Discovery of multiple beta-defensin like homologues in teleost fish

Abstract

Using a database mining approach, multiple defensin like genes have been discovered for the first time in fish, in species including zebrafish Danio rerio and the pufferfish, Takifugu rubripes and Tetraodon nigroviridis. They share the common features of vertebrate defensins, including small size, net cationic charge, and six conserved cysteines in the mature region. Based on their cysteine arrangement, the identified fish defensin like peptides resemble beta-defensin family members in birds and mammals. Computing modelling detected three beta-strands in all three zebrafish defensins and an extra N-terminal alpha-helix in one of the peptides. The coding regions of the fish genes contain three exons and two introns, the same as avian defensin genes. In zebrafish and tetraodon, two defensin genes identified are located in the same chromosome. An additional locus containing a third defensin gene has also been found in a different chromosome in zebrafish, demonstrating that multiple defensin loci may be present in fish. Comparative studies suggest that β-defensins may represent the primitive form of the defensin family, which expanded during evolution by gene or genome duplication. In healthy zebrafish, constitutive expression of defensins was detected by RT-PCR in gill, gonad, gut, kidney, muscle, skin and spleen but the levels and patterns varied for individual defensin genes.

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.