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Expression of genes encoding antimicrobial peptides in the Harderian gland of the bullfrog Lithobates catesbeianus
Comparative Biochemistry and Physiology, Part C 152 (2010) 301–305
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Comparative Biochemistry and Physiology, Part C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p c
Expression of genes encoding antimicrobial peptides in the Harderian gland of the bullfrog Lithobates catesbeianus Itaru Hasunuma a, Shawichi Iwamuro b,⁎, Tetsuya Kobayashi c, Kazuhiko Shirama d, J. Michael Conlon e, Sakae Kikuyama a,b a Department of Biology, Faculty of Education and Integrated Arts and Sciences, Center for Advanced Biomedical Sciences, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan b Department of Biology, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan c Department of Regulatory Biology, Faculty of Sciences, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan d Department of Histology and Neuroanatomy, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-0023, Japan e Department of Biochemistry, Faculty of Medicine and Health Sciences, United Arab Emirates University, 17666 Al-Ain, United Arab Emirates
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Article history: Received 6 May 2010 Received in revised form 18 May 2010 Accepted 18 May 2010 Available online 25 May 2010 Keywords: Harderian gland Bullfrog Antimicrobial peptide Temporin Chensirin-2
a b s t r a c t The Harderian gland is an orbital gland found in many tetrapod species that possess a nictitating membrane. While the main role of the Harderian gland is lubrication of the eyeballs, numerous other functions are attributed to this gland. In amphibians, mast cells have been detected in the Harderian gland, suggesting that the gland is involved in the host's system of innate immunity defending against microbial invasions. Using reverse-transcription polymerase chain reaction, we cloned from the bullfrog Harderian gland total RNA preparations, cDNAs encoding biosynthetic precursors for the antimicrobial peptides temporin-CBa (FLPIASLLGKYL-NH2), previously isolated from an extract of bullfrog skin, and chensirin-2CBa (IIPLPLGYFAKKP) that contained the amino acid substitution Thr13 → Pro compared with chensirin-2 from the Chinese brown frog, Rana chensinensis. By means of in situ hybridization using digoxigenin-labeled cRNA probes for preprotemporin-CBa and preprochensirin-2CBa, we have demonstrated for the first time in an amphibian the presence of mRNAs encoding these two precursors in the cytoplasm of the glandular cells in the bullfrog Harderian gland. © 2010 Elsevier Inc. All rights reserved.
1. Introduction The vertebrate eye is situated at the interface between the organism and its environment and as so requires defensive systems to protect against invasion by pathogenic microorganisms in the environment. Amphibians have a worldwide distribution, occupying aquatic, semiaquatic, and terrestrial environments and thus are exposed to a wide range of microorganisms. The Harderian gland (HG) is an orbital gland found in many tetrapod species that possess a nictitating membrane (Harder, 1694). Although the function of HG is still not completely clear, its size and widespread occurrence among vertebrates suggest potentially important roles. In fact, numerous functions are attributed to this gland such as lubrication of the eye and nictitating membrane, a source of pheromones and growth factors, as well as osmoregulatory, thermoregulatory and immunoregulatory functions (Payne, 1994; Chieffi et al., 1996). In amphibians, the HG is a seromucoid acinar gland located at the medial corner of the orbit and consists of a single type of epithelial cells (Chieffi Baccari et al., 1991; Payne 1994). It is known to show seasonal
⁎ Corresponding author. Tel.: + 81 47 472 5206; fax: + 81 47 472 1188. E-mail address: [email protected] (S. Iwamuro). 1532-0456/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2010.05.005
changes in secretory activity in a temperature-dependent manner (Minucci et al., 1990). Because of these seasonal changes and its spatial localization in the orbit, it is speculated that the amphibian HG may be involved in the innate immune system of the host's eyes, defending against pathogenic microorganisms encountered in the environment. Antimicrobial peptides (AMPs) are gene-encoded polypeptides of various lengths and structures found in all organisms including vertebrates, invertebrates, plants, and bacteria. In vertebrates, these molecules are important components of the innate immune system of the organisms and protect against microbial infections before adaptive immunity is activated (Auvynet and Rosenstein, 2009). AMPs can target and neutralize a broad range of microorganisms by mechanisms that involve non-specific interactions with the cell membrane. Although there are no obvious conserved amino acid sequences among these peptides, most AMPs are hydrophobic and cationic, and have a propensity to form an amphipathic helical conformation in a membrane-mimetic environment (Brogden et al., 2005). Amphibians are a promising source of AMPs. Based on limited amino acid sequence similarities, AMPs in ranid frogs are classified into several well-established groups that include the temporin, brevinin, ranatuerin, palustrin, esculentin, nigrocin, and japonicin families (Conlon et al., 2004). Although extensive amino acid sequence variations are observed
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among these AMPs, their precursor proteins show a high degree of sequence similarity suggesting that they have arisen from a common ancestral gene (Nicolas et al., 2003). Molecular cloning studies have demonstrated that the biosynthetic precursors of these peptides comprise three domains: a signal peptide region, an intervening sequence region, and an AMP region (Simmaco et al., 1996). While nucleotide sequences of the AMP-coding regions of ranid AMP precursors are hypervariable, those of the signal peptide and the intervening sequence regions are moderately to strongly conserved among different AMP families. In addition, nucleotide sequences of the 3′-untranslated region (UTR) of amphibian AMP precursor mRNAs are also well conserved among each AMP family (Ohnuma et al., 2010). Using these nucleotide sequence similarities, we have successfully amplified several cDNAs encoding biosynthetic precursors of AMPs from total RNA samples prepared, not only from skin, but also from several extradermal tissues of ranid frogs by a one-step reverse-transcription polymerase chain reaction (RT-PCR) procedure (Iwamuro et al. 2006; Suzuki et al., 2007b; Ohnuma et al., 2010). In the present study, we have employed this protocol using a set of preprotemporin genes-specific primers to clone cDNAs encoding AMPs from the bullfrog HG and then performed in situ hybridization analyses in order to clarify whether the AMP genes are expressed in the gland. Nomenclature adopted for AMPs from frogs of the Ranidae family follows recent guidelines (Conlon, 2008), and species nomenclature follows the taxonomic recommendations of Frost (2010). 2. Materials and methods 2.1. Tissue collection and total RNA extraction Adult male and female bullfrogs, Lithobates catesbeianus (formerly Rana catesbeiana) were captured in Ibaraki Prefecture, Japan, in July for molecular cloning studies, and in November for morphological investigations and in situ hybridization experiments. Specimens were purchased from Ouchi Aquatic Animal Supply (Saitama, Japan). All experiments were approved by Saitama and Waseda Universities Bioethics and Animal Ethics Committees and carried out by authorized investigators. Animals (n = 45) were anesthetized by immersion in ice water and sacrificed by decapitation. Since the gland was pinkish in color, similar to the color of musculi bulbi, care was taken during sample collection to avoid contamination. HGs were dissected from the orbits under a microscope and immediately frozen on dry ice. Total RNA was extracted by the acid phenol–guanidinium– isothiocyanate procedure (Chomczynski and Sacchi, 1987). RNA concentrations were estimated by measurement of the absorbance at 260 and 280 nm, and quality was checked by the ratio OD260:OD280. Samples were stored at − 80 °C. 2.2. Amplification of open reading frame (ORF) of AMP cDNAs from HG total RNA by RT-PCR ORFs of AMP precursor cDNAs were amplified by RT-PCR in a volume of 50 μL using a One-Step RT-PCR kit (Qiagen, Chatsworth, CA, USA) according to the method described in a previous report (Iwamuro and Kobayashi, 2010). Briefly, aliquots of 100 ng of total RNA along with the set of preprotemporin gene-specific primers were incubated at 50 °C for 30 min for reverse transcription, and then at 95 °C for 15 min for denaturation of reverse transcriptase. Subsequently, PCR was performed under the following conditions: 5 min at 94 °C for DNA denaturation followed by 35 cycles of 30 s at 94 °C, 30 s at 50 °C, and 30 s at 72 °C with a final extension step of 7 min at 72 °C. The forward primer (5′ATGTTCACCTTGAAGAAATC-3′) and reverse primer (5′-AGATGATTTCCAATTCCAT-3′) were designed according to the nucleotide sequence of temporin precursor cDNAs obtained from several Japanese ranid frogs (Ohnuma et al., 2007; Suzuki et al., 2007a; Tazato et al., 2010). Synthetic oligonucleotides were purchased from Sigma Genosys (Ishikari, Japan).
RT-PCR products (10 µL) were separated by electrophoresis on a 1.2% agarose gel, stained with ethidium bromide, and visualized on an UV transilluminator. The DNA bands were excised and purified using a Wizard SV gel and a PCR clean-up system (Promega, Madison, WI, USA) and subcloned into pSTBlue-1 vector using the Acceptor Vector Kit (Novagen, Darmstadt, Germany). Nucleotide sequence analysis was performed by the dideoxy-chain termination method using a Big-Dye Terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) by Biomatrix Company (Chiba, Japan). Nucleotide and amino acid sequence identities and prediction of the secondary structures were analyzed using Genetyx Mac version 15.0.1 (Software Development Corporation, Osaka, Japan). 2.3. In situ hybridization analyses Aliquots of the RT-PCR products of preprotemporin-CBa and preprochensirin-2CBa (Section 2.2) were subcloned into pBluescript II plasmid vector (Stratagene, La Jolla, CA, USA) and subjected to PCR in order to obtain cDNA templates for in vitro transcription. The reaction conditions were as follows: 5 min at 94 °C for DNA denaturation, followed by 30 cycles of 30 s at 94 °C, 30 s at 50 °C, and 30 s at 72 °C, with a final extension step of 7 min at 72 °C. Sets of primers for amplification of cDNA templates for antisense (forward: M13 forward primer; reverse; 5′-GTAAAACGACGGCCAGT-3′) and sense (forward: 5′GGAAACAGCTATGACCATG-3′; reverse: M13 reverse primer) cRNA probes were used. Digoxigenin (DIG)-labeled antisense and sense probes were synthesized using a DIG RNA labeling kit (Roche Diagnostics, Basel, Switzerland). Three fresh HG specimens from adult male bullfrogs were embedded in Tissue-Tek O.C.T. Compound (Sakura Finetechnical, Tokyo, Japan) by immersion in liquid nitrogen-cooled isopentane. Frozen sections (10 µm) were cut and thaw-mounted onto MAS-coated slides (Matsunami, Osaka, Japan). Sections were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, rinsed in PBS, acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0), and washed in 2× saline sodium citrate (SSC; 1× SSC = 150 mM NaCl and 15 mM sodium citrate, pH 7.0). The sections were prehybridized for 2 h at 50 °C in prehybridization buffer (50% formamide, 2× SSC, 1× Denhardt's solution, 500 μg/mL yeast tRNA, 500 μg/mL heparin sodium, 0.1% sodium pyrophosphate). Hybridization was performed at 50 °C for 16 h with DIG-labeled preprotemporin-CBa and/or preprochensirin2CBa cRNA probes diluted with hybridization buffer (prehybridization buffer supplemented with 10% dextran sulfate). After hybridization, the sections were washed in 2× SSC at room temperature, 2× SSC-50% formamide at 50 °C for 1 h, 1× SSC-50% formamide at 50 °C for 1 h, and 2× SSC at room temperature. Slides were rinsed in buffer 1 (100 mM Tris–HCl, 150 mM NaCl, pH7.5), followed by incubation in buffer 1 containing 2% blocking reagent (Roche Diagnostics). Subsequently, sections were incubated with anti-DIG antibody conjugated with alkaline phosphatase (Roche Diagnostics), and then washed with buffer 1 followed by immersion in buffer 2 (100 mM Tris–HCl, 100 mM NaCl, 50 mM MgCl2, pH9.5). The bound antibody-conjugate was visualized with nitro-blue tetrazolium chloride (Sigma-Aldrich, St. Louis, MO, USA) and 5-bromo-4-chloro-3'-indolylphosphatase p-toluidine salt (SigmaAldrich) in buffer 2 at room temperature for 18 h in the dark. These sections were viewed using a Nikon Eclipse E600 microscope (Nikon, Tokyo, Japan), and digital images were captured using an Olympus DP70 CCD camera (Olympus, Tokyo, Japan). 3. Results 3.1. Cloning of AMP precursor cDNAs from the bullfrog HG samples Two cDNA clones (clones 1 and 2) were amplified by RT-PCR using the set of preprotemporin-specific primers. Nucleotide sequence analysis revealed that clone 1 consisted of 209 bp and included an ORF of 186 bp (including a stop codon) and a 3′-UTR of 23 bp (Fig. 1). The deduced
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Fig. 1. Nucleotide and deduced amino acid sequences of AMP precursor cDNAs prepared from the bullfrog HG total RNA samples. In the nucleotide sequences, primer-derived sequences are underlined. In the amino acid sequences, AMP sequences are underlined in bold type, stop codons are marked with an asterisk (*), and cleavage sites by signal peptidase and processing enzymes are marked with an arrow and box, respectively.
amino acid sequence of clone 1 consisted of 61 amino acid residues and included a region identical to that of temporin-CBa (initially described as ranatuerin-5) previously isolated from bullfrog skin extract (Goraya et al., 1998) (Fig. 2). Thus, clone 1 was designated preprotemporin-CBa. CB is derived from the abbreviation of the species name of L. catesbeianus. It is proposed that the penultimate Gly residue in the sequence acts as a substrate for peptidyl-glycine α-amidating monooxygenase to produce a C-terminal α-amidated residue in the secreted peptide (Prigge et al., 2000). Clone 2 consisted of 190 bp and included an ORF of 180 bp (including a stop codon) and a 3′-UTR of 10 bp (Fig. 1). The deduced amino acid sequence of clone 2 consisted of 59 amino acid residues. The predicted amino acid sequence of clone 2 was identical to the chensirin-2 precursor that has been cloned from the skin of the Chinese brown frog R. chensinensis (GenBank accession DQ471290), with the exception of one amino acid substitution (Thr→Pro) at the C-terminus (Fig. 2). Thus, clone 2 was designated preprochensirin-2CBa. Nucleotide sequence identity
between preprotemporin-CBa and preprochensirin-2CBa was 81%. Sequences of preprotemporin-CBa and preprochensirin-2CBa have been deposited in the GenBank/EMBL/DDBJ database (accession numbers are AB558903 and AB558904, respectively). 3.2. In situ hybridization To investigate the expression of AMP genes in the bullfrog HG, in situ hybridization experiments were performed using DIG-labeled antisense and sense cRNA probes for preprotemporin-CBa and preprochensirin2CBa, respectively. The hybridization signals demonstrated the presence of preprotemporin-CBa and preprochensirin-2CBa mRNAs in the cytoplasm of the glandular cells in the bullfrog HG (Fig. 3A, C). More intense signals were obtained when a mixture of both two antisense cRNA probes was used (Fig. 3E). Very weak hybridization signals were detected in the HG using the sense cRNA probes (Fig. 3B, D, F).
Fig. 2. Comparison and schematic alignment of deduced amino acid sequences of the bullfrog HG temporin and chensirin-2 precursor cDNAs with corresponding sequences prepared from the skins of various other ranid species. Residue deletions are denoted by a dash (–). Peptides designated -CB and -C are from L. catesbeianus, -P from L. pipiens, -TG from R. tagoi, -O from R. ornativentris, -PL from L. palustris, -CDY and -ST from R. dybowskii, and -CE from R. chensinensis. Temporin G is from R. temporaria. Chensirin-2 is from R. chensinensis. The GenBank/EMBL/DDBJ accession numbers are in parentheses.
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Fig. 3. In situ hybridization of AMP precursor antisense (A, C, E) and sense (B, D, F) cRNA probes in the bullfrog HG. A and B, preprotemporin-CBa; C and D, preprochensirin-2CBa; E and F, mixture of preprotemporin-CBa and preprochensirin-2CBa. The hybridization signals are localized in the cytoplasm of the glandular cells. L, acinar lumia; bars, 100 μm.
4. Discussion Our recent findings have demonstrated the expression of AMP genes not only in skin but also in various extradermal tissues such as those of liver, kidney, stomach, small intestine, and skeletal muscle of the hind limbs of selected Japanese brown frogs (Suzuki et al., 2007b; Ohnuma et al., 2010; Tazato et al., 2010). In the present investigation, we have extended these studies to include the HG and have performed molecular cloning of cDNAs encoding AMP precursors from total RNA samples prepared from the bullfrog HG. In situ hybridization experiments demonstrated the expression of genes encoding AMPs in the gland. To the best of our knowledge, the present study is the first report describing such expression in the HG of a vertebrate. Currently, several hundred AMPs have been found in the skins of frogs belonging to various families. Problems arise because there is currently no consistent nomenclature to describe the AMPs isolated from the skin of the various species studied. Examples abound of peptides that show clear structural similarity to each other, indicative of a common evolutionary origin, but have been given completely different names (Conlon, 2008). Ranatuerin 5 was first isolated from the skin extract of the American bullfrog, L. catesbeianus, as one of nine ranatuerin-related peptides (Goraya et al., 1998). Based on structural similarities, five of the nine peptides (ranatuerins 5–9 consisting of 12–14 amino acid residues) have been reclassified as members of the temporin family (Conlon, 2008). As shown in Fig. 2, the signal peptide and the intervening sequence regions of preprotemporin-CBa showed high nucleotide sequence similarities to the corresponding regions of the putative temporin precursors cloned from various ranid frog species. Thus, the findings in the present study support the reclassification of ranatuerin 5 to the temporin family. It is recommended that the five nucleotide sequences (GenBank accession FJ830664 to FJ830668) registered as “ranatuerin 5 precursors” in the GenBank database should be renamed as temporin precursors. Temporins are a family of small hydrophobic C-terminal α-amidated peptides first isolated from the Asian frog R. erythraea on the basis of hemolytic activity (Yasuhara et al., 1986) but members of family are known to be widely distributed in the skins of frogs belonging
to the Ranidae (Mangoni, 2006). In most cases, temporins show greater potencies against Gram-positive bacteria than against Gram-negative bacteria (Mangoni, 2006), but temporin-CBa (ranatuerin-5) was not active against the Gram-positive microorganism Staphylococcus aureus at concentrations up to 200 μM (Goraya et al., 1998). Basic Local Alignment Search Tool (BLAST) analysis revealed that clone 2 was orthologous to preprochensirin-2 that has been cloned from the skin of the Chinese brown frog, R. chensinensis. This nucleotide sequence was deposited in the GenBank database in May 2006 as a “direct submission”. Structurally similar cDNAs that have been termed preprodybowskin-1CDYa and preprodybowskin-1ST from the Chinese frog, R. dybowskii, and preprotemporin-1CEc from R. chensinensis, have also been registered in the database (GenBank accession EU827807, GU249565, EU746504, respectively). The deduced amino acid sequences of these precursors are identical, with the exception of the single substitution Ser18 → Asn in preprodybowskin-1CDYa, although they have been given completely different names. Shang et al. (2009) have presented preprotemporin-1CEc from R. chensinensis as a member of the temporin family but the nucleotide sequence within the 3′-UTR of the preprotemporin-1CEc cDNA was not similar to those of typical preprotemporin cDNAs (data not shown). Clone 2 is described as a preprochensirin-2 on the basis of priority of the date of submission to the database. Recently, Jin et al. (2009) have shown antimicrobial activities synthetic replicate of chensirin-2 (IIPLPLGYFAKKT) against both Gram-negative and Gram-positive bacteria. The substitution Thr→ Pro at the C-terminus of chensirin-2CBa does not change the net positive charge at physiological pH and secondary structure prediction of chensirin-2CBa by Genetyx software does not indicate a major change in secondary structure. Consequently, chensirin-2CBa from bullfrog HG is expected to be active against at least some environmental microorganisms. Several species of anurans adopt the strategy of producing multiple structurally similar AMPs in the same tissues/organs in order to increase the effectiveness of the antimicrobial defenses (Ohnuma et al., 2007). Previous studies have shown that some amphibian AMPs act synergistically so that secreted mixtures of AMPs show more potent growthinhibitory activity against environmental microorganisms than individual peptides (reviewed in Mangoni and Shai, 2009). It remains to be established whether temporin-CBa and chensirin-2CBa act in this manner within the HG. In addition to their role as endogenous antibiotics, recent findings have indicated that AMPs act as natural effectors of the innate immune system in vertebrates through their endotoxin detoxification activities, anti-inflammatory, immunostimulatory, and immunomodulatory activities (Tossi et al., 2000; Radek and Gallo, 2007; Mangoni, 2006; Rosenfeld et al., 2008; Mangoni et al., 2008; Capparelli et al., 2009). Mast cells have been detected in the frog HG (Di Matteo et al., 1995), suggesting that the gland has immunological roles in amphibians. Thus, as well as direct antimicrobial functions, AMPs in HG may play additional roles in the complex network of immune responses. In amphibians, the HG displays seasonal changes in morphology and in secretory activity (De Rienzo et al., 2002; Raucci et al., 2005). Secretory activity reaches a maximum during the hottest months, i.e., July–August, with the development of secretory granules in the glandular cells. Secretory activity then drops to medium–low levels in autumn with shortening of the cells and disorganization of the glands, and then increases slowly from mid autumn onward in a recovery phase (Di Matteo et al., 1989; De Rienzo et al., 2002). In the present study, in situ hybridization experiments were performed in late autumn (November). This may be one of the reasons for the somewhat weak signals observed in the in situ hybridization experiments. The HG is found in all vertebrates, except fishes, primates, and some other species (Payne, 1994). The HG appears at the late prometamorphic stage in the genera Rana and Bufo, while in Xenopus, the gland develops around the climax stages although the nictitating membrane is absent even after metamorphosis is completed suggesting that the HG is
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retained during readaptation to water (Shirama et al., 1982; Baccari, 1996). Development of the HG does not occur after hypophysectomy or thyroidectomy in the tadpole but can be induced by the administration of thyroid hormones (THs) or thyroid stimulating hormone (TSH) (Di Matteo et al., 1998). Thus, the HG is considered to be an “adult” organ in amphibians. Most AMPs in amphibians have been isolated and/or cloned from the skins of adult specimens and may not be present in larval specimens. In addition, expression of amphibian skin AMP genes occurs in a metamorphosis-dependent fashion and is stimulated by exogenous THs (Ohnuma et al., 2006, 2009; Suzuki et al., 2007b). The development of the dermis containing the AMP-producing skin glands in amphibians is dependent on metamorphosis and controlled by circulating THs in the developing animals (Seki et al., 1989; Reilly et al., 1994). It is possible, therefore, that THs are factors regulating AMP gene expression in the HG as well as in the skin. Currently, we are performing experiments to examine this hypothesis. Acknowledgments The authors thank Dr. Okada and Mr. Nakano in Saitama University for collection of frog HG samples. This work was supported by Grantin-Aid for Scientific Research from the Japan Society for the Promotion of Science to I.H. (22770060) and to S.I., T.K., and S.K. (21570068) and by Toho University Nukada Memorial Scholarship Fund to S.I. References Auvynet, C., Rosenstein, Y., 2009. Multifunctional host defense peptides: antimicrobial peptides, the small yet big players in innate and adaptive immunity. FEBS J. 276, 6497–6508. Baccari, G.C., 1996. Organogeneis of the Harderian gland: a comparative survey. Microsc. Res. Tech. 34, 6–15. Brogden, K.A., Guthmiller, J.M., Salzet, M., Zasloff, M., 2005. The nervous system and innate immunity: the neuropeptide connection. Nat. Immunol. 6, 558–564. Capparelli, R., Romanelli, A., Iannaccone, M., Nocerino, N., Ripa, R., Pensato, S., Pedone, C., Iannelli, D., 2009. Synergistic antibacterial and anti-inflammatory activity of temporin A and modified temporin B in vivo. PLoS One 4, e7191. Chieffi Baccari, G., Minucci, S., Marmorino, C., Vitiello Izzo, I., 1991. Number of mast cells in the Harderian gland of the green frog, Rana esculenta: the annual cycle and its relation to environmental and hormonal factors. J. Anat. 179, 75–83. Chieffi, G., Chieffi Baccari, G., Di Matteo, L., d'Istria, M., Minucci, S., Varriale, B., 1996. Cell biology of the Harderian gland. Int. Rev. Cytol. 168, 1–80. Chomczynski, P., Sacchi, N., 1987. Single-step of RNA isolation by acid guanidinium thiocyanated-phenol-chloroform extraction. Anal. Biochem. 162, 56–59. Conlon, J.M., 2008. Reflections on a systematic nomenclature for antimicrobial peptides from the skins of frogs of the family Ranidae. Peptides 29, 1815–1819. Conlon, J.M., Kolodziejek, J., Nowotny, N., 2004. Antimicrobial peptides from ranid frogs: taxonomic and phylogenetic markers and a potential source of new therapeutic agents. Biochim. Biophys. Acta 1696, 1–14. De Rienzo, G., Di Sena, R., Ferrara, D., Palmiero, C., Chieffi Baccari, G., Minucci, S., 2002. Temporal and spatial localization of prothymosin alpha transcript in the Harderian gland of the frog, Rana esculenta. J. Exp. Zool. 292, 633–639. Di Matteo, L., Minucci, S., Chieffi Baccari, G., Pellicciari, C., d'Istria, M., Chieffi, G., 1989. The Harderian gland of the frog, Rana esculenta, during the annual cycle: histology, histochemistry and ultrastructure. Basic Appl. Histochem. 33, 93–112. Di Matteo, L., Chieffi Baccari, G., Chieffi, P., Minucci, S., 1995. The effects of testosterone and estradiol on mast cell number in the Harderian gland of the frog, Rana esculenta. Zool. Sci. 12, 457–466. Di Matteo, L., Baccari, G.C., Minucci, S., 1998. TSH and thyroid hormones induce the release of secretory granules in the Harderian gland of hypophysectomized frogs, (Rana esculenta): morphological observations. Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol. 120, 383–387. Frost, D.R., 2010. Amphibian species of the world: an online reference. Version 5.4. American Museum of Natural History, New York, USA. Electronic database accessible at http://www.research.amnh.org/herpetology/amphibia. Goraya, J., Knoop, F.C., Conlon, J.M., 1998. Ranatuerins: antimicrobial peptides isolated from the skin of the American bullfrog, Rana catesbeiana. Biochem. Biophys. Res. Commun. 250, 589–592.
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New insights into copper monooxygenases and peptide amidation: structure, mechanism and function. Cell. Mol. Life Sci. 57, 1236–1259. Radek, K., Gallo, R., 2007. Antimicrobial peptides: natural effectors of the innate immune system. Semin. Immunopathol. 29, 27–43. Raucci, F., Santillo, A., D'Aniello, A., Chieffim, P., Baccari, G.C., 2005. D-Aspartate modulates transcriptional activity in Harderian gland of frog, Rana esculenta: morphological and molecular evidence. J. Cell Physiol. 204, 445–454. Reilly, D.S., Tomassini, N., Zasloff, M., 1994. Expression of magainin antimicrobial peptide genes in the developing granular glands of Xenopus skin and induction by thyroid hormone. Dev. Biol. 162, 123–133. Rosenfeld, Y., Sahl, H.G., Shai, Y., 2008. Parameters involved in antimicrobial and endotoxin detoxification activities of antimicrobial peptides. Biochemistry 47, 6468–6478. Seki, T., Kikuyama, S., Yanaihara, N., 1989. Development of Xenopus laevis skin glands producing 5-hydroxytryptoamine and caerulein. Cell Tissue Res. 258, 483–489. Shang, D., Yu, F., Li, J., Zheng, J., Zhang, L., Li, Y., 2009. Molecular cloning of cDNAs encoding antimicrobial peptide precursors from the skin of the Chinese brown frog, Rana chensinensis. Zool. Sci. 26, 220–226. Shirama, K., Kikuyama, S., Takeo, Y., Shimizu, K., Maekawa, K., 1982. Development of Harderian gland during metamorphosis in anurans. Anat. Rec. 202, 371–378. Simmaco, M., Mignogna, G., Canofeni, S., Miele, R., Mangoni, M.L., Barra, D., 1996. Temporins, antimicrobial peptides from the European red frog Rana temporaria. Eur. J. Biochem. 242, 788–792. Suzuki, H., Iwamuro, S., Ohnuma, A., Coquet, L., Leprince, J., Jouenne, T., Vaudry, H., Taylor, C.K., Abel, P.W., Conlon, J.M., 2007a. Expression of genes encoding antimicrobial and bradykinin-related peptides in skin of the stream brown frog Rana sakuraii. Peptides 28, 505–514. Suzuki, H., Conlon, J.M., Iwamuro, S., 2007b. Evidence that the genes encoding the melittin-related peptides in the skins of the Japanese frogs Rana sakuraii and Rana tagoi are not orthologous to bee venom melittin genes: developmental- and tissuedependent gene expression. Peptides 28, 2061–2068. Tazato, S., Colon, J.M., Iwamuro, S., 2010. Cloning and expression of genes encoding antimicrobial peptides and bradykinin from the skin and brain of Tago's brown frog, Rana tagoi okiensis. Peptides 31, 1480–1487. Tossi, A., Sandri, L., Giangaspero, A., 2000. Amphipathic, α-helical antimicrobial peptides. Biopolymers 55, 4–30. Yasuhara, T., Nakajima, T., Erspamer, V., Falconieri-Erspamer, G., Tukamoto, Y., Mori, M., 1986. Isolation and sequential analysis of peptides in Rana erythraea skin, In: Kiso, Y. (Ed.), Peptide Chemistry, 1985. Protein Research Foundation, Osaka, pp. 363–368.
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Comparative Biochemistry and Physiology, Part C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p c
Expression of genes encoding antimicrobial peptides in the Harderian gland of the bullfrog Lithobates catesbeianus Itaru Hasunuma a, Shawichi Iwamuro b,⁎, Tetsuya Kobayashi c, Kazuhiko Shirama d, J. Michael Conlon e, Sakae Kikuyama a,b a Department of Biology, Faculty of Education and Integrated Arts and Sciences, Center for Advanced Biomedical Sciences, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan b Department of Biology, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan c Department of Regulatory Biology, Faculty of Sciences, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan d Department of Histology and Neuroanatomy, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-0023, Japan e Department of Biochemistry, Faculty of Medicine and Health Sciences, United Arab Emirates University, 17666 Al-Ain, United Arab Emirates
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Article history: Received 6 May 2010 Received in revised form 18 May 2010 Accepted 18 May 2010 Available online 25 May 2010 Keywords: Harderian gland Bullfrog Antimicrobial peptide Temporin Chensirin-2
a b s t r a c t The Harderian gland is an orbital gland found in many tetrapod species that possess a nictitating membrane. While the main role of the Harderian gland is lubrication of the eyeballs, numerous other functions are attributed to this gland. In amphibians, mast cells have been detected in the Harderian gland, suggesting that the gland is involved in the host's system of innate immunity defending against microbial invasions. Using reverse-transcription polymerase chain reaction, we cloned from the bullfrog Harderian gland total RNA preparations, cDNAs encoding biosynthetic precursors for the antimicrobial peptides temporin-CBa (FLPIASLLGKYL-NH2), previously isolated from an extract of bullfrog skin, and chensirin-2CBa (IIPLPLGYFAKKP) that contained the amino acid substitution Thr13 → Pro compared with chensirin-2 from the Chinese brown frog, Rana chensinensis. By means of in situ hybridization using digoxigenin-labeled cRNA probes for preprotemporin-CBa and preprochensirin-2CBa, we have demonstrated for the first time in an amphibian the presence of mRNAs encoding these two precursors in the cytoplasm of the glandular cells in the bullfrog Harderian gland. © 2010 Elsevier Inc. All rights reserved.
1. Introduction The vertebrate eye is situated at the interface between the organism and its environment and as so requires defensive systems to protect against invasion by pathogenic microorganisms in the environment. Amphibians have a worldwide distribution, occupying aquatic, semiaquatic, and terrestrial environments and thus are exposed to a wide range of microorganisms. The Harderian gland (HG) is an orbital gland found in many tetrapod species that possess a nictitating membrane (Harder, 1694). Although the function of HG is still not completely clear, its size and widespread occurrence among vertebrates suggest potentially important roles. In fact, numerous functions are attributed to this gland such as lubrication of the eye and nictitating membrane, a source of pheromones and growth factors, as well as osmoregulatory, thermoregulatory and immunoregulatory functions (Payne, 1994; Chieffi et al., 1996). In amphibians, the HG is a seromucoid acinar gland located at the medial corner of the orbit and consists of a single type of epithelial cells (Chieffi Baccari et al., 1991; Payne 1994). It is known to show seasonal
⁎ Corresponding author. Tel.: + 81 47 472 5206; fax: + 81 47 472 1188. E-mail address: [email protected] (S. Iwamuro). 1532-0456/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2010.05.005
changes in secretory activity in a temperature-dependent manner (Minucci et al., 1990). Because of these seasonal changes and its spatial localization in the orbit, it is speculated that the amphibian HG may be involved in the innate immune system of the host's eyes, defending against pathogenic microorganisms encountered in the environment. Antimicrobial peptides (AMPs) are gene-encoded polypeptides of various lengths and structures found in all organisms including vertebrates, invertebrates, plants, and bacteria. In vertebrates, these molecules are important components of the innate immune system of the organisms and protect against microbial infections before adaptive immunity is activated (Auvynet and Rosenstein, 2009). AMPs can target and neutralize a broad range of microorganisms by mechanisms that involve non-specific interactions with the cell membrane. Although there are no obvious conserved amino acid sequences among these peptides, most AMPs are hydrophobic and cationic, and have a propensity to form an amphipathic helical conformation in a membrane-mimetic environment (Brogden et al., 2005). Amphibians are a promising source of AMPs. Based on limited amino acid sequence similarities, AMPs in ranid frogs are classified into several well-established groups that include the temporin, brevinin, ranatuerin, palustrin, esculentin, nigrocin, and japonicin families (Conlon et al., 2004). Although extensive amino acid sequence variations are observed
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among these AMPs, their precursor proteins show a high degree of sequence similarity suggesting that they have arisen from a common ancestral gene (Nicolas et al., 2003). Molecular cloning studies have demonstrated that the biosynthetic precursors of these peptides comprise three domains: a signal peptide region, an intervening sequence region, and an AMP region (Simmaco et al., 1996). While nucleotide sequences of the AMP-coding regions of ranid AMP precursors are hypervariable, those of the signal peptide and the intervening sequence regions are moderately to strongly conserved among different AMP families. In addition, nucleotide sequences of the 3′-untranslated region (UTR) of amphibian AMP precursor mRNAs are also well conserved among each AMP family (Ohnuma et al., 2010). Using these nucleotide sequence similarities, we have successfully amplified several cDNAs encoding biosynthetic precursors of AMPs from total RNA samples prepared, not only from skin, but also from several extradermal tissues of ranid frogs by a one-step reverse-transcription polymerase chain reaction (RT-PCR) procedure (Iwamuro et al. 2006; Suzuki et al., 2007b; Ohnuma et al., 2010). In the present study, we have employed this protocol using a set of preprotemporin genes-specific primers to clone cDNAs encoding AMPs from the bullfrog HG and then performed in situ hybridization analyses in order to clarify whether the AMP genes are expressed in the gland. Nomenclature adopted for AMPs from frogs of the Ranidae family follows recent guidelines (Conlon, 2008), and species nomenclature follows the taxonomic recommendations of Frost (2010). 2. Materials and methods 2.1. Tissue collection and total RNA extraction Adult male and female bullfrogs, Lithobates catesbeianus (formerly Rana catesbeiana) were captured in Ibaraki Prefecture, Japan, in July for molecular cloning studies, and in November for morphological investigations and in situ hybridization experiments. Specimens were purchased from Ouchi Aquatic Animal Supply (Saitama, Japan). All experiments were approved by Saitama and Waseda Universities Bioethics and Animal Ethics Committees and carried out by authorized investigators. Animals (n = 45) were anesthetized by immersion in ice water and sacrificed by decapitation. Since the gland was pinkish in color, similar to the color of musculi bulbi, care was taken during sample collection to avoid contamination. HGs were dissected from the orbits under a microscope and immediately frozen on dry ice. Total RNA was extracted by the acid phenol–guanidinium– isothiocyanate procedure (Chomczynski and Sacchi, 1987). RNA concentrations were estimated by measurement of the absorbance at 260 and 280 nm, and quality was checked by the ratio OD260:OD280. Samples were stored at − 80 °C. 2.2. Amplification of open reading frame (ORF) of AMP cDNAs from HG total RNA by RT-PCR ORFs of AMP precursor cDNAs were amplified by RT-PCR in a volume of 50 μL using a One-Step RT-PCR kit (Qiagen, Chatsworth, CA, USA) according to the method described in a previous report (Iwamuro and Kobayashi, 2010). Briefly, aliquots of 100 ng of total RNA along with the set of preprotemporin gene-specific primers were incubated at 50 °C for 30 min for reverse transcription, and then at 95 °C for 15 min for denaturation of reverse transcriptase. Subsequently, PCR was performed under the following conditions: 5 min at 94 °C for DNA denaturation followed by 35 cycles of 30 s at 94 °C, 30 s at 50 °C, and 30 s at 72 °C with a final extension step of 7 min at 72 °C. The forward primer (5′ATGTTCACCTTGAAGAAATC-3′) and reverse primer (5′-AGATGATTTCCAATTCCAT-3′) were designed according to the nucleotide sequence of temporin precursor cDNAs obtained from several Japanese ranid frogs (Ohnuma et al., 2007; Suzuki et al., 2007a; Tazato et al., 2010). Synthetic oligonucleotides were purchased from Sigma Genosys (Ishikari, Japan).
RT-PCR products (10 µL) were separated by electrophoresis on a 1.2% agarose gel, stained with ethidium bromide, and visualized on an UV transilluminator. The DNA bands were excised and purified using a Wizard SV gel and a PCR clean-up system (Promega, Madison, WI, USA) and subcloned into pSTBlue-1 vector using the Acceptor Vector Kit (Novagen, Darmstadt, Germany). Nucleotide sequence analysis was performed by the dideoxy-chain termination method using a Big-Dye Terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) by Biomatrix Company (Chiba, Japan). Nucleotide and amino acid sequence identities and prediction of the secondary structures were analyzed using Genetyx Mac version 15.0.1 (Software Development Corporation, Osaka, Japan). 2.3. In situ hybridization analyses Aliquots of the RT-PCR products of preprotemporin-CBa and preprochensirin-2CBa (Section 2.2) were subcloned into pBluescript II plasmid vector (Stratagene, La Jolla, CA, USA) and subjected to PCR in order to obtain cDNA templates for in vitro transcription. The reaction conditions were as follows: 5 min at 94 °C for DNA denaturation, followed by 30 cycles of 30 s at 94 °C, 30 s at 50 °C, and 30 s at 72 °C, with a final extension step of 7 min at 72 °C. Sets of primers for amplification of cDNA templates for antisense (forward: M13 forward primer; reverse; 5′-GTAAAACGACGGCCAGT-3′) and sense (forward: 5′GGAAACAGCTATGACCATG-3′; reverse: M13 reverse primer) cRNA probes were used. Digoxigenin (DIG)-labeled antisense and sense probes were synthesized using a DIG RNA labeling kit (Roche Diagnostics, Basel, Switzerland). Three fresh HG specimens from adult male bullfrogs were embedded in Tissue-Tek O.C.T. Compound (Sakura Finetechnical, Tokyo, Japan) by immersion in liquid nitrogen-cooled isopentane. Frozen sections (10 µm) were cut and thaw-mounted onto MAS-coated slides (Matsunami, Osaka, Japan). Sections were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, rinsed in PBS, acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0), and washed in 2× saline sodium citrate (SSC; 1× SSC = 150 mM NaCl and 15 mM sodium citrate, pH 7.0). The sections were prehybridized for 2 h at 50 °C in prehybridization buffer (50% formamide, 2× SSC, 1× Denhardt's solution, 500 μg/mL yeast tRNA, 500 μg/mL heparin sodium, 0.1% sodium pyrophosphate). Hybridization was performed at 50 °C for 16 h with DIG-labeled preprotemporin-CBa and/or preprochensirin2CBa cRNA probes diluted with hybridization buffer (prehybridization buffer supplemented with 10% dextran sulfate). After hybridization, the sections were washed in 2× SSC at room temperature, 2× SSC-50% formamide at 50 °C for 1 h, 1× SSC-50% formamide at 50 °C for 1 h, and 2× SSC at room temperature. Slides were rinsed in buffer 1 (100 mM Tris–HCl, 150 mM NaCl, pH7.5), followed by incubation in buffer 1 containing 2% blocking reagent (Roche Diagnostics). Subsequently, sections were incubated with anti-DIG antibody conjugated with alkaline phosphatase (Roche Diagnostics), and then washed with buffer 1 followed by immersion in buffer 2 (100 mM Tris–HCl, 100 mM NaCl, 50 mM MgCl2, pH9.5). The bound antibody-conjugate was visualized with nitro-blue tetrazolium chloride (Sigma-Aldrich, St. Louis, MO, USA) and 5-bromo-4-chloro-3'-indolylphosphatase p-toluidine salt (SigmaAldrich) in buffer 2 at room temperature for 18 h in the dark. These sections were viewed using a Nikon Eclipse E600 microscope (Nikon, Tokyo, Japan), and digital images were captured using an Olympus DP70 CCD camera (Olympus, Tokyo, Japan). 3. Results 3.1. Cloning of AMP precursor cDNAs from the bullfrog HG samples Two cDNA clones (clones 1 and 2) were amplified by RT-PCR using the set of preprotemporin-specific primers. Nucleotide sequence analysis revealed that clone 1 consisted of 209 bp and included an ORF of 186 bp (including a stop codon) and a 3′-UTR of 23 bp (Fig. 1). The deduced
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Fig. 1. Nucleotide and deduced amino acid sequences of AMP precursor cDNAs prepared from the bullfrog HG total RNA samples. In the nucleotide sequences, primer-derived sequences are underlined. In the amino acid sequences, AMP sequences are underlined in bold type, stop codons are marked with an asterisk (*), and cleavage sites by signal peptidase and processing enzymes are marked with an arrow and box, respectively.
amino acid sequence of clone 1 consisted of 61 amino acid residues and included a region identical to that of temporin-CBa (initially described as ranatuerin-5) previously isolated from bullfrog skin extract (Goraya et al., 1998) (Fig. 2). Thus, clone 1 was designated preprotemporin-CBa. CB is derived from the abbreviation of the species name of L. catesbeianus. It is proposed that the penultimate Gly residue in the sequence acts as a substrate for peptidyl-glycine α-amidating monooxygenase to produce a C-terminal α-amidated residue in the secreted peptide (Prigge et al., 2000). Clone 2 consisted of 190 bp and included an ORF of 180 bp (including a stop codon) and a 3′-UTR of 10 bp (Fig. 1). The deduced amino acid sequence of clone 2 consisted of 59 amino acid residues. The predicted amino acid sequence of clone 2 was identical to the chensirin-2 precursor that has been cloned from the skin of the Chinese brown frog R. chensinensis (GenBank accession DQ471290), with the exception of one amino acid substitution (Thr→Pro) at the C-terminus (Fig. 2). Thus, clone 2 was designated preprochensirin-2CBa. Nucleotide sequence identity
between preprotemporin-CBa and preprochensirin-2CBa was 81%. Sequences of preprotemporin-CBa and preprochensirin-2CBa have been deposited in the GenBank/EMBL/DDBJ database (accession numbers are AB558903 and AB558904, respectively). 3.2. In situ hybridization To investigate the expression of AMP genes in the bullfrog HG, in situ hybridization experiments were performed using DIG-labeled antisense and sense cRNA probes for preprotemporin-CBa and preprochensirin2CBa, respectively. The hybridization signals demonstrated the presence of preprotemporin-CBa and preprochensirin-2CBa mRNAs in the cytoplasm of the glandular cells in the bullfrog HG (Fig. 3A, C). More intense signals were obtained when a mixture of both two antisense cRNA probes was used (Fig. 3E). Very weak hybridization signals were detected in the HG using the sense cRNA probes (Fig. 3B, D, F).
Fig. 2. Comparison and schematic alignment of deduced amino acid sequences of the bullfrog HG temporin and chensirin-2 precursor cDNAs with corresponding sequences prepared from the skins of various other ranid species. Residue deletions are denoted by a dash (–). Peptides designated -CB and -C are from L. catesbeianus, -P from L. pipiens, -TG from R. tagoi, -O from R. ornativentris, -PL from L. palustris, -CDY and -ST from R. dybowskii, and -CE from R. chensinensis. Temporin G is from R. temporaria. Chensirin-2 is from R. chensinensis. The GenBank/EMBL/DDBJ accession numbers are in parentheses.
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Fig. 3. In situ hybridization of AMP precursor antisense (A, C, E) and sense (B, D, F) cRNA probes in the bullfrog HG. A and B, preprotemporin-CBa; C and D, preprochensirin-2CBa; E and F, mixture of preprotemporin-CBa and preprochensirin-2CBa. The hybridization signals are localized in the cytoplasm of the glandular cells. L, acinar lumia; bars, 100 μm.
4. Discussion Our recent findings have demonstrated the expression of AMP genes not only in skin but also in various extradermal tissues such as those of liver, kidney, stomach, small intestine, and skeletal muscle of the hind limbs of selected Japanese brown frogs (Suzuki et al., 2007b; Ohnuma et al., 2010; Tazato et al., 2010). In the present investigation, we have extended these studies to include the HG and have performed molecular cloning of cDNAs encoding AMP precursors from total RNA samples prepared from the bullfrog HG. In situ hybridization experiments demonstrated the expression of genes encoding AMPs in the gland. To the best of our knowledge, the present study is the first report describing such expression in the HG of a vertebrate. Currently, several hundred AMPs have been found in the skins of frogs belonging to various families. Problems arise because there is currently no consistent nomenclature to describe the AMPs isolated from the skin of the various species studied. Examples abound of peptides that show clear structural similarity to each other, indicative of a common evolutionary origin, but have been given completely different names (Conlon, 2008). Ranatuerin 5 was first isolated from the skin extract of the American bullfrog, L. catesbeianus, as one of nine ranatuerin-related peptides (Goraya et al., 1998). Based on structural similarities, five of the nine peptides (ranatuerins 5–9 consisting of 12–14 amino acid residues) have been reclassified as members of the temporin family (Conlon, 2008). As shown in Fig. 2, the signal peptide and the intervening sequence regions of preprotemporin-CBa showed high nucleotide sequence similarities to the corresponding regions of the putative temporin precursors cloned from various ranid frog species. Thus, the findings in the present study support the reclassification of ranatuerin 5 to the temporin family. It is recommended that the five nucleotide sequences (GenBank accession FJ830664 to FJ830668) registered as “ranatuerin 5 precursors” in the GenBank database should be renamed as temporin precursors. Temporins are a family of small hydrophobic C-terminal α-amidated peptides first isolated from the Asian frog R. erythraea on the basis of hemolytic activity (Yasuhara et al., 1986) but members of family are known to be widely distributed in the skins of frogs belonging
to the Ranidae (Mangoni, 2006). In most cases, temporins show greater potencies against Gram-positive bacteria than against Gram-negative bacteria (Mangoni, 2006), but temporin-CBa (ranatuerin-5) was not active against the Gram-positive microorganism Staphylococcus aureus at concentrations up to 200 μM (Goraya et al., 1998). Basic Local Alignment Search Tool (BLAST) analysis revealed that clone 2 was orthologous to preprochensirin-2 that has been cloned from the skin of the Chinese brown frog, R. chensinensis. This nucleotide sequence was deposited in the GenBank database in May 2006 as a “direct submission”. Structurally similar cDNAs that have been termed preprodybowskin-1CDYa and preprodybowskin-1ST from the Chinese frog, R. dybowskii, and preprotemporin-1CEc from R. chensinensis, have also been registered in the database (GenBank accession EU827807, GU249565, EU746504, respectively). The deduced amino acid sequences of these precursors are identical, with the exception of the single substitution Ser18 → Asn in preprodybowskin-1CDYa, although they have been given completely different names. Shang et al. (2009) have presented preprotemporin-1CEc from R. chensinensis as a member of the temporin family but the nucleotide sequence within the 3′-UTR of the preprotemporin-1CEc cDNA was not similar to those of typical preprotemporin cDNAs (data not shown). Clone 2 is described as a preprochensirin-2 on the basis of priority of the date of submission to the database. Recently, Jin et al. (2009) have shown antimicrobial activities synthetic replicate of chensirin-2 (IIPLPLGYFAKKT) against both Gram-negative and Gram-positive bacteria. The substitution Thr→ Pro at the C-terminus of chensirin-2CBa does not change the net positive charge at physiological pH and secondary structure prediction of chensirin-2CBa by Genetyx software does not indicate a major change in secondary structure. Consequently, chensirin-2CBa from bullfrog HG is expected to be active against at least some environmental microorganisms. Several species of anurans adopt the strategy of producing multiple structurally similar AMPs in the same tissues/organs in order to increase the effectiveness of the antimicrobial defenses (Ohnuma et al., 2007). Previous studies have shown that some amphibian AMPs act synergistically so that secreted mixtures of AMPs show more potent growthinhibitory activity against environmental microorganisms than individual peptides (reviewed in Mangoni and Shai, 2009). It remains to be established whether temporin-CBa and chensirin-2CBa act in this manner within the HG. In addition to their role as endogenous antibiotics, recent findings have indicated that AMPs act as natural effectors of the innate immune system in vertebrates through their endotoxin detoxification activities, anti-inflammatory, immunostimulatory, and immunomodulatory activities (Tossi et al., 2000; Radek and Gallo, 2007; Mangoni, 2006; Rosenfeld et al., 2008; Mangoni et al., 2008; Capparelli et al., 2009). Mast cells have been detected in the frog HG (Di Matteo et al., 1995), suggesting that the gland has immunological roles in amphibians. Thus, as well as direct antimicrobial functions, AMPs in HG may play additional roles in the complex network of immune responses. In amphibians, the HG displays seasonal changes in morphology and in secretory activity (De Rienzo et al., 2002; Raucci et al., 2005). Secretory activity reaches a maximum during the hottest months, i.e., July–August, with the development of secretory granules in the glandular cells. Secretory activity then drops to medium–low levels in autumn with shortening of the cells and disorganization of the glands, and then increases slowly from mid autumn onward in a recovery phase (Di Matteo et al., 1989; De Rienzo et al., 2002). In the present study, in situ hybridization experiments were performed in late autumn (November). This may be one of the reasons for the somewhat weak signals observed in the in situ hybridization experiments. The HG is found in all vertebrates, except fishes, primates, and some other species (Payne, 1994). The HG appears at the late prometamorphic stage in the genera Rana and Bufo, while in Xenopus, the gland develops around the climax stages although the nictitating membrane is absent even after metamorphosis is completed suggesting that the HG is
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retained during readaptation to water (Shirama et al., 1982; Baccari, 1996). Development of the HG does not occur after hypophysectomy or thyroidectomy in the tadpole but can be induced by the administration of thyroid hormones (THs) or thyroid stimulating hormone (TSH) (Di Matteo et al., 1998). Thus, the HG is considered to be an “adult” organ in amphibians. Most AMPs in amphibians have been isolated and/or cloned from the skins of adult specimens and may not be present in larval specimens. In addition, expression of amphibian skin AMP genes occurs in a metamorphosis-dependent fashion and is stimulated by exogenous THs (Ohnuma et al., 2006, 2009; Suzuki et al., 2007b). The development of the dermis containing the AMP-producing skin glands in amphibians is dependent on metamorphosis and controlled by circulating THs in the developing animals (Seki et al., 1989; Reilly et al., 1994). It is possible, therefore, that THs are factors regulating AMP gene expression in the HG as well as in the skin. Currently, we are performing experiments to examine this hypothesis. Acknowledgments The authors thank Dr. Okada and Mr. Nakano in Saitama University for collection of frog HG samples. This work was supported by Grantin-Aid for Scientific Research from the Japan Society for the Promotion of Science to I.H. (22770060) and to S.I., T.K., and S.K. (21570068) and by Toho University Nukada Memorial Scholarship Fund to S.I. References Auvynet, C., Rosenstein, Y., 2009. Multifunctional host defense peptides: antimicrobial peptides, the small yet big players in innate and adaptive immunity. FEBS J. 276, 6497–6508. Baccari, G.C., 1996. Organogeneis of the Harderian gland: a comparative survey. Microsc. Res. Tech. 34, 6–15. Brogden, K.A., Guthmiller, J.M., Salzet, M., Zasloff, M., 2005. The nervous system and innate immunity: the neuropeptide connection. Nat. Immunol. 6, 558–564. Capparelli, R., Romanelli, A., Iannaccone, M., Nocerino, N., Ripa, R., Pensato, S., Pedone, C., Iannelli, D., 2009. Synergistic antibacterial and anti-inflammatory activity of temporin A and modified temporin B in vivo. PLoS One 4, e7191. Chieffi Baccari, G., Minucci, S., Marmorino, C., Vitiello Izzo, I., 1991. Number of mast cells in the Harderian gland of the green frog, Rana esculenta: the annual cycle and its relation to environmental and hormonal factors. J. Anat. 179, 75–83. Chieffi, G., Chieffi Baccari, G., Di Matteo, L., d'Istria, M., Minucci, S., Varriale, B., 1996. Cell biology of the Harderian gland. Int. Rev. Cytol. 168, 1–80. Chomczynski, P., Sacchi, N., 1987. Single-step of RNA isolation by acid guanidinium thiocyanated-phenol-chloroform extraction. Anal. Biochem. 162, 56–59. Conlon, J.M., 2008. Reflections on a systematic nomenclature for antimicrobial peptides from the skins of frogs of the family Ranidae. Peptides 29, 1815–1819. Conlon, J.M., Kolodziejek, J., Nowotny, N., 2004. Antimicrobial peptides from ranid frogs: taxonomic and phylogenetic markers and a potential source of new therapeutic agents. Biochim. Biophys. Acta 1696, 1–14. De Rienzo, G., Di Sena, R., Ferrara, D., Palmiero, C., Chieffi Baccari, G., Minucci, S., 2002. Temporal and spatial localization of prothymosin alpha transcript in the Harderian gland of the frog, Rana esculenta. J. Exp. Zool. 292, 633–639. Di Matteo, L., Minucci, S., Chieffi Baccari, G., Pellicciari, C., d'Istria, M., Chieffi, G., 1989. The Harderian gland of the frog, Rana esculenta, during the annual cycle: histology, histochemistry and ultrastructure. Basic Appl. Histochem. 33, 93–112. Di Matteo, L., Chieffi Baccari, G., Chieffi, P., Minucci, S., 1995. The effects of testosterone and estradiol on mast cell number in the Harderian gland of the frog, Rana esculenta. Zool. Sci. 12, 457–466. Di Matteo, L., Baccari, G.C., Minucci, S., 1998. TSH and thyroid hormones induce the release of secretory granules in the Harderian gland of hypophysectomized frogs, (Rana esculenta): morphological observations. Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol. 120, 383–387. Frost, D.R., 2010. Amphibian species of the world: an online reference. Version 5.4. American Museum of Natural History, New York, USA. Electronic database accessible at http://www.research.amnh.org/herpetology/amphibia. Goraya, J., Knoop, F.C., Conlon, J.M., 1998. Ranatuerins: antimicrobial peptides isolated from the skin of the American bullfrog, Rana catesbeiana. Biochem. Biophys. Res. Commun. 250, 589–592.
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