Discovery of multiple beta-defensin like homologues in teleost fish
Introduction
Defensins are a group of small antimicrobial peptides (AMP), which are a first line of host defense against invading pathogens. They are present in plants, invertebrates and vertebrates and have a wide spectrum of antimicrobial activities against Gram positive and Gram negative bacteria, fungi and certain enveloped viruses (Bulet and Stocklin, 2005, Castro and Fontes, 2005, Ganz, 2003, Zhang et al., 2002). In general, they are less than 100 amino acids and have a high degree of degeneracy in amino acid sequence. Regardless of their origin, defensins share some common features including net cationic charge, polarized hydrophobic and hydrophilic domains, 6–8 cysteines forming intracellular disulphide bonds, and a β-sheet structure. Plant defensins contain eight cysteines in the mature peptide region whilst insect and vertebrate defensins contain six cysteines. Vertebrate defensins can be classified as three sub families, α, β, and θ, based on the cysteine pairing to form intramolecular disulphide bonds (Ganz, 2003, Lynn et al., 2004, Xiao et al., 2004). In α-defensins, three disulphide bonds are formed by linkage of C1–C6, C2–C4 and C3–C5, whilst in β-defensins six cysteines are linked in a pattern of C1–C5, C2–C4, and C3–C6. θ-Defensin, a cyclic peptide also containing three pairs of disulphide bonds, is only found in certain primates and is believed to arise from peptide splicing of two-hemi α-defensins (Tran et al., 2002).
In humans and mice, more than 30 defensins exist in five different regions scattered over four chromosomes (Schutte et al., 2002). Chromosomes 8 and 20 in the human genome have been well analysed. Chromosome 8 contains clustered α-, β- and θ-defensin genes whilst chromosome 20 contains several β-defensins (Liu et al., 1997). In birds, some 13 β-defensin homologues have been discovered recently by in silico analysis and are clustered in a 86 kb region in chromosome 3 (Lynn et al., 2004, Xiao et al., 2004). Reptiles are also known to possess defensin like peptides, with two chromatin toxin peptides being identified in rattlesnake, which contain the same six cysteine composition as β-defensins (Radis-Baptista et al., 1999). Such studies strongly suggest that the defensin family has been expanded throughout vertebrate evolution and may have arisen from a common ancestor by gene or genome duplication events.
Defensins are expressed in a wide range of tissues and organs involved in host defense against invading microbes. Mucosal tissues such as intestine and epithelia are the notable sources of defensins (Diamond et al., 2000, O’Neil et al., 1999, Zhao et al., 1996). They are synthesised in many cell types including lymphocytes, monocytes, macrophages, neutrophils, epithelial cells, keratinocytes etc. as prepropeptides and stored in granules in the cytoplasmic region, allowing swift release of active peptides from the granules upon microbial infection. Some defensins are constitutively expressed whilst others can be induced. Differential expression is seen during different stages of cell development. For example, α-defensins are synthesised in neutrophil precursor cells and stored in the granules of the mature cells, which no longer have the ability to produce them. The processing mechanism of defensins is not yet fully elucidated but proteases such as trypsin are known to be involved in cleavage of defensin propeptides.
In recent years several AMPs have been discovered in a variety of fish species. These include: perforin in the Japanese flounder (Hwang et al., 2004), pleurocidin in the winter flounder (Cole et al., 1997), moronecidin in hybrid striped bass (Lauth et al., 2002), histone H1 in Atlantic salmon (Richards et al., 2001), oncorhyncin II and oncorhyncin III in rainbow trout (Fernandes et al., 2003, Fernandes et al., 2004), liver expressed antimicrobial peptides in rainbow trout, hybrid striped bass, zebrafish, red sea bream (Chen et al., 2005, Shike et al., 2002, Shike et al., 2004, Zhang et al., 2004) and cathelicidin in hagfish and rainbow trout (Chang et al., 2005). To date, no defensin family members have been identified in fish though recent studies have demonstrated other classic antimicrobial families found in higher vertebrates are present in lower vertebrates. Taking advantage of the expressed sequence tag (EST) databases and the completed fish genomes including zebrafish, Fugu, and tetraodon, we have identified multiple β-defensin like peptides in such fish species, the first defensin homologues reported in lower vertebrates.
Section snippets
Database mining of fish defensin sequences
By keyword searching of the Pfam database of protein families (http://pfam.wustl.edu), the alignment of the domain sequences of the beta-defensin peptides were retrieved. Since β-defensin peptides are small and their amino acid sequences are divergent, the individual domain sequence in the alignment was then used to search the TIGR expressed sequence tag (EST) database (http://tigrblast.tigr.org) using TBLASTN (blosum 45, cut-off E-value: 10). A search with one of the defensin domain sequences,
Sequence analysis
Using a database mining approach, multiple defensin like genes were discovered for the first time in fish. The identified fish defensin like peptides resemble β-defensins of birds and mammals, and fall into two subgroups based on homology comparison and phylogenetic analysis. They all have the characteristic features of vertebrate defensins including net cationic charge, small size and six conserved cysteines in the mature region. Computer modelling reveals fish defensins contain three
Discussion
In the present study, we describe for the first time the identification of multiple β-defensin like homologues in fish by analysing fish EST and genome databases. These homologues share the characteristic features of beta-defensins, that include small size, six conserved cysteines that form intramolecular disulphide bonds and a net cationic charge. Like their counterparts in higher vertebrates, fish defensins are present as multiple copy genes and some are clustered in fish genomes.
The defensin
Acknowledgements
This study was funded by a Scottish Higher Education Funding Council RDG award and a BBSRC CASE award to C.M.
References (37)
- et al.
Basic local alignment search tool
J. Mol. Biol.
(1990) - et al.
hBD-1: a novel beta-defensin from human plasma
FEBS Lett.
(1995) - et al.
Isolation and characterization of pleurocidin, an antimicrobial peptide in the skin secretions of winter flounder
J. Biol. Chem.
(1997) - et al.
Isolation and characterisation of oncorhyncin II, a histone H1-derived antimicrobial peptide from skin secretions of rainbow trout, Oncorhynchus mykiss
Dev. Comp. Immunol.
(2004) - et al.
Nuetrophil defensins: purification, characterization, and antimicrobial testing
Methods Enzymol.
(1994) - et al.
Discovery and characterization of two isoforms of moronecidin, a novel antimicrobial peptide from hybrid striped bass
J. Biol. Chem.
(2002) - et al.
The human beta-defensin-1 and alpha-defensins are encoded by adjacent genes: two peptide families with differing disulfide topology share a common ancestry
Genomics
(1997) - et al.
SCOP: a structural classification of proteins database for the investigation of sequences and structures
J. Mol. Biol.
(1995) - et al.
Nucleotide sequence of crotamine isoform precursors from a single South American rattlesnake (Crotalus durissus terrificus)
Toxicon
(1999) - et al.
Histone H1: an antimicrobial protein of Atlantic salmon (Salmo salar)
Biochem. Biophys. Res. Commun.
(2001)
Comparative protein modelling by satisfaction of spatial restraints
J. Mol. Biol.
Organization and expression analysis of the zebrafish hepcidin gene, an antimicrobial peptide gene conserved among vertebrates
Dev. Comp. Immunol.
Homodimeric theta-defensins from rhesus macaque leukocytes: isolation, synthesis, antimicrobial activities, and bacterial binding properties of the cyclic peptides
J. Biol. Chem.
Discovery and characterization of two types of liver-expressed antimicrobial peptide 2 (LEAP-2) genes in rainbow trout
Vet. Immunol. Immunopathol.
Widespread expression of beta-defensin hBD-1 in human secretory glands and epithelial cells
FEBS Lett.
Insect antimicrobial peptides: structures, properties and gene regulation
Protein Pept. Lett.
MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences
BMC Bioinform.
Plant defense and antimicrobial peptides
Protein Pept. Lett.
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