Identication of cathelicidin gene from Hoplobatrachus rugulosus and the antioxidant capacity of PC29 peptide

Cathelicidins, a group of vertebrate multifunctional molecules, play a role in innate immunity. Cathelicidins are antimicrobial peptides (AMPs) that are involved in protection against microbial invasion. Presently, cathelicidin peptides have been identied from only 14 amphibian species. In the study, a novel cathelicidin was identied from the lungs of frogs, Hoplobatrachus rugulosus. A 474 base pairs (bp) complementary DNA (cDNA) sequence encoded a 157 amino acid residue prepropeptide of H. rugulosus cathelicidin (cathelicidin-HR), which consisting of a 20-residue signal peptide sequence, a 108-residue cathelin region, and a 29-residue cathelicidin peptide (PC29). Amino acid sequence alignment and cladogram analysis illustrated that cathelicidin-HR have a high degree of similarity to further amphibian cathelicidins. The PC29 peptide displays antimicrobial activity only against Bacillus subtilis and Enterococcus faecalis. However, the PC29 peptide performed dose-dependent antioxidant activity. This is the rst cathelicidin antioxidant peptide identied from the lung which provided a template for the development of potent bi-functional peptide therapeutic agents.


Introduction
Amphibians can survive in a broad range of environmental systems due to the containing pharmacological substances to combat environmental factors such as microbes and ultraviolet radiation [1]. They have abundant peptides that play a role in defense mechanisms [2]. Amphibian bioactive peptides have been proven a variety of biological functions, including antimicrobial, antioxidant, and immunomodulatory activities [3]. Many studies have demonstrated that amphibian skin peptides have the potential for drug development [4,5]. However, not only skin peptides play the role in a protective mechanism but other organ peptides, especially those found in amphibian lungs, also function in an important organ in the respiratory system which not well understood.
Cathelicidin is a class of multi-functional peptides found in almost all vertebrates [6]. The cathelicidins are synthesized as prepropeptides containing the conserve N-terminus cathelin domain and the variable C-terminus active peptide region [7]. Moreover, not only relates antimicrobial activity but cathelicidin is also associated in other biological roles including angiogenesis, induction of immune cytolysis, and immune cells chemotaxis [8].

Materials And Methods
Rapid ampli cation of cDNA ends (RACE) reaction Total RNA was extracted from frog lungs by using GF-1 Total RNA extraction Kit (Vivantis, USA). RNA concentration was measured by spectrophotometer. The experimental procedure was approved by the Institutional Animal Care and Use Committee of Khon Kaen University (Record number IACUC-KKU-10162). The rst-strand cDNA was produced by M-MuLV reverse transcriptase reaction (Vivantis, USA) with oligonucleotide d(T). The cDNA template was magni ed with the forward primer rcCATH-F (5'-ATGAAGATCTGGCAGTGTGTG-3') and the reverse primer rcCATH-R (5'-GGTCAGGCTGACGCACTTC-3') with designed based on the conserved signal peptide sequence and C-terminal cathelin domain of R. catesbiena cathelicidin (cathelicidin-RC) genes, respectively [20]. Furthermore, the 3'-RACE was synthesized using a 5'-speci c forward primer (hrCATH34-F: 5'-GCAATCACATTGCAGTCAGC-3') and oligonucleotide d(T) primer. The polymerase chain reaction (PCR) was conducted by T100 Thermal Cycler (Bio-Rad, USA). The PCR product puri cation was carried out by gel electrophoresis with a GF-1 AmbiClean kit (Vivantis, USA). The puri ed PCR products were sequenced by Sanger sequencing (Macrogen, South Korea).
The secondary structure was performed by PEP-FOLD servers [24]. The structure graphic was hence produced in PyMol (Schrödinger LLC). The prediction of antimicrobial region was calculated through Antimicrobial Sequence Scanning System (AMPA) server (http://tcoffee.crg.cat/apps/ampa/) [25]. The peptide sequences were computed for the physicochemical properties analysis via APD2 database Peptide synthesis PC29 peptide was produced by GenScript (NJ, USA). The amino acid sequencing was validated by electrospray ionization mass spectrometry while peptide purity was checked by reverse-phase high performance liquid chromatography (RP-HPLC).

Antimicrobial activity
The antimicrobial activity assay of peptides was determined using the broth assay. Brie y, bacterial cells were cultured in nutrient broth until the mid-log phase at 37 °C. Then cultured cells were diluted to 10 4 CFU/ml. The 50 µl of diluted cells was aliquoted into microcentrifuge tubes then mixed with 50 µl of 4 mg/ml PC29 peptide, followed by the incubation at 37 °C for 16-18 h. the bacterial growth was observed by spectrophotometer. A decrease in optical density at 600 nm indicated the antimicrobial activity of the peptide. The melittin and double-distilled water (DW) were used as positive and negative controls, respectively. The percentage of bacterial growth inhibition was calculated the following formula: [(OD600 nm, control − OD600 nm, peptide) / OD600 nm, control] × 100.
Antioxidant activity assay A 2, 2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) scavenging activity was determined as previously described [14]. Brie y, the ABTS radical solution was prepared by mixing 2.8 mM potassium persulfate with 7 mM ABTS in water, followed by incubating for 6 h in the dark. The ABTS solution was diluted 50-fold with DW. Samples dissolved in water were added to the diluted stock solution, and the same volume of solvent was used as the negative control. The reactions were kept from light for 30 min.
A decrease in absorbance at 415 nm indicated the antioxidant activity of the samples. The rate of free radical scavenging (%) was calculated by [(A415 nm, blank − A415 nm, sample) /A415 nm, blank] × 100.
The reaction contained 190 μl of 50 μM DPPH radical dissolved in ethanol and 10 μl of 2-fold dilution peptide. The mixture was incubated at room temperature for 30 min in the dark. Then, the absorbance was measured against a blank at 517 nm. The percentage of DPPH free radical scavenging activity was calculated the following formula: [(A517 nm, blank − A517 nm, sample) /A517 nm, blank] × 100.

Hemolytic activity assay
The hemolytic activity of the PC29 peptide was investigated against the de brinated sheep red blood cells (shRBCs). Brie y, the shRBCs were washed with phosphate-buffered saline (PBS), pH 7.4, and then diluted to 0.5 % (v/v) in PBS. The 100 ml of shRBCs solution was divided into microcentrifuge tubes. The 10 ml of 2-fold serial dilution of PC29 peptide was added, followed by incubated at 37 °C for 1 h. The reactions were centrifuged at 1,000 g for 5 min. The 100 ml supernatants have measured absorbance at 415 nm with a spectrophotometer. The 1 % (v/v) Triton X-100 and DW were used as positive and negative controls, respectively. The percentage of hemolysis was computed as [(A415 nm, peptide)/(A415 nm, 1 % (v/v) Triton X-100)] × 100.

Identi cation of cathelicidin-HR
To identify the cathelicidin gene in H. rugulosus, the frog lung was collected then analyzed by RT-PCR with the conserved R. catesbiena cathelicidin gene primer. The 300 bp PCR product was obtained from the reaction with 80% sequence similarity to the cathelicidin-PY1 precursor (AFX61592) from N. yunnanensis (Supplement Data Fig 1a). This nucleotide sequence was further used as a template to obtain the complete 3´ ends of the cathelicidin-HR gene by RACE-PCR. The 800 bp 3´ RACE-PCR product was ampli ed by using speci c cathelicidin-HR primers combined with oligonucleotide d(T) primer (Supplement Data Fig 1b). The complete prepropeptide cathelicidin-HR cDNA sequence was presented in Analysis of cathelicidin-HR amino acid sequence The amino acid sequence analysis with ExPASy showed that the molecular weight of prepro-cathelicidin-HR was 17.97 kDa with the 6.59 pI. Moreover, SignalP 4.0 sequence analysis indicated that the rst 20 amino acid residues domain on the N-terminus site was the signal peptide region. The conserved cathelin region comprised 108 residues whereas the C-terminal end indicated the putative mature cathelicidin-HR (PC29) peptide comprised 29 amino acid residues with a molecular weight of 3.18 kDa and 10.86 of pI ( Table 1). The amino acid sequence alignment illustrated that prepro-cathelicidin-HR showed 66 % similarity with Lf-CATH1 (L. fragilis) and performed 64% and 60% with the cathelicidin-NV precursor (N. ventripunctata) and OL-CATH1 from O. livida, respectively (Fig. 2a). Among amphibian species, the Nterminus cathelin region performed a highly conserved sequence. Whereas, the C-terminus mature cathelicidin peptide presented a peptide sequences variation. The phylogenetic tree analysis of amphibian cathelicidins was divided into three clusters (Fig. 2b). Cluster I was the largest group of amphibian cathelicidin which can split into two sub-groups (cluster I-a) and cluster I-b). Cluster I-a comprised cathelicidin-HR, Lf-CATH1, cathelicidin-NV, and OL-CATH1. In addition, cluster I-b included cathelicidin-RC1-2, OL-CATH 2, PN-CATH1-2, cathelicidin-PP, cathelicidin-PY1, and Lf-CATH2. Besides, cluster II had two cathelicidins from toads, cathelicidin-BG and cathelicidin-DM. Cluster III presented the diversity of amphibian cathelicidin, consisting of cathelicidin-OA1 and cathelicidin-AL.

Analysis of candidate cathelicidin-HR peptide
The AMPA server analysis indicated that the region from Pro129 to Arg140 represented the bactericidal stretch with 13 % probability (data not shown) which is located on the mature cathelicidin peptide domain. Therefore, the candidate putative mature peptide denoted as PC29; NH2-PCRGIFCRTGSRSPIAKPSKDNLVRMSLS-COOH derived from the C-terminus site. The PC29 peptide comprised 29 amino acid residues and performed the cationic peptide property +5 net charge. However, PC29 peptide expressed a positive total net charge which presented a 34% of hydrophobic ratio with lower than at the level of a major group of AMPs. The predicted PC29 peptide secondary structure was showed in Supplement Data Fig. 3. The secondary structure modeling illustrated that the PC29 peptide exhibited the b-strand model containing one salt bridge between Arg12 and Asp21.

Antimicrobial activity of the cathelicidin-HR peptide
The antimicrobial activity of the PC29 peptide was investigated by the broth assay ( Table 2). The results performed that a high concentration of PC29 peptide could inhibit the growth of only Bacillus subtilis TISTR124 and Enterococcus faecalis TISTR927. However, the minimum inhibition concentration of this peptide was performed at 1 mg/ml for both bacterial strains (data not shown). These results indicated that PC29 peptide exhibited low antimicrobial activity.

Antioxidant activity of PC29 peptide
The antioxidant activity of the PC29 peptide was investigated by the ABTS and DPPH scavenging activity (Fig. 5). The results clearly showed that the PC29 peptide performs antioxidant capacity against both ABTS and DPPH scavenging activity. Wherewith, the PC29 peptide exerts the free radical scavenging activity of both ABTS and DPPH with the dose-dependent characteristic. These results illustrated that the PC29 peptide presented antioxidant properties.

Hemolysis of PC29 peptide against red blood cell
The hemolytic of PC29 peptide on sheep erythrocyte presented that PC29 peptide expressed insigni cant hemolysis activity between range 10 % to 18 % even at a concentration of 400 mg/ml (Fig. 6). Whereas, the negative control (DW) also showed hemolysis against red blood cells about 10 %. Besides, the positive control (1% Triton-X 100) destroyed red blood cells about 100 %. These results concluded that the PC29 peptide presented a low toxicity activity.

Discussion
Amphibians were the rst to evolve as land and water connectors. These animals are in direct contact and respond to a wide range of ecological and physical factors such as a microbe, parasite, and environment [1]. Therefore, it has a potent defense system consisting of a variety of gene expression and bioactive peptides [27]. Over the past decades, bioactive peptides from amphibian skin have been extensively studied [1], and more than a hundred peptides have been identi ed from amphibians [1].
Especially, the Ranidae frogs that contain large amounts of biological peptides in their skin. However, most previous studied peptides only presented peptides from the skin which containing antimicrobial activity. While frog peptide from other organs has not been considerably investigated. Cathelicidins are a class of antimicrobial peptides found extensively in vertebrates. Since their rst discovery isolated from granules of bovine neutrophils [28]. Currently, over hundreds of cathelicidins have been identi ed from various vertebrates [29]. For amphibians, to date, a total of 19 cathelicidin sequences have been identi ed from 14 different species.
The cathelicidin-HR was identi ed from the lung as the previous reports of cathelicidin-RC1 and cathelicidin-RC2 from R. catesbeiana, FM-CATH1-2 from F. multistriata, and OL-CATH1-2 from O. livida [20][21][22]. The 157 amino acid residues of cathelicidin-HR contained the 20 amino acid residues signal peptide following the 108 residues conserved cathelin domain and the PC29 putative mature peptide end (Fig. 1). The sequence alignment elucidated that among the group of amphibian cathelicidin has a highly similar degree of cathelin sequence region which not only four conserved cysteine residues (Fig. 2a).
However, except for cathelicidin-AL and cathelicidin-OA1 which exhibit sequence similarities to reptilian cathelicidin [9,14]. The phylogenetic analysis illustrated that the cathelicidin-HR relate to other amphibian cathelicidins which mostly located in cluster I (Fig. 2b). On the other hand, cathelicidin-AL and cathelicidin-OA1 presented that both sequences are distinct to amphibian sequence clusters that might be the connecting link between the amphibian and reptilia cathelicidin [9,14] (Fig. 2b). However, the sequence similarity result indicated that the cathelicidin-HR sequence showed the closest sequence relates to Lf-CATH1 which was isolated from the spleen of L. fragilis (Fig. 2b).
In general, cathelicidin was essential enzymatically cleaved for proteolytic maturation. Enzymatic processing in most of the cathelicidins was mediated by elastase. This enzyme was typically sensitive to valine or an alanine residue. However, the proteolytic processing for amphibian cathelicidins is remaining unclear. In this study, based on antimicrobial peptide domain prediction and amino acid sequence alignment results, PC29 peptide (PCRGIFCRTGSRSPIAKPSKDNLVRMSLS) was estimated as the mature peptide released from cathelicidin-HR. The cationic PC29 peptide comprised 29 residues with +5 net charge and b-strand characteristic. As described earlier, some amphibian cathelicidin could not exhibit antimicrobial activity but we cannot refuse that this peptide lacks this property. Since the PC29 peptide performed antimicrobial activity against gram-positive bacteria B. subtilis TISTR124 and E. faecalis TISTR927 although at high concentration. The b-strand character of the PC29 peptide might be an uncommon AMPs structure. Although the PC29 peptide presented the positive total net charge this peptide expressed the hydrophobic ratio percentage at 34 % which lower than the range of a main group of AMPs which commonly hydrophobic ratio percentage between range 40 % to 50 % (APD). The optimum hydrophobicity of peptides may lead to the e cient action of the peptide on membranes especially penetration and disruption [30]. In cluster I, only Lf-CATH1 (40 % hydrophobic ratio) exhibits antimicrobial activity but OL-CATH1 and cathelicidin-NV with hydrophobic ratio percentage between range 30 % to 33 % were lacking antimicrobial activity although all peptides presented +5 net charge (Supplement Data Table 1).
PC29 peptide performed obvious ABTS+ and DPPH scavenging activity (Fig. 3). The two cysteine residues of PC29 peptide were estimated that might play a role in the free radicals scavenging activity. However, the antioxidant activity of cathelicidin peptide was disrupted by the formation of an intramolecular disul de bridge [14]. Thus, it is assumed that two cysteine residues probably were free cysteine and performed the scavenging activity. Furthermore, the predicted PC29 peptide secondary structure also established that no disul de bond formation (Supplement Data Fig. 3). According to the results, the conclusion can be made that the PC29 peptide expressed the bi-functional peptide with antimicrobial and antioxidant activities. Recently, PN-CATHs from P. nigromaculata also showed both antimicrobial and antioxidant activities [17]. However, here is the rst report of a non-skin cathelicidin antioxidant peptide identi ed from an amphibian.

Conclusions
In summary, a cathelicidin peptide was identi ed from H. rugolosus that offers the diversity role of cathelicidin in amphibian. The amino acid sequence analysis revealed cathelicidin-HR shares the common conserved sequence among the amphibian cathelicidins. The PC29 peptide was predicted as mature peptide released from cathelicidin-HR exhibit low antimicrobial activity whereas performing the antioxidant activity in a dose-dependent manner. The results provide a new template peptide for the development of potent two modes peptide employing antimicrobial and antioxidant activities.

Declarations
Funding This work was supported by Thailand Research Fund (Grant No. MRG6280081)

Con ict of interest
The authors have no con icts of interest to declare that are relevant to the content of this article.

Informed consent
The authors declare that they consent to participate to this study. CK, and AP performed experiments. AT and PW analyzed the data and wrote the manuscript. NS and AlT revised the manuscript. SD and SK supervised the study. All authors read the paper and approved the nal manuscript.

Ethical approval
This experiment was approved by T by the animal ethics committee of Khon Kaen University (Record number IACUC-KKU-10162). Tables   Table 1 Physicochemical Figure 1 The cDNA sequence and the predicted prepropeptide sequence of cathelicidin-HR. The predicted mature peptide is displayed in bold. The predicted signal peptides are underlined. An asterisk (*) indicates the stop codon.  The antioxidant activity of PC29 peptide. The ABTS+ (a.) and DPPH (b.) scavenging activity of PC29 peptide.