Mature Peptide Region Research Articles

Characterisation of a novel crustin isoform from mud crab, Scylla serrata (Forsskål, 1775) and its functional analysis in silico.

A 336-base pair (bp) sized mRNA sequence encoding 111 amino acid size crustin isoform (MC-crustin) was obtained from the gill sample of the green mud crab, Scylla serrata. MC-crustin possessed an N-terminal signal peptide region comprising of 21 amino acid residues, followed by a 90 amino acid mature peptide region having a molecular weight of 10.164kDa, charge + 4.25 and theoretical pI of 8.27. Sequence alignment and phylogenetic tree analyses revealed the peptide to be a Type I crustin, with four conserved cysteine residues forming the cysteine rich region, followed by WAP domain. MC-crustin was cationic with cysteine/proline rich structure and was predicted with antimicrobial, anti-inflammatory, anti-angiogenic and anti-hypertensive property making it a potential molecule for possible therapeutic applications.

A β-defensin isoform from the Flathead grey mullet, Mugil cephalus (Linnaeus 1758): Structural and functional characterisation in silico

Defensins are an important class of AMPs, rich in cysteine with antibacterial, antifungal and antiviral functions. The present study identified and functionally characterised a β-defensin gene from Grey mullet, Mugil cephalus, termed as Mc-Def. A 192 base pair complementary DNA with an open reading frame encoding 63 amino acids comprising 20 amino acid signal and 43 amino acid mature peptide region was obtained. Mc-Def has a net charge of 3.9 and a molecular weight of 7.27 kDa with an isoelectric point of 8.88. It possessed six cysteines forming 3 disulphide bonds between C1–C5, C2–C4 and C3–C6. Three dimensional structure identified a short N-terminal α helix followed by β sheet strands stabilized by disulfide bonds. The mRNA structure was predicted by Biomodel server with significant MEF score. Multiple sequence alignment and phylogeny revealed the sequence similarity of Mc-Def with fish β-defensin 2 type of AMPs. Molecular diversity and evolution of β-defensin provided immunity to host system against various infections. In silico analysis identified its potential as antibacterial, antifungal, anticancer, antioxidant and non-hemolytic peptide activity. These intriguing results firmly suggest that the Mc-Def can be used as a potent drug molecule in fish health management.

A hepatic antimicrobial peptide, hepcidin from Indian major carp, Catla catla: molecular identification and functional characterization

BackgroundIncrease of antibiotic resistance in pathogenic microbes necessitated novel molecules for curing infection. Antimicrobial peptides (AMPs) are the gene-encoded evolutionarily conserved small molecules with therapeutic value. AMPs are considered as an alternative drug for conventional antibiotics. Hepcidin, the cysteine-rich antimicrobial peptide, is an important component in innate immune response. In this study, we identified and characterized hepcidin gene from the fish, Catla catla (Indian major carp) and termed it as Cc-Hep. ResultsOpen reading frame of Cc-Hep consists of 261 base pair that encodes 87 amino acids. Cc-Hep is synthesized as a prepropeptide consisting of 24 amino acid signal peptide, 36 amino acid propeptide, and 26 amino acid mature peptide. Sequence analysis revealed that Cc-Hep shared sequence similarity with hepcidin from Sorsogona tuberculata. Phylogenetic analysis indicated that Cc-Hep was grouped with HAMP2 family. Structure analysis of mature Cc-Hep identified two antiparallel beta sheets stabilized by four disulphide bonds and a random coil. The mature peptide region of Cc-Hep has a charge of + 2, isoelectric value 8.23 and molecular weight 2.73 kDa. ConclusionFunctional characterization predicted antibacterial, antioxidant, and anticancer potential of Cc-Hep, which can be explored in aquaculture or human health care.

Analysis of the B-Type Natriuretic Peptide and the Aminoterminal-Pro-B-Type Natriuretic Peptide in Different Parrot, Raptor and Owl Species.

The B-type natriuretic peptide (BNP), a member of the natriuretic peptide family and a cardiac hormone, is produced mainly in the ventricular myocytes and released into the circulation due to mechanical stimuli during an increasing cardiac wall stretch. BNP has a significant role in the regulation of the cardiovascular system and body fluid. The concentration of this hormone and of the biologically inactive amino-terminal-prohormone in the blood plasma is a helpful diagnostic tool for detecting cardiovascular diseases in human medicine and can be used as a prognostic marker for the risk of mortality, whilst such a tool does not exist for avian medicine. To date, the amino acid sequence of BNP is not known for many of the species commonly presented in avian consultation. In this study, the amino acid sequence of BNP and the prepropeptide was described for 12 parrot species as well as 3 raptor and 3 owl species by polymerase chain reaction (PCR) after RNA isolation from the heart. The results showed a high similarity between the amino acid sequences in the mature peptide region of the BNP. The prepropeptide showed several differences between the examined species, some of them shared by closely related species.

The B-type natriuretic peptide of the Congo and Timneh grey parrot

In captivity, cardiovascular diseases are common in grey parrots. The diagnosis of these diseases in living birds is difficult, and new diagnostic possibilities would be desirable. The heart is an important endocrine organ in which cardiomyocytes synthetise B-type natriuretic peptide (BNP) and release it into the bloodstream. This hormone has a significant role in cardiovascular and body fluid regulation. The blood concentration of BNP is used in human medicine and small animal medicine as a diagnostic tool in the identification of heart diseases and as a prognostic marker for the risk of mortality. The nucleotide and amino acid sequence of BNP was described in Congo (n = 4) and Timneh (n = 3) grey parrots by PCR after RNA isolation from the atria and ventricles. The results showed a high similarity between the nucleotide sequences of the grey parrots’ BNP and the already known sequence of this hormone in chickens. The amino acid sequence of the mature peptide region is consistent in these three species. BNP plasma concentration could be a possible blood parameter for identifying clinically manifest cardiovascular diseases in grey parrots as it is in other species.

β-Defensin from the Asian Sea Bass, Lates calcarifer: Molecular Prediction and Phylogenetic Analysis

Antimicrobial peptides (AMPs) are an important element of the innate immune system of all living organisms and serve as a barrier that safeguards the organisms against a wide range of pathogens. Fishes are proven to be a prospective source of AMPs, and β-defensins form an important family of AMPs with potent antimicrobial, chemotactic and immunomodulatory activities. The present study reports a β-defensin AMP sequence (Lc-BD) from the Asian sea bass, Lates calcarifer, a commercially important fish species in tropical and subtropical regions of Asia and the Pacific. A 202-bp cDNA fragment with an open reading frame encoding 63 amino acids (aa) was obtained from the mRNA of gill tissue by RT-PCR. The deduced aa sequence of Lc-BD possessed a signal and a mature peptide region with 20 and 43 aa residues, respectively. Lc-BD was characterized at the molecular level, and a molecular weight of 5.24 kDa and a net charge of +4.5 was predicted for the mature peptide. The molecular characterization of Lc-BD revealed the presence of three intramolecular disulphide bonds involving the sixconserved cysteine residues in the sequence, and the phylogenetic analysis of Lc-BD showed a close relationship with β-defensins from fishes like Siniperca chuatsi, Argyrosomus regius, Trachinotus ovatus and Oplegnathus fasciatus.

Expression of Ietalurus punetaus β-defensin based on recombinant Pichia pastoris

β-defensin is a primary protein immune factor in channel catfish's (Ietalurus punetaus) resistance to pathogenic microorganisms. Its primary structure contains a signal peptide composed of 24 amino acid residues at the N-terminal and a mature peptide composed of 43 amino acid residues at the C-terminal. The mature peptide region is responsible for the biological activity of β-defensin. In the present study, a recombinant strain of Pichia pastoris that produces channel catfish β-defensin, was constructed to realize the biosynthesis of channel catfish β-defensin based on eukaryotic expression. First, the β-defensin gene "IPBD" was isolated from the skin of channel catfish by RT-PCR. After linking it with the expression vector pPICZA, pPICZA-IPBD was transferred into competent P. pastoris X-33 cells to obtain recombinant P. pastoris strains. The yeast transformants with multi-copy gene inserts were obtained by using the culture medium containing 1 000 μg/mL zeocin. Using BMM culture medium (without amino nitrogen culture medium) instead of BMMY culture medium (with amino nitrogen culture medium), the fermentation and culture conditions of the recombinant strain were optimized, and the optimal conditions for producing channel catfish β-defensin were determined as follows: the expression was induced for 96 h with 1.0% methanol at 28 °C , 250 r/min. Purified protein with molecular weight of 5.98 kDa was obtained by nickel affinity chromatography, and MALDI-TOF/TOF mass spectrometry proved that it was the expected recombinant IPBD. The antibacterial test results showed that the inhibitory rates of recombinant IPBD on Gram-positive Staphylococcus aureus and Listeria monocytogenes and Gram-negative Pseudomonas aeruginosa were 69.6%, 71.6% and 65.8%, respectively. This study provides a recombinant DNA technique for the development of small molecule natural antibacterial peptide from fish.

Transcriptomic Profiling Reveals Extraordinary Diversity of Venom Peptides in Unexplored Predatory Gastropods of the Genus Clavus.

Predatory gastropods of the superfamily Conoidea number over 12,000 living species. The evolutionary success of this lineage can be explained by the ability of conoideans to produce complex venoms for hunting, defense, and competitive interactions. Whereas venoms of cone snails (family Conidae) have become increasingly well studied, the venoms of most other conoidean lineages remain largely uncharacterized. In the present study, we present the venom gland transcriptomes of two species of the genus Clavus that belong to the family Drilliidae. Venom gland transcriptomes of two specimens of Clavus canalicularis and two specimens of Clavus davidgilmouri were analyzed, leading to the identification of a total of 1,176 putative venom peptide toxins (drillipeptides). Based on the combined evidence of secretion signal sequence identity, entire precursor similarity search (BLAST), and the orthology inference, putative Clavus toxins were assigned to 158 different gene families. The majority of identified transcripts comprise signal, pro-, mature peptide, and post-regions, with a typically short (<50 amino acids) and cysteine-rich mature peptide region. Thus, drillipeptides are structurally similar to conotoxins. However, convincing homology with known groups of Conus toxins was only detected for very few toxin families. Among these are Clavus counterparts of Conus venom insulins (drillinsulins), porins (drilliporins), and highly diversified lectins (drillilectins). The short size of most drillipeptides and structural similarity to conotoxins were unexpected, given that most related conoidean gastropod families (Terebridae and Turridae) possess longer mature peptide regions. Our findings indicate that, similar to conotoxins, drillipeptides may represent a valuable resource for future pharmacological exploration.

Recombinant expression of Crassostrea gigas defensin in Pichia pastoris and its antibacterial activity

Defensins are endogenous cationic antimicrobial peptides rich in arginine and cysteine residues. They are important immune factors resisting pathogenic bacteria infection for mollusks. The 43 amino acid residues near the carboxyl terminal for Crassostrea gigas defensin (CgD) form its mature peptide region, responsible for the biological activity of CgD. First, two target genes, CgDH⁺ (with 6×His-tag at 3' end) and CgDH- (without 6×His-tag at 3' end) were separated and amplified by RT-PCR with specific primers from Crassostrea gigas mantle. These two target genes were ligated to the expression vector pPICZαA to construct recombinant expression vectors, pPICZαA-CgDH⁺ and pPICZαA-CgDH-, which were transformed into competent Pichia pastoris X-33 cells by electroporation respectively. The recombinant target proteins, CgDH⁺ and CgDH-, were induced for 72 h with 1% methanol at 29 °C and 250 r/min. The recombinant CgDH⁺ (5.78 kDa) was purified by immobilized metal affinity chromatography (IMAC), and identified by MALDI-TOF-TOF analysis, demonstrating that it was the expected target protein. Based on the concentration of the purified product, the estimated yield of recombinant CgDH⁺ was 2.32 mg/L. Antimicrobial assay showed that the culture medium supernatant containing recombinant CgDH⁺ and recombinant CgDH-, respectively, had activities against Staphylococcus aureus and Pseudomonas aeruginosa, indicating that the existence of 6×His tag in the recombinant proteins do not affect their biological activities.

Copy number variation of PR-39 cathelicidin, and identification of PR-35, a natural variant of PR-39 with reduced mammalian cytotoxicity

Proline-arginine-rich (PR)-39 is neutrophil antimicrobial peptide that has potent antimicrobial activity against a broad spectrum of microorganisms, including bacteria, fungi, and some enveloped viruses as a part of the innate immune system. We analyzed the nucleotide sequence variations of PR-39 exon 4, which is the mature peptide region responsible for antimicrobial activity, from 48 pigs of six breeds using sequence-based typing. The analysis identified four alleles including allele PR-35 with a 12-bp deletion near the N-terminus. Interestingly, 16.7% of individuals showed the presence of three alleles per individual, but only in the Berkshire and Duroc breeds. We further analyzed the genetic diversity of PR-39 for the entire genomic region of the gene from PR-39 exon 1 to the 3′ untranslated region for different alleles by PCR amplification and cloning. The antimicrobial activity of chemically synthesized PR-35 was similar to that of PR-39, but the level of mammalian cell cytotoxicity was lower than the wild type. Better knowledge of the genetic diversity of PR-39 among different individuals and breeds may contribute to improved immune defense of pigs. PR-35, as a natural antimicrobial peptide variant, could be an interesting candidate for the development of peptide antibiotics.

Genome-wide analysis of the ovodefensin gene family: Monophyletic origin, independent gene duplication and presence of different selection patterns

Ovodefensins (OvoDs) represent a group of cysteine-rich host defense peptides that are abundant in the egg white. Recent studies have found that ovodefensins are specific to birds and reptiles. However, the entire repertoire and evolutionary relationships of this gene family have not been thoroughly elucidated to date. Following our cross-species and genome-wide computational study, a total of 94 ovodefensin genes with multiple novel cysteine sequence motifs were identified from 22 phylogenetically divergent species. Phylogenetic analysis suggests that a large number of OvoDs evolved by gene duplication after species divergence. Furthermore, the OvoD genes in each species trend to be clustered densely in a syntenic region flanked by the XKR6 and MTMR9 genes, indicating that they are of monophyletic origin and appear to have emerged via independent gene duplication events in snakes, turtles, crocodiles, birds and the green lizard. Furthermore, positive selection sites are located primarily in the mature peptide region of the turtle, lizard and snake OvoD genes. Moreover, the duplicate OvoDAs in birds seem to be maintained in almost identical sequences and functions by strong purifying selection. Genome-wide identification and analyses of the OvoD gene family may greatly improve our understanding of the potential evolutionary relationship scenario of the OvoD gene family. Continued sequence mining and functional studies of OvoDs will be helpful in shedding light on the relationships between OvoDs and other defensin-related gene families.

Genes Encoding Hevein-Like Antimicrobial Peptides from Elytrigia repens (L.) Desv. ex Nevski

Earlier, we discovered a gene family encoding hevein-like antimicrobial peptides (WAMP) in the highly resistant wheat species Triticum kiharae Dorof. et Migusch. and related species of the Triticum and Aegilops genera. These peptides suppress growth and development of fungi and bacteria by inhibition of secreted metalloproteinases of the pathogens. In this study, we analyzed wamp homologs in the wild cereal Elytrigia repens Desv. ex Nevski, which is an invasive weed. The wamp homologs were isolated by PCR with E. repens genomic DNA or cDNA and primers specific to the wheat wamp genes. The nucleotide sequences of three novel E. repenswamp genes encoding the precursors of the antimicrobial peptides named ERAMP-1, ERAMP-2 and ERAMP-3 were determined. The mature peptide regions of the precursors differed in single amino acid substitutions. It was shown that ERAMP-2 and ERAMP-3 have valine at position 34 affecting the degree of fungal proteinase inhibition, which has not been found in other WAMP homologs. To elucidate the role of the valine residue in the E. repens peptide antifungal activity, the recombinant peptide was expressed in E. coli and its antifungal activity was assayed against a range of phytopathogenic fungi belonging to ascomycetes. The peptide was more active than the wheat WAMP-1 peptide against three of four tested fungi infecting cereals and other plant species. The results obtained contribute to our knowledge of the biodiversity of wamp genes in Poaceae. In addition, they expand our understanding of the repertoire of defensive genes in E. repens responsible for its enhanced pathogen resistance.

Plant thionins: structure, biological functions and potential use in biotechnology

Antimicrobial peptides (AMPs) are important components of defense system in both plants and animals. They represent an ancient mechanism of innate immunity providing rapid first line of defense against pathogens. Plant AMPs are classified into several families: thionins, defensins, nonspecific lipid-transfer proteins, hevein- and knottin-type peptides, hairpinins and macrocyclic peptides (cyclotides). The review focuses on the thionin family. Thionins comprise a plant-specific AMP family that consists of short (~5 kDA) cysteine-rich peptides containing 6 or 8 cysteine residues with antimicrobial and toxic properties. Based on similarity in amino acid sequences and the arrangement of disulphide bonds, five structural classes of thionins are discriminated. The three-dimensional structures of a number of thionins were determined. The amphipathic thionin molecule resembles the Greek letter Г, in which the long arm is formed by two antiparallel α-helices, while the short one, by two parallel β-strands. The residues responsible for the antimicrobial activity of thionins were identified. Thionins are synthesized as precursor proteins consisting of a signal peptide, the mature peptide region and the C-terminal prodomain. Thionins protect plants from pathogenic bacteria and fungi acting directly on the membranes of microorganisms at micromolar concentrations, although their precise mode of action remains unclear. In addition to plant pathogens, thionins inhibit growth of a number of human pathogens and opportunistic microorganisms, such as Candida spp., Saccharomyces cerevisiae, Fusarium solani, Staphylococcus aureus and Escherichia coli. Thionins are toxic to different types of cells including mammalian cancer cell lines. Transgenic plants expressing thionin genes display enhanced resistance to pathogens. A wide range of biological activities makes thionins promising candidates for practical application in agriculture and medicine.

Motif analysis of avian liver expressed antimicrobial peptide 2 (LEAP2)

Liver-expressed antimicrobial peptide 2 (LEAP2) is the second blood-derived peptide identified from human blood and shows antimicrobial activity and predominant expression in the liver. It is a cysteine-rich antimicrobial peptide characterized by the presence of a four cystein conserved motifs and two intracellular disulphide bridges. In silico analyses of avian LEAP2 sequences were performed in the study. Mature peptide regions were found to be highly conserved in comparison to pro-domain and signal sequence. Majority of the avian samples analyzed possessed RQRR motif at the cleavage site between pro-region and mature region. Beyond microbial killing, LEAP2 of Aquila chrysaetos canadensis has been found to play additional roles in signaling pathway, oocyte, follicular development, cytokine activity and in apoptosis regulation. Higher conservation in the amino acid sequences of LEAP2 among birds suggests the significant role of this molecule in avian evolution.

Genes encoding hevein-like antimicrobial peptides WAMPs in the species of the genus Aegilops L.

Earlier, in the wheat Triticum kiharae Dorof. et Migusch., a new family of genes coding for the hevein-like antimicrobial peptides WAMPs, involved in the protection of wheat plants against pathogens, was discovered. In the present study, we examined the wamp homologs in plants belonging to ten di-, tetra-, and hexaploid species of the genus Aegilops L., among which there are donors of polyploid wheat genomes, as well as of the resistance genes to the most important wheat pathogens. Using PCR amplification with genomic DNA as a template and primers specific to the sequences of the wheat wamp genes, for the first time, nucleotide sequences of the protein-coding regions of wamp homologs were determined in the species of the genus Aegilops L. The wamp homologs were found in all species studied. It was demonstrated that the WAMP peptide precursors encoded by them differed in single nucleotide substitutions, as well as deletions/insertions of amino acid sequences. The most conserved region of the precursor is the mature peptide region, where, in addition to the variable position 34, deletions of amino acid sequences were found in a number of peptides. To elucidate the role of deletions in the antimicrobial activity of WAMPs, a recombinant WAMP-3 peptide with a deletion in the N-terminal region was produced by expression in E. coli cells, and it was shown that antimicrobial activity of the peptide was preserved. It was demonstrated that all the discovered wamp genes were expressed in seedlings of the studied Aegilops species. The results shed new light on the structural diversity of genes encoding the hevein-like antimicrobial peptides WAMPs.

Molecular Characterisation of a Novel Isoform of Hepatic Antimicrobial Peptide, Hepcidin (Le-Hepc), from Leiognathus equulus and Analysis of Its Functional Properties In Silico.

Hepcidin represents a family of cysteine-rich antimicrobial peptides that are mainly expressed in the liver of living organisms. In this study, we have identified and characterised a novel isoform of hepcidin from the common pony fish, Leiognathus equulus (Le-Hepc). A 261-bp fragment cDNA coding for 86 amino acids was obtained. Homologous analysis showed that Le-Hepc belongs to the hepcidin super family and shares sequence identity with other known fish pre-propeptide hepcidin sequences. The ORF encodes for a 24-amino acid (aa) signal peptide coupled to a 36-aa prodomain followed by a 26-aa mature peptide. The mature peptide region has a calculated molecular weight of 2.73kDa, a net positive charge of +2 and a theoretical pI of 8.23. Phylogenetic analysis of Le-Hepc showed a strong relationship with other fish hepcidin sequences and clustered into HAMP2 group hepcidins. Secondary structural analysis indicated that Le-Hepc mature peptide contains two antiparallel β-sheets strengthened by four disulphide bonds formed by eight conserved cysteine residues. The physicochemical properties of the peptide and its structural parameters are in agreement with characteristic features of an antimicrobial peptide. This is the first report of an antimicrobial peptide from the common pony fish, L. equulus.

A CXCL ortholog from Hippocampus abdominalis: Molecular features and functional delineation as a pro-inflammatory chemokine

Chemokines are a family of chemotactic cytokines that regulate leukocyte migration. They are classified into four groups namely, CXC, CC, C and CX3C, based on the formation of a disulfide bridge. Among these, CXC chemokines have been identified as the largest group of chemokines in humans. In this study, we identified and functionally characterized a homolog of CXC chemokine from the big-belly seahorse, Hippocampus abdominalis, and designated it as ShCXCL. The cDNA of ShCXCL composed of a 342-bp open reading frame encoding 113 amino acids (aa). The CXC family-specific small cytokine domain (SCY) was identified from the mature peptide region, which comprised of a conserved CXC motif. As ShCXCL lacks an ELR (Glutamic acid-Leucine-Arginine) motif, it belongs to ELR− subfamily. The recombinant ShCXCL protein strongly induced the nitric oxide (NO) production in macrophage cells (RAW 264.7 cell line) and showed the chemotactic effect on flounder peripheral blood leukocytes. Tissue profiling showed a ubiquitous expression pattern in all examined tissues, with a high abundance in spleen. The up-regulated mRNA expression pattern of ShCXCL was observed in blood and kidney tissues after immune stimulation by live bacteria, such as Streptococcus iniae and Edwardsiella tarda, and mitogens, such as lipopolysaccharides (LPS) and polyinosinic:polycytidylic acid (poly I:C), suggesting its important role in host immune defense against microbial infection.

Antimicrobial activity of recombinant E. coli expressed chicken AvBD-2 and its mRNA expression in Indian native Aseel and Kadaknath chicken breeds

Avian b defensins (AvBD) are antimicrobial peptides that play a crucial role in the innate immune response in chickens. In the present study, chicken AvBD2 gene was cloned, expressed in E. coli system and the in vitro antimicrobial activity of recombinant peptide was evaluated. The entire mature peptide region of chicken AvBD2 region was amplified and cloned in pUC29 cloning vector. Further, the coding region was sub cloned in pET-28A expression vector. After transformation in E. coli cells, the peptide synthesis was induced and recombinant protein (7.7 kDa) was purified by using Ni-NTA affinity column. The recombinant chicken AvBD2 showed antibacterial activity against S. Pullorum. The minimum bactericidal concentration (MBC) of recombinant chicken AvBD2 evaluated by micro-broth dilution assay was 35 µg/ ml. We also quantified the expression of AvBD2 transcript expression in day-old spleen tissue of Indian native chicken breeds (Aseel and Kadaknath) and White Leghorn. Measurable AvBD2 mRNA expression was found in the spleen of all three breeds. However, no significant difference was found in AvBD2 gene expression between native chickens and White Leghorn.

A Comprehensive Analysis of RALF Proteins in Green Plants Suggests There Are Two Distinct Functional Groups

Rapid Alkalinization Factors (RALFs) are small, cysteine-rich peptides known to be involved in various aspects of plant development and growth. Although RALF peptides have been identified within many species, a single wide-ranging phylogenetic analysis of the family across the plant kingdom has not yet been undertaken. Here, we identified RALF proteins from 51 plant species that represent a variety of land plant lineages. The inferred evolutionary history of the 795 identified RALFs suggests that the family has diverged into four major clades. We found that much of the variation across the family exists within the mature peptide region, suggesting clade-specific functional diversification. Clades I, II, and III contain the features that have been identified as important for RALF activity, including the RRXL cleavage site and the YISY motif required for receptor binding. In contrast, members of clades IV that represent a third of the total dataset, is highly diverged and lacks these features that are typical of RALFs. Members of clade IV also exhibit distinct expression patterns and physico-chemical properties. These differences suggest a functional divergence of clades and consequently, we propose that the peptides within clade IV are not true RALFs, but are more accurately described as RALF-related peptides. Expansion of this RALF–related clade in the Brassicaceae is responsible for the large number of RALF genes that have been previously described in Arabidopsis thaliana. Future experimental work will help to establish the nature of the relationship between the true RALFs and the RALF-related peptides, and whether they function in a similar manner.

Characterization and expression of non‐polymorphic liver expressed antimicrobial peptide 2: LEAP‐2 in the Japanese quail, Coturnix japonica

Liver-expressed antimicrobial peptide 2 (LEAP-2) is a cationic peptide that plays an important role in innate immunity for host defense. The aim of this study was to characterize the LEAP-2 gene in the Japanese quail (Coturnix japonica). Japanese quail LEAP-2 (CjLEAP-2) was identified from the Japanese quail draft genome database by a local BLAST analysis using chicken LEAP-2 (GgLEAP-2). The exon-intron structure of CjLEAP-2, analyzed from three quails, is composed of three exons, as is the chicken LEAP-2 homolog (GgLEAP-2). An analysis of the coding sequence revealed that CjLEAP-2 is 231 bp long, like GgLEAP-2, and 93% identical to GgLEAP-2 at the nucleic acid level. The predicted amino acid sequence of CjLEAP-2 contained the liver-expressed antimicrobial peptide 2-precursor domain and four cysteine residues characteristic of the LEAP-2 protein. The amino acid sequence of the mature peptide of CjLEAP-2 was 100% identical to that of GgLEAP-2. We confirmed that CjLEAP-2 was transcribed in at least seven tissues, including the digestive system. Additionally, the mature peptide region of CjLEAP-2 exhibited no polymorphisms in 99 quails from six strains. Taken together, these findings indicate that CjLEAP-2 is non-polymorphic and therefore, it likely plays an important role in the innate immunity of quail as it does in chicken.