coli) with minimum inhibitory concentration (MIC) of 1 and 8 AM, respectively. However, it had no effect on tested fungal stain C. albicans.
Y. Zhao et al. / Comparative Biochemistry and Physiology, Part C 142 (2006) 46 – 52
49
Fig. 2. Effects of pH on enzyme activity. Lysozyme activity was determined in 0.1 M PBS buffer at the indicated pH, and is shown as % of the highest activity.
3.4. Cloning of cDNA encoding BA-lysozyme and conformation of deduced amino acid sequence by peptide mass fingerprinting The cDNA sequence of BA-lysozyme obtained is 751-base long, including a 438-bp opening reading frame and 3V noncoding region. The cDNA codes a polypeptide of 146 amino acid residues (Fig. 4). According to the determined N-terminal sequence of the purified protein, the mature protein of BAlysozyme is composed of 130 amino acid residues, beginning with Gln. The endoproteinase trypsin digests of BA-lysozyme were analyzed by MALDI-TOF mass spectrometer. The obtained trypsin-digested peptide masses match well with calculated theoretical masses for BA-lysozyme internal peptides, thereby confirming the identity of the obtained cDNA (Table 1). Its molecular mass, computed from the amino acid sequence, is 14745.54 Da, and the theoretic isoelectric point is 9.1. 3.5. Multiple alignment and phylogenetic sequences analysis A BLAST search with the sequence of BA-lysozyme in the sequence database of the National Center for Biotechnology
Fig. 4. Nucleotide sequence and deduced amino acid sequence of the BAlysozyme precursor protein. The nucleotides of protein coding region are shown by uppercase letters and those of noncoding region by lowercase letters.
Information (NCBI) shows that the most similar protein is the lysozyme of the soft-shelled turtle Trionyx sinensis japonicus, the only identified reptile lysozyme with complete protein sequence (Araki et al., 1998). BA-lysozyme also exhibits a medium degree of sequence identity with lysozymes from African clawed frog (61%), human (56%) and chicken eggwhite (56%) (Fig. 5). A phylogenetic tree based on the multiple sequence alignment (data not shown) was constructed as shown in Fig. 6. 4. Discussion Antimicrobial peptides play an important role in the innate immunity that constitutes the first-line defense against invading pathogens for a wide range of vertebrate and invertebrate species (Hoffmann et al., 1999). The synthesis of such peptides in granular glands located in the skin is detected in several anuran species. These peptides have been isolated from the frogs belonging to the families Bombinatoridae, Hylidae, Hyperoliidae, Myobatrachidae, Pipidae, and Ranidae (Simmaco et al., 1998; Duda et al., 2002; Apponyi et al., 2004; Bevier et al., 2004; Conlon et al., 2004a). In contrast, isolation of skin antimicrobial peptide from Bufonidae species has not been reported. Recently, comparative investigations on skin Table 1 Theoretical and observed peptide masses of BA-lysozyme
Fig. 3. Effects of temperature on enzyme activity. BA-lysozyme was incubated in 0.1 M PBS buffer (pH 6.0) at a temperature range from 20 -C to 80 -C for 30 min. Enzyme activity determined at pH 6.0 and 30 -C is shown as % of the highest activity.
Sequence
Theoretical masses (Da)
Observed masses (Da)
51 – 62: STDYGIFQINSR 70 – 82: TPRSKNTCNIDCK 83 – 94 VLLGDDISPAIK 99 – 113: VVSDPNGMGAWVAWK 120 – 129: NLSQWTQGCK
1400.6804 1479.7042 1240.7147 1616.7889 1164.5466
1400.6989 1479.7124 1240.7058 1616.7973 1164.5335
50
Y. Zhao et al. / Comparative Biochemistry and Physiology, Part C 142 (2006) 46 – 52
Fig. 5. Amino acid sequence comparison of chicken, human, turtle and toad lysozymes. Identical residues in four lysozymes are indicated by asterisk (*) and conservative residues are indicated by colon (:). BAL: BA-lysozyme; HEL: chicken lysozyme; HL: human lysozyme; TL: turtle lysozyme.
Fig. 6. Phylogenetic tree of c-type lysozymes. A distance tree (neighbor joining) is presented. Numbers at the nodes represent bootstrap proportions on 1000 replicates. The sequences analyzed here are lysozymes from baboon (Papio anubis, GenBank accession no. P61629), monkey (Macaca mulatta, GenBank accession no. CAA42796), human (Homo sapiens, GenBank accession no. AAA36188), seal (Phoca vitulina, GenBank accession no. CAH39863), bovine (Bos Taurus, GenBank accession no. AAT92538), rabbit (Oryctolagus cuniculus, GenBank accession no. JX0367), sheep (Ovis aries, GenBank accession no. P80190), pig (Sus scrofa, GenBank accession no. NP_999557), rat (Rattus norvegicus, GenBank accession no. NP_036903), mouse (Mus musculus, GenBank accession no. NP_059068), rainbow trout (Oncorhynchus mykiss, GenBank accession no. AAL48290), halibut (Paralichthys olivaceus, GenBank accession no. Q9DD65), torafugu (Takifugu rubripes, GenBank accession no. P61944), Chinese toad (Bufo andrewsi this work), Africa clawed frog (Xenopus laevis, GenBank accession no. AAH72985), turtle (Trionyx gangeticus, PIR Entry JC5493), hen (Gallus gallus, GenBank accession no. 630460A), pheasant (Lophura leucomelanos, GenBank accession no.P24364), goose (Alopochen aegyptiacus, GenBank accession no. JC7953), duck (Aix sponsa, GenBank accession no. Q7LZQ2), fly (Musca domestica, GenBank accession no. AAQ20048), moth (Hyalophora cecropia, GenBank accession no. P05105) and mosquito (Anopheles gambiae, GenBank accession no. XP_308448).
Y. Zhao et al. / Comparative Biochemistry and Physiology, Part C 142 (2006) 46 – 52
secretions of toads of Bombinatoridae and Bufonoidae have been conducted in our lab. Many bioactive peptides, most of which are antimicrobial peptides, have been identified in the skin secretions of Bombina maxima, a Bombinatoridae species (Lai et al., 2002, 2003; Lee et al., 2005a,b; Wang et al., 2005; Zhang et al., 2005). On the contrary, no antimicrobial peptide, as well as other bioactive peptide present in B. maxima skin secretions, has been detected in the skin secretions of B. andrewsi, a Bufonoidae species under the present experimental conditions. Besides antimicrobial peptides, as an immuno-protective polypeptide, lysozyme was also present in the skin of an amphibian species, R. pipiens (Ostrovsky et al., 1976). In this study, an antimicrobial component with a molecular weight of 15 kDa, that is similar to the molecular weight of many lysozymes, was detected and isolated from B. andrewsi skin secretions. The enzymactic assay and sequence determination confirmed its identity as a lysozyme, and then the component was designed as BA-lysozyme. Lysozome belongs to a class of enzymes lyse the cell wall of certain Gram-positive bacteria by splitting h-1,4-glycosidic linkages between N-acetylmuramic acid and N-acetylglucosamine of the peptidoglycan. Due to its catalytic characteristics, lysozyme is much more effective against some Gram-positive bacteria than against Gramnegative bacteria (Hasselberger, 1978; Hughey and Johnson, 1987). Recently, it has been reported that some lysozymes contain peptide sequences which can induce non-catalytic bacterial inhibition (During et al., 1999; Ibrahim et al., 2001; Mine et al., 2004). For examples, the peptides Ile98 – Trp108 and Ala107 – Arg112 in chicken egg-white lysozyme exhibited inhibitory activity against Gram-negative bacterial E.coliK12 (Mine et al., 2004; Hunter et al., 2005). These peptide sequences are conservative in the sequence of BA-lysozyme (Fig. 5). Results of BA-lysozyme antimicrobial activity showed that it has potent bactericidal effect against both Gram-positive and Gram-negative bacteria. This observation indicates that the bactericidal activity of BA-lysozyme might be partially independent of the enzymatic activity, like that of T4 phage lysozyme (During et al., 1999). In the known lysozymes, two acidic residues are essential for lysozyme activity. The first is a glutamate, locating at position 35 (according to hen egg-white lysozyme numbering). The second is the aspartate at position 52, which was supposed to stabilize the intermediate during the enzymatic reaction (Imoto, 1996). Both the catalytic residues are conserved in the sequence of BA-lysozyme. Besides these, residues at some other positions also take roles in lysozyme’s functions, namely, substrate binding, oligosaccharide hydrolysis and transglycosylation (Inoue et al., 1992a,b; Kumagai et al., 1992). For example, Phe34, Arg45, Thr47 and Arg114 are the amino acids contribute to the substrate binding of chicken lysozyme at E and F binding sites. Compared to chicken lysozyme, these positions in BA-lysozyme are substituted to be His34, Tyr45, Pro47, and Tyr114, mostly like that of turtle lysozyme (Araki et al., 1998). To evaluate the evolution relationship between BA-lysozyme and other organism c-type lysozymes, a phylogenetic tree
51
was constructed. In this tree, toad is close to reptile lysozyme and avian lysozymes. According to the tree, c-type lysozymes are clearly classified into six groups, roughly similar to the morphological classification (Fig. 6). In conclusion, a lysozyme has been isolated from Chinese toad B. andrewsi. It is the first time that amphibian skin lysozyme with complete primary structure was reported. Based on its potent bacterialcidal activity, lysozyme, in combination with other antimicrobial organic molecules (Cunha Filho et al., 2005), might play important roles in innate defense system in bufonoid toad skin like antimicrobial peptides in other amphibian skin. Acknowledgements This work was supported by the grants of ‘‘Western Light’’ Projects from The Chinese Academy of Sciences to Dr. Zhang and Dr. Lee; and grants from National Natural Science Foundation (30170195, 30470380, 30570359) and Yunnan Science and Technology Commission (2003C0066M). References Apponyi, M.A., Pukala, T.L., Brinkworth, C.S., Maselli, V.M., Bowie, J.H., Tyler, M.J., Booker, G.W., Wallace, J.C., Carver, J.A., Separovic, F., Doyle, J., Llewellyn, L.E., 2004. Host-defence peptides of Australian anurans: structure, mechanism of action and evolutionary significance. Peptides 25, 1035 – 1054. Araki, T., Yamamoto, T., Torikata, T., 1998. Reptile lysozyme: the complete amino acid sequence of soft-shelled turtle lysozyme and its activity. Biosci. Biotechnol. Biochem. 62, 316 – 324. Bevier, C.R., Sonnevend, A., Kolodziejek, J., Nowotny, N., Nielsen, P.F., Conlon, J.M., 2004. Purification and characterization of antimicrobial peptides from the skin secretions of the mink frog (Rana septentrionalis). Comp. Biochem. Physiol. C 139, 31 – 38. Bevins, C.L., Zasloff, M., 1990. Peptides from frog skin. Annu. Rev. Biochem. 59, 395 – 414. Canfield, R.E., 1963. The amino acid sequence of egg white lysozyme. J. Biol. Chem. 238, 2698 – 2707. Clarke, B.T., 1997. The natural history of amphibian skin secretions, their normal functioning and potential medical applications. Biol. Rev. Camb. Philos. Soc. 72, 365 – 379. Conlon, J.M., Kolodziejek, J., Nowotny, N., 2004a. Antimicrobial peptides from ranid frogs: taxonomic and phylogenetic markers and a potential source of new therapeutic agents. Biochim. Biophys. Acta 1696, 1 – 14. Conlon, J.M., Seidel, B., Nielsen, P.F., 2004b. An atypical member of the brevinin-1 family of antimicrobial peptides isolated from the skin of the European frog Rana dalmatina. Comp. Biochem. Physiol. C 137, 191 – 196. Cunha Filho, G.A., Schwartz, C.A., Resck, I.S., Murta, M.M., Lemos, S.S., Castro, M.S., Kyaw, C., Pires, O.R., Leite Jr., J.R., Bloch, C., Schwartz Jr., E.F., 2005. Antimicrobial activity of the bufadienolides marinobufagin and telocinobufagin isolated as major components from skin secretion of the toad Bufo rubescens. Toxicon 45, 777 – 782. Daly, J.W., 1995. The chemistry of poisons in amphibian skin. Proc. Natl. Acad. Sci. U. S. A. 92, 9 – 13. Duda, T.F., Vanhoye Jr., D., Icolas, N.P., 2002. Roles of diversifying selection and coordinated evolution in the evolution of amphibian antimicrobial peptides. Mol. Biol. Evol. 19, 858 – 864. During, K., Porsch, P., Mahn, A., Brinkmann, O., Gieffers, W., 1999. The nonenzymatic microbicidal activity of lysozymes. FEBS Lett. 449, 93 – 100. Erspamer, V., Melchiorri, P., Falconieri Erspamer, G., Montecucchi, P.C., de Castiglione, R., 1985. Phyllomedusa skin: a huge factory and store-house of a variety of active peptides. Peptides 6, 7 – 12.
52
Y. Zhao et al. / Comparative Biochemistry and Physiology, Part C 142 (2006) 46 – 52
Hasselberger, F.X., 1978. Uses of Enzymes and Immobilized Enzymes. NelsonHall Inc., Chicago. Hoffmann, J.A., Kafatos, F.C., Janeway, C.A., Ezekowitz, R.A., 1999. Phylogenetic perspectives in innate immunity. Science 284, 1313 – 1318. Hughey, V.L., Johnson, E.A., 1987. Antimicrobial activity of lysozyme against bacteria involved in food spoilage and food-borne disease. Appl. Environ. Microbiol. 53, 2165 – 2170. Hunter, H.N., Jing, W., Schibli, D.J., Trinh, T., Park, I.Y., Kim, S.C., Vogel, H.J., 2005. The interactions of antimicrobial peptides derived from lysozyme with model membrane systems. Biochim. Biophys. Acta 1668, 175 – 189. Ibrahim, H.R., Thomas, U., Pellegrini, A., 2001. A helix-loop-helix peptide at the upper lip of the active site cleft of lysozyme confers potent antimicrobial activity with membrane permeabilization action. J. Biol. Chem. 276, 43767 – 43774. Imoto, T., 1996. Engineering of lysozyme. EXS 75, 163 – 181. Inoue, M., Yamada, H., Yasukochi, T., Miki, T., Horiuchi, T., Imoto, T., 1992a. Left-sided substrate binding of lysozyme: evidence for the involvement of asparagine-46 in the initial binding of substrate to chicken lysozyme. Biochemistry 31, 10322 – 10330. Inoue, M., Yamada, H., Yasukochi, T., Kuroki, R., Miki, T., Horiuchi, T., Imoto, T., 1992b. Multiple role of hydrophobicity of tryptophan108 in chicken lysozyme: structural stability, saccharide binding ability, and abnormal pKa of glutamic acid-35. Biochemistry 31, 5545 – 5553. Ito, Y., Yoshikawa, A., Hotani, T., Fukuda, S., Sugimura, K., Imoto, T., 1999. Amino acid sequences of lysozymes newly purified from invertebrates imply wide distribution of a novel class in the lysozyme family. Eur. J. Biochem. 259, 456 – 461. King, J.D., Al-Ghaferi, N., Abraham, B., Sonnevend, A., Leprince, J., Nielsen, P.F., Conlon, J.M., 2005. Pentadactylin: an antimicrobial peptide from the skin secretions of the South American bullfrog Leptodactylus pentadactylus. Comp. Biochem. Physiol. C 141 (4), 393 – 397. Kumagai, I., Sunada, F., Takeda, S., Miura, K., 1992. Redesign of the substratebinding site of hen egg white lysozyme based on the molecular evolution of C-type lysozymes. J. Biol. Chem. 267, 4608 – 4612. Kumar, S., Tamura, K., Nei, M., 2004. MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief. Bioinform. 5, 150 – 163. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 – 685. Lai, R., Zheng, Y.T., Shen, J.H., Liu, G.J., Liu, H., Lee, W.H., Tang, S.Z., Zhang, Y., 2002. Antimicrobial peptides from skin secretions of Chinese red belly toad Bombina maxima. Peptides 23, 427 – 435.
Lai, R., Liu, H., Lee, W.H., Zhang, Y., 2003. Bombinakinin M gene associated peptide, a novel bioactive peptide from skin secretions of the toad Bombina maxima. Peptides 24, 199 – 204. Lee, W.H., Zhuang, Q.Y., Zhang, Y., 2003. Cloning and characterization of a blood coagulation factor IX-binding protein from the venom of Trimeresurus stejnegeri. Toxicon 41, 765 – 772. Lee, W.H., Liu, S.B., Shen, J.H., Jin, Y., Zhang, Y., 2005a. Cloning of bradykinin precursor cDNAs from skin of Bombina maxima reveals novel Bombinakinin M antagonists and a bradykinin potential peptide. Regul. Pept. 127, 207 – 215. Lee, W.H., Li, Y., Lai, R., Li, S., Zhang, Y., Wang, W., 2005b. Variety of antimicrobial peptides in the Bombina maxima toad and evidence of their rapid diversification. Eur. J. Immunol. 35, 1220 – 1229. Mine, Y., Ma, F., Lauriau, S., 2004. Antimicrobial peptides released by enzymatic hydrolysis of hen egg white lysozyme. J. Agric. Food Chem. 52, 1088 – 1094. Ostrovsky, D.S., Snyder, J.A., Iwata, T., Izaka, K.I., Maglott, D.S., Nace, G.W., 1976. Frog lysozyme: I. Its identification, occurrence as isozymes, and quantitative distribution in tissues of the leopard frog, Rana pipiens. J. Exp. Zool. 195, 279 – 290. Shugar, D., 1952. The measurement of lysozyme activity and the ultra-violet inactivation of lysozyme. Biochim. Biophys. Acta 8, 302 – 309. Simmaco, M., Mignogna, G., Barra, D., 1998. Antimicrobial peptides from amphibian skin: what do they tell us? Biopolymers 47, 435 – 450. Simpson, R.J., Begg, G.S., Dorow, D.S., Morgan, F.J., 1980. Complete amino acid sequence of the goose-type lysozyme from the egg white of the black swan. Biochemistry 19, 1814 – 1819. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673 – 4680. Wang, T., Zhang, J., Shen, J.H., Jin, Y., Lee, W.H., Zhang, Y., 2005. Maximins S, a novel group of antimicrobial peptides from toad Bombina maxima. Biochem. Biophys. Res. Commun. 327, 945 – 951. Zhang, Y., Wisner, A., Xiong, Y., Bon, C., 1995. A novel plasminogen activator from snake venom. Purification, characterization, and molecular cloning. J. Biol. Chem. 270, 10246 – 10255. Zhang, J., Zhang, Y., Wan, S.G., Wei, S.S., Lee, W.H., Zhang, Y., 2005. BmTFF2, a trefoil factor protein with platelet activation activity from frog Bombina maxima skin secretions. Biochem. Biophys. Res. Commun. 330, 1027 – 1033. Zhao, Y., Jin, Y., Lee, W.H., Zhang, Y., 2005. Isolation and preliminary characterization of a 22-kDa protein with trypsin inhibitory activity from toad Bufo andrewsi skin. Toxicon 46, 277 – 281.
Purification of a lysozyme from skin secretions of Bufo andrewsi Yu Zhao a,b, Yang Jin a, Wen-Hui Lee a, Yun Zhang a,* a
Department of Animal Toxinology, Kunming Institute of Zoology, The Chinese Academy of Sciences, 32 East Jiao Chang Road Kunming, Yunnan 650223, China b Graduate School of the Chinese Academy of Sciences, Beijing 100039, China Received 2 July 2005; received in revised form 2 October 2005; accepted 3 October 2005 Available online 23 November 2005
Abstract A novel toad lysozyme (named BA-lysozyme) was purified from skin secretions of Bufo andrewsi by a three-step chromatography procedure. BA-lysozyme is a single chain protein and the apparent molecular weight is about 15 kDa as judged by SDS-PAGE. The specific lytic activity against Micrococcus lysodeikticus of BA-lysozyme is 2.7 105 units/mg, indicating that it is a potent lysozyme. It displayed potent bactericidal activity against Staphylococcus aureus and Escherichia coli with minimal inhibitory concentrations (MIC) of 1 and 8 AM, respectively. The deduced primary structure of BA-lysozyme from cloned cDNA was confirmed by N-terminal sequencing and peptide mass fingerprinting. Its amino acid sequence shares 56.5% identity with that of chicken egg-white lysozyme. Phylogenetic analysis indicates that B. andrewsi lysozyme is closely related to that of turtle. This is the first report on the isolation and primary structure determination of amphibian lysozyme. D 2005 Elsevier Inc. All rights reserved. Keywords: Lysozyme; cDNA; Bufo andrewsi; Skin secretions
1. Introduction Amphibian skin is a morphologically, biochemically and physiologically complex organ which fulfills a wide range of functions necessary for amphibian survival, including respiration, water regulation, anti-predator, antimicrobial defense, excretion, temperature control, etc. (Clarke, 1997). Skin gland secretions are a primary source of amphibian biochemical, and numerous peptides with diverse biological activities have been isolated from amphibian skin (Erspamer et al., 1985; Bevins and Zasloff, 1990). In particular, numerous antimicrobial peptides have been found in the skin secretions of many amphibian species that are responsible for the innate immune defense, and most of them have a molecular weight less than 5000 (Simmaco et al., 1998; Apponyi et al., 2004; Bevier et al., 2004; Conlon et al., 2004a,b; King et al., 2005). Bufonidae is a major family of Anura and widely distributed in the world. Unlike other families of anura, bufonoid toad skin secretions contain much more organic molecules (e.g. bufadienolides or bufadienolide-like steroids, * Corresponding author. Tel.: +86 871 5198515; fax: +86 871 5191823. E-mail address: [email protected] (Y. Zhang). 1532-0456/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2005.10.001
alkaloids and biogentic amines), but less peptides or proteins (Daly, 1995; Clarke, 1997). Recently, investigations on the proteins of bufonoid toad skin secretions have been conducted in our lab, and some bioactive proteins, such as protease inhibitor have been isolated and characterized (Zhao et al., 2005). Lysozyme (EC.3.2.1.17) is widely distributed among eukaryotes and prokaryotes and takes part in protecting microbial infections or digestion. Lysozyme kills bacteria by hydrolyzing h-1,4-glycosidic linkages between N-acetylglucosamine and N-acetylmuramic acid of the peptidoglycan layer in the bacterial cell wall. Lysozymes are classified into three major types: chicken type (c-type) (Canfield, 1963), goose type (g-type) (Simpson et al., 1980) and invertebrate type (i-type) (Ito et al., 1999). The c-type lysozyme has been found in many organisms including virus, bacteria, plants, insects, reptiles, birds and mammals. Lysozyme was also found in the skin of leopard frog (Rana pipiens) two decades ago (Ostrovsky et al., 1976). Since then, there have had no further investigations on amphibian skin lysozyme. In this study, we firstly report the isolation and complete amino acid sequence of an amphibian lysozyme from the skin secretions of a common bufonoid toad, Bufo andrewsi, in China.
Y. Zhao et al. / Comparative Biochemistry and Physiology, Part C 142 (2006) 46 – 52
2. Materials and methods
47
but without DTT). The gel was stained with Coomassie brilliant blue R-250 (Sigma).
2.1. Materials 2.5. Amino acid sequence determination Adult specimens of B. andrewsi of both sexes were collected in Qujing county, Yunnan province of China. DEAE-Sephadex A-50 and Sephadex G-50 (superfine) were purchased from Pharmacia (Sweden). Trifluoroacetic acid (HPLC grade) was from Perkin Elmer. Acetonitrile (HPLC grade) was from Fisher Chemicals. Zorbax 300 SB-C8 reversephase HPLC (RP-HPLC) column (5 Am; 25 0.46 cm) was from Agilent. Micrococcus lysodeikticus was obtained from Institute of Microbiology, the Chinese Academy of Sciences. Chicken lysozyme (EC 3.2.1.17) was purchased from Fluka and bovine serum albumin (BSA) was from Sigma-Aldrich. RNeasy Midi Kit was purchased from Qiagen and SuperScripti Plasmid System was from Invitrogen. All other reagents used were of the highest purity available. 2.2. Animals and secretions collection Adult specimen of B. andrewsi of both sexes (n = 50; weight range 80– 120 g) were used. The toads were electrically stimulated three times (24 V, 50 Hz) for 5 –10 s each and washed in 0.9% NaCl. The secretions were centrifuged at 10,000g at 4 -C for 30 min. The supernatant was filtered and lyophilized and stored at 80 -C. 2.3. Purification of BA-lysozyme The lyophilized skin secretions were dissolved in 50 mL of 50 mM Tris– HCl, pH 7.8 buffer, dialyzed against the same buffer and then applied on a DEAE-Sephadex A-50 ion exchanger chromatography column (3.5 50 cm), equilibrated with the same buffer. The elution was firstly performed with two column volumes of the same buffer without NaCl gradient. Proteins were monitored at 280 nm. The fractions containing lysozyme activity were collected and concentrated, then applied on a Sephadex G-50 gel filtration column (2.6 100 cm) pre-equilibrated with 50 mM PBS, pH 6.0 buffer, containing 100 mM NaCl. Fractions with lysozyme activity were lyophilized, then dissolved in water and applied to a Zorbax 300 SB-C8 reverse-phase HPLC (RP-HPLC) column (5 Am; 25 0.46 cm) equilibrated with 0.1% (v/v) trifluoroacetic acid/water. Elution was carried out with an acetonitrile linear gradient at a flow rate of 0.7 mL/min. Proteins were detected by monitoring absorbance at 220 nm. 2.4. SDS-polyacrylamide gel electrophoresis Fifteen percentage of non-continuous SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was used to determine the apparent molecular weight of BA-lysozyme according to the method of Laemmli (1970). Proteins were mixed with 6 reducing loading buffer (1% SDS, 30% glycerol, 0.28 M Tris – HCl, pH 6.8, 0.001% bromphenol blue, 0.5 M DTT) or 6 non-reducing loading buffer (same as reducing loading buffer,
The amino acid sequence of N-terminal was determined by automated Edman degradation on an Applied Biosystems sequencer (model 476A). 2.6. Enzyme assays Lysozyme activity was measured with a continuous spectrophotometric assay using M. lysodeikticus bacterial suspension as substrate, as described by Shugar (1952). The initial decrease in 450 nm of the suspensions caused by the lysis of M. lysodeikticus cells was measured at 30 -C for 2 min. A decrease of 0.001 in OD was defined as 1 unit of lysozyme activity. The concentration of the protein was determined by a protein assay kit (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as standard. 2.7. Effects of pH and temperature on enzyme activity Lysozyme activity was determined in 0.1 M PBS buffer at different pHs (5.0 – 8.0). For measuring the effects of temperature on enzyme activity, BA-lysozyme was incubated in 0.1 M PBS buffer (pH 6.0) at a temperature range from 20 -C to 80 -C for 30 min. Enzyme activity was determined at pH 6.0 and 30 -C, using M. lysodeikticus suspension as substrate. 2.8. Antimicrobial assays Standard bacterial and fungal strains used in antimicrobial assays are gram-positive bacterial strain Staphylococcus aureus (ATCC2592), gram-negative bacterial strain Escherichia coli (ATCC25922) and fungal strain Candida albicans (ATCC2002). The purified protein was examined for antimicrobial activity according to the method described before (Lai et al., 2002). Briefly, bacteria were first grown in LB (Luria – Bertani) broth to an OD 600 nm of 0.8. A 10-AL aliquot of the bacteria was then taken and added to 8 mL of fresh LB broth with 0.7% agar and poured over on a 90-mm Petri dish containing 25 mL of 1.5% agar in LB broth. After the top agar hardened, a 20-AL aliquot of the sample (0.5 mg/mL) filtered on a 0.22-Am Millipore filter was dropped onto the surface of the top agar and completely dried before incubated overnight at 37 -C. If the sample contains antimicrobial activity, a clear zone formed on the surface of the top agar representing inhibition of bacterial growth. Minimal inhibitory concentration (MIC) was determined in liquid LB medium by incubating the bacteria in LB broth with various amounts of the sample tested. The MIC at which no visible growth occurred was recorded. 2.9. Construction and screening of a cDNA library mRNAs were prepared from the total RNA extracted from the skin of a single B. andrewsi by oligo(dT) cellulose
48
Y. Zhao et al. / Comparative Biochemistry and Physiology, Part C 142 (2006) 46 – 52
chromatography with a RNA purification kit. A directional cDNA library was constructed with plasmid cloning kit (SuperScripti Plasmid System, Invitrogen) with some modifications as described previously (Zhang et al., 1995). Briefly, linkers containing a NotI restriction site and a SalI site were added to size-selected cDNAs. These cDNAs were cloned into the pSPORT1 vector (Gibco-BRL) by insertion at the NotI and SalI site of pSPORT1 vector arms and used to transform E. coli DH52 competent cells, producing a library of about 4 107 independent colonies. Degenerate primer (5V-GCNAARGCTYTGAARAARGG-3V) was designed according to N-terminal sequence of BAlysozyme (AKALKKG) and a vector SP6 promoter primer (5V-CATACGATTTAGGTGACACTATAG-3V, in the antisense direction) located in 3Vof the cloned insert, were used in PCR screening in the cDNA library of B. andrewsi skin. Positive clones were picked and applied to DNA sequencing. 2.10. Confirmation of the deduced protein sequences by peptide mass fingerprinting Peptide mass fingerprinting analysis was conducted as described previously (Lee et al., 2003). Briefly, purified BAlysozyme (10 Ag) was run on a 15% SDS-PAGE gel and the corresponding band was cut and the rest of the steps were according to the standard procedure provided by the manufacture. Proteomics Solution I system (PSI) (Applied Biosystems) was used to detect the corresponding trypsin-digested peptide masses. Peptide mass detecting range was among 800 –3000 Da. 2.11. Multiple alignment and phylogenetic sequence analyses The amino acid sequences of BA-lysozyme and other reported c-type lysozymes of various organisms from the Protein Information Resource and Swiss-PROT were aligned using Clustal W (Thompson et al., 1994). Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 3.0 with neighbor-joining method (Kumar et al., 2004).
Fig. 1. Purification of BA-lysozyme from Bufo andrewsi skin secretions. (A) G50 gelfiltration chromatography. The NaCl-free fractions of DEAE Sephadex A50 were collected and concentrated, then applied on a Sephadex G-50 gel filtration column (2.6 100 cm) pre-equilibrated with 0.05 M PBS, pH 6.0 buffer, containing 0.1 M NaCl. Elution was achieved with the same buffer at a flow rate of 22 mL/h. Four protein peaks were observed and peak III showed lysozyme activity. (B) The pooled peak III from Sephadex G-50 was concentrated and applied to a Zorbax 300 SB-C8 reverse-phase HPLC (RPHPLC) column (5 Am; 25 0.46 cm), equilibrated with 0.1% (v/v) trifluoroacetic acid/water. Elution was performed with an acetonitrile linear gradient at a flow rate of 0.7 mL/min. Lysozyme activity was found in the peak indicated by the arrow. Insert: SDS-PAGE profile of BA-lysozyme. Lane 1, molecular mass markers. Lane 2, reducing BA-lysozyme. Lane 3, non-reducing BA-lysozyme.
3.2. Lysozyme activity, optimum pH and thermal stability 3. Results 3.1. Purification of BA-lysozyme The secretions dissolved in 50 mM Tris – HCl (pH 7.8) were applied to the DEAE-Sephadex A-50 column pre-equilibrated by the same buffer. The lysozyme activity was detected in the unabsorbed fractions. These fractions were collected, concentrated, and loaded on a gel filtration column (Sephadex G-50). This chromatography step resolved in four peaks and lysozyme activity was concentrated in peak III (Fig. 1A). Then the peak of interest was pooled and applied to a reverse-phase C8 column. Fraction with lysozyme activity was eluted at 35% acetonitrile (Fig. 1B). The final preparation was subjected to SDS-PAGE analysis and a single band corresponding to a molecular mass of ¨ 15 kDa was detected after Coomassie blue staining (Fig. 1B: insert). The N-terminal amino acid sequence of BA-lysozyme was determined to be QKYERXELAKALKKGGL.
The activity of purified BA-lysozyme, as determined by the turbidometric method of Shugar (1952), was determined to be 2.7 105 units/mg protein. The lytic activity of BA-lysozyme against M. lysodeikticus was examined at various pHs (pH 5.0– pH 8.0). As shown in Fig. 2, BA-lysozyme displayed highest lytic activity at pH 6.0. The thermal stability of BAlysozyme lytic activity was measured in the range of 20 -C – 80 -C (Fig. 3). It was stable between 20 and 70 -C, and it was significantly inactivated at 80 -C. 3.3. Antimicrobial assays BA-lysozyme exerted antibacterial activity towards both gram-positive (S. aureus) and gram-negative bacteria (E. coli) with minimum inhibitory concentration (MIC) of 1 and 8 AM, respectively. However, it had no effect on tested fungal stain C. albicans.
Y. Zhao et al. / Comparative Biochemistry and Physiology, Part C 142 (2006) 46 – 52
49
Fig. 2. Effects of pH on enzyme activity. Lysozyme activity was determined in 0.1 M PBS buffer at the indicated pH, and is shown as % of the highest activity.
3.4. Cloning of cDNA encoding BA-lysozyme and conformation of deduced amino acid sequence by peptide mass fingerprinting The cDNA sequence of BA-lysozyme obtained is 751-base long, including a 438-bp opening reading frame and 3V noncoding region. The cDNA codes a polypeptide of 146 amino acid residues (Fig. 4). According to the determined N-terminal sequence of the purified protein, the mature protein of BAlysozyme is composed of 130 amino acid residues, beginning with Gln. The endoproteinase trypsin digests of BA-lysozyme were analyzed by MALDI-TOF mass spectrometer. The obtained trypsin-digested peptide masses match well with calculated theoretical masses for BA-lysozyme internal peptides, thereby confirming the identity of the obtained cDNA (Table 1). Its molecular mass, computed from the amino acid sequence, is 14745.54 Da, and the theoretic isoelectric point is 9.1. 3.5. Multiple alignment and phylogenetic sequences analysis A BLAST search with the sequence of BA-lysozyme in the sequence database of the National Center for Biotechnology
Fig. 4. Nucleotide sequence and deduced amino acid sequence of the BAlysozyme precursor protein. The nucleotides of protein coding region are shown by uppercase letters and those of noncoding region by lowercase letters.
Information (NCBI) shows that the most similar protein is the lysozyme of the soft-shelled turtle Trionyx sinensis japonicus, the only identified reptile lysozyme with complete protein sequence (Araki et al., 1998). BA-lysozyme also exhibits a medium degree of sequence identity with lysozymes from African clawed frog (61%), human (56%) and chicken eggwhite (56%) (Fig. 5). A phylogenetic tree based on the multiple sequence alignment (data not shown) was constructed as shown in Fig. 6. 4. Discussion Antimicrobial peptides play an important role in the innate immunity that constitutes the first-line defense against invading pathogens for a wide range of vertebrate and invertebrate species (Hoffmann et al., 1999). The synthesis of such peptides in granular glands located in the skin is detected in several anuran species. These peptides have been isolated from the frogs belonging to the families Bombinatoridae, Hylidae, Hyperoliidae, Myobatrachidae, Pipidae, and Ranidae (Simmaco et al., 1998; Duda et al., 2002; Apponyi et al., 2004; Bevier et al., 2004; Conlon et al., 2004a). In contrast, isolation of skin antimicrobial peptide from Bufonidae species has not been reported. Recently, comparative investigations on skin Table 1 Theoretical and observed peptide masses of BA-lysozyme
Fig. 3. Effects of temperature on enzyme activity. BA-lysozyme was incubated in 0.1 M PBS buffer (pH 6.0) at a temperature range from 20 -C to 80 -C for 30 min. Enzyme activity determined at pH 6.0 and 30 -C is shown as % of the highest activity.
Sequence
Theoretical masses (Da)
Observed masses (Da)
51 – 62: STDYGIFQINSR 70 – 82: TPRSKNTCNIDCK 83 – 94 VLLGDDISPAIK 99 – 113: VVSDPNGMGAWVAWK 120 – 129: NLSQWTQGCK
1400.6804 1479.7042 1240.7147 1616.7889 1164.5466
1400.6989 1479.7124 1240.7058 1616.7973 1164.5335
50
Y. Zhao et al. / Comparative Biochemistry and Physiology, Part C 142 (2006) 46 – 52
Fig. 5. Amino acid sequence comparison of chicken, human, turtle and toad lysozymes. Identical residues in four lysozymes are indicated by asterisk (*) and conservative residues are indicated by colon (:). BAL: BA-lysozyme; HEL: chicken lysozyme; HL: human lysozyme; TL: turtle lysozyme.
Fig. 6. Phylogenetic tree of c-type lysozymes. A distance tree (neighbor joining) is presented. Numbers at the nodes represent bootstrap proportions on 1000 replicates. The sequences analyzed here are lysozymes from baboon (Papio anubis, GenBank accession no. P61629), monkey (Macaca mulatta, GenBank accession no. CAA42796), human (Homo sapiens, GenBank accession no. AAA36188), seal (Phoca vitulina, GenBank accession no. CAH39863), bovine (Bos Taurus, GenBank accession no. AAT92538), rabbit (Oryctolagus cuniculus, GenBank accession no. JX0367), sheep (Ovis aries, GenBank accession no. P80190), pig (Sus scrofa, GenBank accession no. NP_999557), rat (Rattus norvegicus, GenBank accession no. NP_036903), mouse (Mus musculus, GenBank accession no. NP_059068), rainbow trout (Oncorhynchus mykiss, GenBank accession no. AAL48290), halibut (Paralichthys olivaceus, GenBank accession no. Q9DD65), torafugu (Takifugu rubripes, GenBank accession no. P61944), Chinese toad (Bufo andrewsi this work), Africa clawed frog (Xenopus laevis, GenBank accession no. AAH72985), turtle (Trionyx gangeticus, PIR Entry JC5493), hen (Gallus gallus, GenBank accession no. 630460A), pheasant (Lophura leucomelanos, GenBank accession no.P24364), goose (Alopochen aegyptiacus, GenBank accession no. JC7953), duck (Aix sponsa, GenBank accession no. Q7LZQ2), fly (Musca domestica, GenBank accession no. AAQ20048), moth (Hyalophora cecropia, GenBank accession no. P05105) and mosquito (Anopheles gambiae, GenBank accession no. XP_308448).
Y. Zhao et al. / Comparative Biochemistry and Physiology, Part C 142 (2006) 46 – 52
secretions of toads of Bombinatoridae and Bufonoidae have been conducted in our lab. Many bioactive peptides, most of which are antimicrobial peptides, have been identified in the skin secretions of Bombina maxima, a Bombinatoridae species (Lai et al., 2002, 2003; Lee et al., 2005a,b; Wang et al., 2005; Zhang et al., 2005). On the contrary, no antimicrobial peptide, as well as other bioactive peptide present in B. maxima skin secretions, has been detected in the skin secretions of B. andrewsi, a Bufonoidae species under the present experimental conditions. Besides antimicrobial peptides, as an immuno-protective polypeptide, lysozyme was also present in the skin of an amphibian species, R. pipiens (Ostrovsky et al., 1976). In this study, an antimicrobial component with a molecular weight of 15 kDa, that is similar to the molecular weight of many lysozymes, was detected and isolated from B. andrewsi skin secretions. The enzymactic assay and sequence determination confirmed its identity as a lysozyme, and then the component was designed as BA-lysozyme. Lysozome belongs to a class of enzymes lyse the cell wall of certain Gram-positive bacteria by splitting h-1,4-glycosidic linkages between N-acetylmuramic acid and N-acetylglucosamine of the peptidoglycan. Due to its catalytic characteristics, lysozyme is much more effective against some Gram-positive bacteria than against Gramnegative bacteria (Hasselberger, 1978; Hughey and Johnson, 1987). Recently, it has been reported that some lysozymes contain peptide sequences which can induce non-catalytic bacterial inhibition (During et al., 1999; Ibrahim et al., 2001; Mine et al., 2004). For examples, the peptides Ile98 – Trp108 and Ala107 – Arg112 in chicken egg-white lysozyme exhibited inhibitory activity against Gram-negative bacterial E.coliK12 (Mine et al., 2004; Hunter et al., 2005). These peptide sequences are conservative in the sequence of BA-lysozyme (Fig. 5). Results of BA-lysozyme antimicrobial activity showed that it has potent bactericidal effect against both Gram-positive and Gram-negative bacteria. This observation indicates that the bactericidal activity of BA-lysozyme might be partially independent of the enzymatic activity, like that of T4 phage lysozyme (During et al., 1999). In the known lysozymes, two acidic residues are essential for lysozyme activity. The first is a glutamate, locating at position 35 (according to hen egg-white lysozyme numbering). The second is the aspartate at position 52, which was supposed to stabilize the intermediate during the enzymatic reaction (Imoto, 1996). Both the catalytic residues are conserved in the sequence of BA-lysozyme. Besides these, residues at some other positions also take roles in lysozyme’s functions, namely, substrate binding, oligosaccharide hydrolysis and transglycosylation (Inoue et al., 1992a,b; Kumagai et al., 1992). For example, Phe34, Arg45, Thr47 and Arg114 are the amino acids contribute to the substrate binding of chicken lysozyme at E and F binding sites. Compared to chicken lysozyme, these positions in BA-lysozyme are substituted to be His34, Tyr45, Pro47, and Tyr114, mostly like that of turtle lysozyme (Araki et al., 1998). To evaluate the evolution relationship between BA-lysozyme and other organism c-type lysozymes, a phylogenetic tree
51
was constructed. In this tree, toad is close to reptile lysozyme and avian lysozymes. According to the tree, c-type lysozymes are clearly classified into six groups, roughly similar to the morphological classification (Fig. 6). In conclusion, a lysozyme has been isolated from Chinese toad B. andrewsi. It is the first time that amphibian skin lysozyme with complete primary structure was reported. Based on its potent bacterialcidal activity, lysozyme, in combination with other antimicrobial organic molecules (Cunha Filho et al., 2005), might play important roles in innate defense system in bufonoid toad skin like antimicrobial peptides in other amphibian skin. Acknowledgements This work was supported by the grants of ‘‘Western Light’’ Projects from The Chinese Academy of Sciences to Dr. Zhang and Dr. Lee; and grants from National Natural Science Foundation (30170195, 30470380, 30570359) and Yunnan Science and Technology Commission (2003C0066M). References Apponyi, M.A., Pukala, T.L., Brinkworth, C.S., Maselli, V.M., Bowie, J.H., Tyler, M.J., Booker, G.W., Wallace, J.C., Carver, J.A., Separovic, F., Doyle, J., Llewellyn, L.E., 2004. Host-defence peptides of Australian anurans: structure, mechanism of action and evolutionary significance. Peptides 25, 1035 – 1054. Araki, T., Yamamoto, T., Torikata, T., 1998. Reptile lysozyme: the complete amino acid sequence of soft-shelled turtle lysozyme and its activity. Biosci. Biotechnol. Biochem. 62, 316 – 324. Bevier, C.R., Sonnevend, A., Kolodziejek, J., Nowotny, N., Nielsen, P.F., Conlon, J.M., 2004. Purification and characterization of antimicrobial peptides from the skin secretions of the mink frog (Rana septentrionalis). Comp. Biochem. Physiol. C 139, 31 – 38. Bevins, C.L., Zasloff, M., 1990. Peptides from frog skin. Annu. Rev. Biochem. 59, 395 – 414. Canfield, R.E., 1963. The amino acid sequence of egg white lysozyme. J. Biol. Chem. 238, 2698 – 2707. Clarke, B.T., 1997. The natural history of amphibian skin secretions, their normal functioning and potential medical applications. Biol. Rev. Camb. Philos. Soc. 72, 365 – 379. Conlon, J.M., Kolodziejek, J., Nowotny, N., 2004a. Antimicrobial peptides from ranid frogs: taxonomic and phylogenetic markers and a potential source of new therapeutic agents. Biochim. Biophys. Acta 1696, 1 – 14. Conlon, J.M., Seidel, B., Nielsen, P.F., 2004b. An atypical member of the brevinin-1 family of antimicrobial peptides isolated from the skin of the European frog Rana dalmatina. Comp. Biochem. Physiol. C 137, 191 – 196. Cunha Filho, G.A., Schwartz, C.A., Resck, I.S., Murta, M.M., Lemos, S.S., Castro, M.S., Kyaw, C., Pires, O.R., Leite Jr., J.R., Bloch, C., Schwartz Jr., E.F., 2005. Antimicrobial activity of the bufadienolides marinobufagin and telocinobufagin isolated as major components from skin secretion of the toad Bufo rubescens. Toxicon 45, 777 – 782. Daly, J.W., 1995. The chemistry of poisons in amphibian skin. Proc. Natl. Acad. Sci. U. S. A. 92, 9 – 13. Duda, T.F., Vanhoye Jr., D., Icolas, N.P., 2002. Roles of diversifying selection and coordinated evolution in the evolution of amphibian antimicrobial peptides. Mol. Biol. Evol. 19, 858 – 864. During, K., Porsch, P., Mahn, A., Brinkmann, O., Gieffers, W., 1999. The nonenzymatic microbicidal activity of lysozymes. FEBS Lett. 449, 93 – 100. Erspamer, V., Melchiorri, P., Falconieri Erspamer, G., Montecucchi, P.C., de Castiglione, R., 1985. Phyllomedusa skin: a huge factory and store-house of a variety of active peptides. Peptides 6, 7 – 12.
52
Y. Zhao et al. / Comparative Biochemistry and Physiology, Part C 142 (2006) 46 – 52
Hasselberger, F.X., 1978. Uses of Enzymes and Immobilized Enzymes. NelsonHall Inc., Chicago. Hoffmann, J.A., Kafatos, F.C., Janeway, C.A., Ezekowitz, R.A., 1999. Phylogenetic perspectives in innate immunity. Science 284, 1313 – 1318. Hughey, V.L., Johnson, E.A., 1987. Antimicrobial activity of lysozyme against bacteria involved in food spoilage and food-borne disease. Appl. Environ. Microbiol. 53, 2165 – 2170. Hunter, H.N., Jing, W., Schibli, D.J., Trinh, T., Park, I.Y., Kim, S.C., Vogel, H.J., 2005. The interactions of antimicrobial peptides derived from lysozyme with model membrane systems. Biochim. Biophys. Acta 1668, 175 – 189. Ibrahim, H.R., Thomas, U., Pellegrini, A., 2001. A helix-loop-helix peptide at the upper lip of the active site cleft of lysozyme confers potent antimicrobial activity with membrane permeabilization action. J. Biol. Chem. 276, 43767 – 43774. Imoto, T., 1996. Engineering of lysozyme. EXS 75, 163 – 181. Inoue, M., Yamada, H., Yasukochi, T., Miki, T., Horiuchi, T., Imoto, T., 1992a. Left-sided substrate binding of lysozyme: evidence for the involvement of asparagine-46 in the initial binding of substrate to chicken lysozyme. Biochemistry 31, 10322 – 10330. Inoue, M., Yamada, H., Yasukochi, T., Kuroki, R., Miki, T., Horiuchi, T., Imoto, T., 1992b. Multiple role of hydrophobicity of tryptophan108 in chicken lysozyme: structural stability, saccharide binding ability, and abnormal pKa of glutamic acid-35. Biochemistry 31, 5545 – 5553. Ito, Y., Yoshikawa, A., Hotani, T., Fukuda, S., Sugimura, K., Imoto, T., 1999. Amino acid sequences of lysozymes newly purified from invertebrates imply wide distribution of a novel class in the lysozyme family. Eur. J. Biochem. 259, 456 – 461. King, J.D., Al-Ghaferi, N., Abraham, B., Sonnevend, A., Leprince, J., Nielsen, P.F., Conlon, J.M., 2005. Pentadactylin: an antimicrobial peptide from the skin secretions of the South American bullfrog Leptodactylus pentadactylus. Comp. Biochem. Physiol. C 141 (4), 393 – 397. Kumagai, I., Sunada, F., Takeda, S., Miura, K., 1992. Redesign of the substratebinding site of hen egg white lysozyme based on the molecular evolution of C-type lysozymes. J. Biol. Chem. 267, 4608 – 4612. Kumar, S., Tamura, K., Nei, M., 2004. MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief. Bioinform. 5, 150 – 163. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 – 685. Lai, R., Zheng, Y.T., Shen, J.H., Liu, G.J., Liu, H., Lee, W.H., Tang, S.Z., Zhang, Y., 2002. Antimicrobial peptides from skin secretions of Chinese red belly toad Bombina maxima. Peptides 23, 427 – 435.
Lai, R., Liu, H., Lee, W.H., Zhang, Y., 2003. Bombinakinin M gene associated peptide, a novel bioactive peptide from skin secretions of the toad Bombina maxima. Peptides 24, 199 – 204. Lee, W.H., Zhuang, Q.Y., Zhang, Y., 2003. Cloning and characterization of a blood coagulation factor IX-binding protein from the venom of Trimeresurus stejnegeri. Toxicon 41, 765 – 772. Lee, W.H., Liu, S.B., Shen, J.H., Jin, Y., Zhang, Y., 2005a. Cloning of bradykinin precursor cDNAs from skin of Bombina maxima reveals novel Bombinakinin M antagonists and a bradykinin potential peptide. Regul. Pept. 127, 207 – 215. Lee, W.H., Li, Y., Lai, R., Li, S., Zhang, Y., Wang, W., 2005b. Variety of antimicrobial peptides in the Bombina maxima toad and evidence of their rapid diversification. Eur. J. Immunol. 35, 1220 – 1229. Mine, Y., Ma, F., Lauriau, S., 2004. Antimicrobial peptides released by enzymatic hydrolysis of hen egg white lysozyme. J. Agric. Food Chem. 52, 1088 – 1094. Ostrovsky, D.S., Snyder, J.A., Iwata, T., Izaka, K.I., Maglott, D.S., Nace, G.W., 1976. Frog lysozyme: I. Its identification, occurrence as isozymes, and quantitative distribution in tissues of the leopard frog, Rana pipiens. J. Exp. Zool. 195, 279 – 290. Shugar, D., 1952. The measurement of lysozyme activity and the ultra-violet inactivation of lysozyme. Biochim. Biophys. Acta 8, 302 – 309. Simmaco, M., Mignogna, G., Barra, D., 1998. Antimicrobial peptides from amphibian skin: what do they tell us? Biopolymers 47, 435 – 450. Simpson, R.J., Begg, G.S., Dorow, D.S., Morgan, F.J., 1980. Complete amino acid sequence of the goose-type lysozyme from the egg white of the black swan. Biochemistry 19, 1814 – 1819. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673 – 4680. Wang, T., Zhang, J., Shen, J.H., Jin, Y., Lee, W.H., Zhang, Y., 2005. Maximins S, a novel group of antimicrobial peptides from toad Bombina maxima. Biochem. Biophys. Res. Commun. 327, 945 – 951. Zhang, Y., Wisner, A., Xiong, Y., Bon, C., 1995. A novel plasminogen activator from snake venom. Purification, characterization, and molecular cloning. J. Biol. Chem. 270, 10246 – 10255. Zhang, J., Zhang, Y., Wan, S.G., Wei, S.S., Lee, W.H., Zhang, Y., 2005. BmTFF2, a trefoil factor protein with platelet activation activity from frog Bombina maxima skin secretions. Biochem. Biophys. Res. Commun. 330, 1027 – 1033. Zhao, Y., Jin, Y., Lee, W.H., Zhang, Y., 2005. Isolation and preliminary characterization of a 22-kDa protein with trypsin inhibitory activity from toad Bufo andrewsi skin. Toxicon 46, 277 – 281.