Identification and characterisation of the BPI/LBP/PLUNC-like gene repertoire in chickens reveals the absence of a LBP gene

Abstract

Palate, lung and nasal epithelial clone (PLUNC) proteins are structural homologues to the innate defence molecules LPS-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI). PLUNCs make up the largest portion of the wider BPI/LBP/PLUNC-like protein family and are amongst the most rapidly evolving mammalian genes. In this study we systematically identified and characterised BPI/LBP/PLUNC-like protein-encoding genes in the chicken genome. We identified eleven complete genes (and a pseudogene). Five of them are clustered on a >50 kb locus on chromosome 20, immediately adjacent to BPI. In addition to BPI, we have identified presumptive orthologues LPLUNCs 2, 3, 4 and 6, and BPIL-2. We find no evidence for the existence of single domain containing proteins in birds. Strikingly our analysis also suggests that there is no LBP orthologue in chicken. This observation may in part account for the relative resistance to LPS toxicity observed in birds. Our results indicate significant differences between the avian and mammalian repertoires of BPI/LBP/PLUNC-like genes at the genomic and transcriptional levels and provide a framework for further functional analyses of this gene family in chickens.

Fig. 1

Organisation of the BPI/LBP/PLUNC-like gene locus in chickens. Schematic representation of the human PLUNC and chicken BPI/LBP/PLUNC-like loci. The lower panel indicates the organisation of the chicken locus on Chromosome 20, whereas the upper panel represents the human locus (adapted from Wheeler et al., 2007). Arrows between the two loci indicate orthologous relationships described in the text. The position of the locus on the chromosome is indicated by the grey arrow-heads and nucleotide numbers of the assembled chicken chromosome, obtained from the UCSC BLAT genome browser (http://genome.ucsc.edu/). The solid bars above the locus indicate the positions of gaps in the assembly that were closed in this study. The cross hatches at either end of the locus indicate breaks in the sequence introduced for clarity. The arrowed end of each gene indicates the direction of the ORF. Intact BPI/LBP/PLUNC-like (BPIL) genes are shaded while the pseudogene (P10) is hatched. The unrelated gene CDK5RAP1 is not filled. The black scale bars indicate either 50 kb or 10 kb.

Fig. 2

Unrooted phylogenetic tree of BPI/LBP/PLUNC-like family members. An unrooted phylogenetic tree of chicken BPI/LBP/PLUNC-like proteins was generated as described using additional two-domain containing proteins from a range of species. Where a chicken protein is identified as a 1:1(or a 2:1 for cBPIL2a/2b) orthologue of a human protein the position of the branchpoint marking the most recent ancestral protein of the pair is marked with a small filled circle. The closer the circle is to the centre of figure, the more dissimilar is the orthologous pair. The distances that must be traced along the tree to connect two proteins are related to the sequence similarity of the proteins. Example sequence identities to provide scale are hLPLUNC4: mLPLUNC4 at 86%, hLPLUNC4: cLPLUNC4 at 58%, cLPLUNC4: cLPLUNC3 at 40% and cLPLUNC4; cLBP/BPI at 20%. Branches with bootstrap values <95% are coloured red; the detailed branching order is uncertain in these regions. The following sequences were retrieved from public databases and used for the analysis. Zebra finch (Taeniopygia guttata). tgBPI, XP_002192404.1; tgCETP, XP_002197982.1; tgPLTP, XP_002191962.1. Zebra fish (Danio rerio). drPLTP, NP_001003519.1; drCETP, NP_001007361.1. Puffer fish (Tetraodon nigroviridis). tnBPILBP, CAF96904.1; tn1, CAG03233.1; tnPLTP, CAF90160.1. Atlantic cod (Gadus morhua). gmBPILBP1, AAM52335.1; gmBPILBP2, AAM52336.1. Trout (Oncorhynchus mykiss). omLBPBPI1, NP_001118057.1; omLBPBPI2, NP_001117670.1. Catfish (Ictalurus punctatus). ipBPILBP, AAX20011.1. Pacific oyster (Crassostrea gigas) cgLBP/BPI, AAN84552.1. Pipid frog (Xenopus laevis). xlCETP, NP_001088804.1; xlBPI1a, NP_001089628.1; xlBPI1b, NP_001086208.1; xlPLTP, NP_001087767.1; xl2, NP_001088621.1. Cow (Bos taurus). bBPI3, XP_592903.2; bLBP, NP_001033763.1; bBPI, NP_776320.1. Human (Homo sapiens). hLBP, NP_004130.2; hBPI, NP_001716.2; hCETP, NP_000069.2; hPLTP, AAH19847.1; hLPLUNC1, AAH08429.1; hLPLUNC4, NP_872325.2; hLPLUNC3, NP_872599.1; hLPLUNC2, NP_079503.1; hLPLUNC6, NP_777557.1; hBPIL2, NP_777592.1. Mouse (Mus musculus). mBPI, Q67E05.1; mLPLUNC1, NP_700467.2; mLPLUNC5, NP_659139.2; mLPLUNC3, NP_919338.2; mLPLUNC2, NP_079907.2; mLPLUNC6, Q8BU51.2; mBPIL2, NP_808440.2; mPLTP, AAH03782.1; mLPLUNC4, NP_001030047.2; mLBP, NP_032515.2. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

Fig. 3

Multiple sequence analysis of chicken BPI/LBP/PLUNC-like proteins. The predicted amino acid sequences of chicken BPI/LBP/PLUNC proteins were aligned using CLUSTALX. In the alignment, identical residues (found in >7 sequences) are indicated by the white on black background, whereas similar residues (conserved in >7sequences) have the grey background. Spaces introduced for maximum alignments are indicated by a -. The positions of the conserved cysteine residues and the disulphide bond are indicated by *----*. The numbers indicate the amino acids.

Fig. 3

Multiple sequence analysis of chicken BPI/LBP/PLUNC-like proteins. The predicted amino acid sequences of chicken BPI/LBP/PLUNC proteins were aligned using CLUSTALX. In the alignment, identical residues (found in >7 sequences) are indicated by the white on black background, whereas similar residues (conserved in >7sequences) have the grey background. Spaces introduced for maximum alignments are indicated by a -. The positions of the conserved cysteine residues and the disulphide bond are indicated by *----*. The numbers indicate the amino acids.

Fig. 4

Expression analysis of chicken BPI/LBP/PLUNC-like proteins. Expression of chicken BPI/LBP/PLUNC-like genes was studied by PCR in multiple tissue samples using exon spanning primer pairs to each individual gene as described in the materials and methods section. Expression of β-actin (as a PCR control) was also monitored in the same samples.

Fig. 5

Schematic representation of the evolution of the BPI/LBP//PLUNC-like genes. The tree illustrates the evolution of the wider BPI/PLUNC-like gene family during vertebrate evolution. Fish represent the most divergent vertebrate ancestors in which clear orthologues exist. Black filled circles represent the earliest origin of clearly defined orthologues. Open circles with crossed out symbols represent gene loss in a lineage.