Expansion of the Bactericidal/Permeability Increasing-like (BPI-like) protein locus in cattle
© Wheeler et al; licensee BioMed Central Ltd. 2007
Received: 08 December 2006
Accepted: 15 March 2007
Published: 15 March 2007
Cattle and other ruminants have evolved the ability to derive most of their metabolic energy requirement from otherwise indigestible plant matter through a symbiotic relationship with plant fibre degrading microbes within a specialised fermentation chamber, the rumen. The genetic changes underlying the evolution of the ruminant lifestyle are poorly understood. The BPI-like locus encodes several putative innate immune proteins, expressed predominantly in the oral cavity and airways, which are structurally related to Bactericidal/Permeability Increasing protein (BPI). We have previously reported the expression of variant BPI-like proteins in cattle (Biochim Biophys Acta 2002, 1579, 92–100). Characterisation of the BPI-like locus in cattle would lead to a better understanding of the role of the BPI-like proteins in cattle physiology
We have sequenced and characterised a 722 kbp segment of BTA13 containing the bovine BPI-like protein locus. Nine of the 13 contiguous BPI-like genes in the locus in cattle are orthologous to genes in the human and mouse locus, and are thought to play a role in host defence. Phylogenetic analysis indicates the remaining four genes, which we have named BSP30A, BSP30B, BSP30C and BSP30D, appear to have arisen in cattle through a series of duplications. The transcripts of the four BSP30 genes are most abundant in tissues associated with the oral cavity and airways. BSP30C transcripts are also found in the abomasum. This, as well as the ratios of non-synonymous to synonymous differences between pairs of the BSP30 genes, is consistent with at least BSP30C having acquired a distinct function from the other BSP30 proteins and from its paralog in human and mouse, parotid secretory protein (PSP).
The BPI-like locus in mammals appears to have evolved rapidly through multiple gene duplication events, and is thus a hot spot for genome evolution. It is possible that BSP30 gene duplication is a characteristic feature of ruminants and that the BSP30 proteins contribute to an aspect of ruminant-specific physiology.
Ruminants have acquired a number of physiological and anatomical specialisations in order to adapt to a lifestyle in which pasture is the predominant source of metabolic energy. Most notably ruminants have a fore-stomach, the rumen, in which pasture polysaccharides are broken down by microbial β-glycosidases in a neutral pH anaerobic environment. In addition, ruminants have other adaptations, including a markedly different saliva composition compared with monogastric mammals [1, 2]. It is assumed that these physiological adaptations must be accompanied by genetic changes, however, there have been few reports of changes in the genomes of ruminants, which facilitate a specialised ruminant physiological function. Virtually the only such report is of the expansion of the lysozyme locus in cattle . The recent availability of a draft cattle genome sequence, the first for a ruminant, provides an opportunity to discover additional genetic characteristics that facilitate the ruminant lifestyle.
The Bactericidal/Permeability Increasing protein (BPI) plays an important role in host-defence in mammals. BPI is found in the secretory granules of neutrophils and is secreted in response to activation of Toll-like receptor (TLR)-mediated signalling, whereupon it acts as an innate immune effector protein by permeabilising the plasma membrane of Gram negative bacteria as well as attenuating the TLR response [4, 5]. Three well-characterised proteins have some sequence conservation with BPI. Lipopolysaccharide binding protein (LBP) is secreted from the liver into the circulation where it appears to act as a sensor for the presence of bacteria . LBP acts as an opsin, binding lipopolysaccharide (LPS) derived from the outer membrane of Gram negative bacteria, and thence stimulating a TLR-mediated innate immune response . Phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP) function as lipid transport proteins in the blood (reviewed in [8, 9]). Recent reports have shown the existence of at least 10 additional genes in humans and mice, which are related to BPI through sequence similarity, exon segmentation and predicted secondary structure [10, 11]. All but two of these are found as a gene cluster at a single locus on human chromosome 20 or the syntenic region of mouse chromosome 2. The similarity of the products of these genes to BPI and LBP, their expression in oral cavity and airways tissues [12–16] and evidence for the antimicrobial activity of at least one of them  suggests that they play a role in host defence.
We have previously characterised two closely related members of the expanded BPI-like protein family in cattle. These proteins, BSP30A and BSP30B, are expressed in saliva and are both most closely related to human and mouse PSP [18–20]. We have now characterised the entire BPI-like protein locus in cattle in order to understand the relationship of the BSP30A and BSP30B genes to one another and to PSP, and to understand the evolutionary events that have occurred in the locus in cattle, particularly gene duplication. Here we report that the bovine locus contains 13 BPI-like genes, comprising nine homologues of BPI-like genes from mouse and human as well as four paralogues of PSP, two of which have not been previously described. These appear to have arisen from a series of gene duplication events. Their distinct patterns of transcript abundance, and their presence in at least some other ruminant species is consistent with the multiple BSP30 genes having a specific role in ruminant physiology.
Characterisation of the bovine BPI-like locus
Six bovine BACs spanning the BPI-like locus were identified by alignment of bovine BAC end sequences with the region of the human genome sequence containing the BPI-like locus. These BACs were subjected to random shotgun high throughput sequencing. These sequences, as well as sequence contigs from the bovine assembly (BosTau 2.0, current at the time study was undertaken) were used to create a single 721,869 bp contig as described in the Methods [GenBank: DQ667137].
Genes comprising the bovine BPI-like locus
start of first exon
end of last exon
% aa identity2
73 (h), 63 (m)
79 (h), 68 (m)
80 (h), 82 (m)
84 (h), 85 (m)
67 (h), 63 (m)
73 (h), 65 (m)
56 (h), 49 (m)
50 (h), 59 (m)
alignment of BSP30 and PSP amino acid sequences (% aa identity)
Expression of the BPI-like genes in cattle
Expression of bovine BPI-like genes (Y indicates expression observed)
TIGR Gene Indices
Evolution of the BPI-like locus
dN/dS ratios (± SE) for the BPI-like protein genes
0.92 (± 0.31)
0.51 (± 0.19)
0.48 (± 0.17)
0.16 (± 0.13)
0.22 (± 0.13)
0.26 (± 0.11)
0.25 (± 0.11)
0.12 (± 0.05)
0.12 (± 0.05)
0.06 (± 0.03)
0.10 (± 0.04)
0.04 (± 0.02)
0.90 (± 0.35)
0.66 (± 0.23)
0.54 (± 0.18)
0.54 (± 0.19)
0.31 (± 0.11)
0.22 (± 0.08)
0.39 (± 0.19)
0.37 (± 0.15)
0.28 (± 0.11)
0.22 (± 0.08)
dN/dS ratios (± SE) for the BSP30 and PSP genes
0.57 (± 0.32)
1.90 (± 0.60)
1.13 (± 0.38)
1.13 (± 0.34)
1.33 (± 0.40)
1.95 (± 0.62)
1.37 (± 0.48)
1.12 (± 0.34)
1.25 (± 0.39)
2.73 (± 0.90)
1.33 (± 0.38)
1.54 (± 0.47)
1.16 (± 0.34)
1.40 (± 0.42)
0.96 (± 0.29)
Amino acid sites under positive selection
Number of Parameters
Parameter Estimates a
Sites under positive selection
þ0 = 0.114
ω0 = 0.185
þ0 = 0.031
þ1 = 0.771
ω0 = 0
ω2 = 3.881
80, 118, 182, 198, 214 & 233
þ = 0.166 q = 0.012
þ0 = 0.793
þ = 0.171
q = 0.011
ωs = 3.612
80, 118, 182, 198, 214 & 233
A segment of the genomic contig from the RY2G5 to the BASE gene was used as query in a BLAST search of a bovine repetitive sequence database  to search for the presence of repeat elements within it. The search returned two Long Interspersed Nuclear Elements (LINEs), L1Bt and RTEBt1. In total, significant alignments of greater than 800 bp were obtained at ten locations along the genomic segment. All but one of these were positioned within or very close to the gaps between the genomic duplications (Fig. 7). It is possible that these LINE elements could have contributed to the genome instability in this region of the locus, resulting in the multiple gene duplications, in a manner analogous to what has previously been suggested for Alu sequences in the human genome .
This report provides the first characterisation of the locus encoding the BPI-like proteins in a ruminant species. The analyses have shed light on how the locus has evolved as well as raised possibilities regarding the function of the BPI-like proteins in ruminants. The structural similarity and clustering of the individual genes in the BPI-like locus in the bovine, mouse, and human genomes suggests evolution from a single ancestral gene, with gene duplication followed by divergent evolution giving rise to the differences between the family members. This appears to be particularly prevalent in the bovine BPI-like protein locus compared with the other species. Gene duplication has been previously noted in ruminants. Duplication of a single ancestral secretory RNAse gene appears to have given rise to the separate pancreatic, seminal and brain RNAse genes found in ruminants . Ruminants and other artiodactyls contain a large number of genes encoding the pregnancy-associated glycoproteins, which appear to have arisen by gene duplication . Duplication of the lysozyme gene is a feature in its recruitment as a digestive enzyme in ruminant artiodactyls . The orientation of the genes in the BPI-like cluster is consistent with gene amplification by a series of unequal crossovers . The presence of multiple LINE elements within the expanded BSP30 region of the locus provides one possible mechanism for the initiation step of the expansion.
The family of proteins in the cluster is divided into those with one or two BPI domains (the long and short PLUNCs ). The single-domain genes are contiguous within the cluster. It is possible that the ancestral BPI-like gene was a two-domain lipid binding protein. The six two-domain proteins then arose through a series of gene duplications. The first single-domain protein may have been created through duplication of the N-terminal portion of one of the two-domain proteins. The phylogenetic analysis together with their position in the locus suggests that this is most likely to have been the VEMSGP gene giving rise to the PLUNC gene. Under this scenario, subsequent duplication of PLUNC would have given rise to additional single-domain proteins, some of which could have been duplicated in turn, as appears to be the case with PSP giving rise to the four BSP30 genes. The most recent duplication appears to have been that giving rise to BSP30A and B. The data indicates that duplication events have occurred more frequently than the minimal number of times required for the observed gene duplications. Interestingly, it appears that there have been distinct duplications in the mouse lineage giving rise to SPLUNC5 and SPLUNC6.
The duplications giving rise to the four BSP30 genes in cattle may have occurred after divergence of cattle from other mammals. Examination of genome assemblies from other species indicates that human, dog and chimpanzee have only a single PSP ortholog. Furthermore, a search of EST databases revealed only single PSP orthologs in human, dog and pig [GenBank:CB986486, DN405753 and CJ027526, respectively]. Analysis of an in-house sheep EST database revealed four ESTs [GenBank: EE792560, EE792910, EE794201, and EE794362] that align closely with both BSP30A and BSP30B. This indicates that homologs of BSP30A and/or B exist in a second ruminant species, the sheep, consistent with the possibility that the BSP30 gene expansion may have coincided with the radiation of ruminant species. Further analyses are required to confirm this.
The analyses reported here raise some intriguing questions regarding the function of the BPI-like proteins. All of the greater family of BPI-like proteins characterised to date share a common biochemical property in binding complex lipids. These proteins have distinct sites of expression and diverse functions such as lipid transport and innate immunity. This and other reports [14, 16, 20, 30] show that most of the BPI-like proteins are most abundant in tissues associated with the oral cavity and airways. Here we show that at least one family member is also expressed at a lower level in the digestive tract. It is likely the BPI-like proteins function in epithelial mucosa after being secreted.
The question of whether the BSP30 proteins have a similar lipid binding or bactericidal activity to that of BPI awaits experimental verification. In support of this, recombinant human PSP has been reported to inhibit the growth of P. aeruginosa , and human PLUNC, has been shown to bind LPS . If these activities for the BSP30 proteins are confirmed, a possible biological role for them could be in modulating the microbial ecology in the bovine oral cavity so as to maintain optimal digestive function or prevent pathological infection. The results presented here suggest that the shape of the hydrophobic pocket may be important for determining functional differences among the BSP30 proteins.
The bovine BPI-like locus of cattle features expansion of the single PSP gene present in human and mouse into four distinct BSP30 genes. The dN/dS ratio data are consistent with evolutionary pressure for conservation of protein structure between all the orthologous pairs of BPI-like genes in cattle, human and mouse. However, this pressure is absent between the BSP30 genes, and the data suggests there is pressure for sequence divergence between BSP30C and the other BSP30 proteins. This, as well as its distinct expression profile, is consistent with BSP30C having acquired a distinct function from the other BSP30 proteins. The most likely biological role of the BPI-like proteins, including the BSP30 proteins, is as either detector or effector proteins in innate immune host defence . While their precise biological roles are unknown, one can speculate that the BSP30 proteins may influence the host response to the commensal microbial ecosystem in cattle, including that of the rumen. BSP30A and B comprise approximately 30% of total salivary protein in cattle , thus resulting in up to 150 g per day of BSP30 proteins being delivered into the rumen. A focus for future investigations is to determine whether the BSP30 gene duplications and subsequent divergence observed in cattle could have been a key step in the evolution of ruminants by facilitating adaptation to a ruminant lifestyle.
BAC screening, sequencing and assembly
A 1 Mbp region of the human assembly containing the PSP region was downloaded from UCSC (hg16:Chr20:32–33 Mbp lower case masked) and compared against 292,638 reads from the ends of individual random BAC clones derived from two bovine genomic DNA BAC libraries (downloaded NCBI Nov 2003) using BLASTN and the following options -m 8 -e 1e-2 -U T. The output was then processed and ranked for high quality paired end hits in the appropriate orientation and estimated size. Over 30 BAC clones mapped to the human BPI-like locus on chromosome 20. These were screened for the presence of known BPI-like genes by PCR using primer sequences derived from the previously determined bovine cDNA sequences of BPIL1, VEMSGP, PLUNC, BPIL3 and RYA3. The primer pair for BSP30B was derived from sequencing of a segment of the BSP30B promoter contained within a bovine genomic DNA clone in a cosmid vector. The following primer pairs were used. BSP30B: CACATCCTCACCACACACCTGGA and CAGACTGTCTGTGTCCAGTTCTGC; BPIL1: AGTTTCCCGAGCCCATGCCT and GGACTGGAAAGCCGAGTTGGAG; VEMSGP; GCCAGGTTGTTCAACTCAGAA and GTGAGTTTTCCCGAATGG; PLUNC: CTCTCAGCAATGGCCTGCTCT and GGAGAGGGGTGAGTGAAGTCACTT; BPIL3: TGCTGGCTTCTCCAGGCTGT and AAGCAGCCCCCACCACTCAA; RYA3: ACACTGCCTCTCATCTCCAACCA and AGGTTTAGCCAAGTAGAGGCCATT. The PCR products were gel purified, cloned into pGEM-Teasy vector (Promega) and sequenced to confirm the specificity of the PCR screen.
Six BAC clones that were confirmed by PCR to contain parts of the BPI-like locus (CH240_399M6, CH240_104J7, CH240_90E15, CH240_3F14, CH240_477F3, and CH240_253F4) were selected for high throughput shotgun sequencing. This was performed by the TIGR library construction, random sequencing, and closure teams as follows. BAC DNA was isolated, nebulized, the ends polished, and adaptors added. The DNA were size-selected (2–3 kbp and 8–10 kbp), ligated to a modified pBR322 vector, and transformed into E. coli . The libraries were checked for insert size, bovine origin, randomness, and overlap between clones prior to high throughput sequencing. Templates were then prepared from the shotgun clones using an automated production pipeline. Sequencing reactions were carried out on plasmid templates with MJ Research thermocyclers using Applied Biosystems PRISM Big Dye™ Terminator Cycle Sequencing Ready Reaction Kits. Reactions were set-up by Beckman Multimek automated pipetting robotic workstations combining templates and reaction mixes. Thirty to forty consecutive cycles of linear amplification steps were performed. The reactions were then cleaned up by ethanol precipitation and analysed on Applied Biosystems 3730xl DNA Analyzers. Base calling was performed with phred and Paracel TraceTuner that had been trained with TIGR trace data. Sequence trimming was conducted using LUCY – a program developed at TIGR  with a trimming standard of an overall base call error rate of <1%, free of vector- and E. coli sequences, and a trimmed sequence read length of > 100 bp.
A total of 11,445 vector screened and clipped shotgun sequences, with Phred quality scores, were assembled using Phrap resulting in eight contigs ranging in size from 2 kbp to 158 kbp. The contigs were then mapped on to the version 2.0 of the bovine assembly (BosTau2.0, The Bovine Genome Sequencing Project Consortium) and the length of the between-contig gaps were estimated (which ranged between 79 to 519 bp, while one gap could not be estimated with the current depth of the genomic assembly). The final sequence was submitted to HTG part of the GenBank (accession number DQ667137). The ver 2.0 of the bovine genomic assembly was found to have order and orientation anomalies, which is quite common in the draft assemblies. However, the underlying contigs were found to be assembled correctly. Thus the between contig gaps were filled in from the bovine genomic assembly.
Known cDNAs and protein sequences of the BPI-like genes from human, mouse and cattle from the GenBank, Ensembl bovine gene and protein predictions of the BPI-like genes  the human and bovine RefSeq nucleotide and protein sequences (release 13) and the TIGR bovine Gene Indices (release 11.0) , were mapped on to the 722 kbp bovine genomic contig. The nucleotide sequences were mapped using the GMAP program , while the protein sequences were mapped using protein2genome model of Exonerate . The bovine genomic contig was also used as query to predict additional genes within the BPI-like locus using the Genescan gene prediction program .
The 722 kbp genomic contig was used as a backbone on an in-house installation of the Generic genome Browser, . The contigs resulting from the BAC sequencing, gene mappings as well as the gene predictions were then put as tracks on the GBrowse to facilitate identification of members of this family.
Sequencing of SPLUNC3, BSP30A and BSP30C cDNA
The predicted cDNA sequences for SPLUNC3, BSP30A and BSP30C were used to query an in-house database of over 200,000 bovine ESTs. These searches resulted contigs that matched very well to each of the genes, but whose sequence differed slightly from the predicted sequence. For SPLUNC3 and BSP30C, the contig sequence was extended using 5' and 3' RACE [GenBank: DQ677839 and DQ835286]. For BSP30A, additional clones were obtained by RT-PCR and the region of sequence divergence with the previous GenBank entry [U79413] was sequenced. These additional clones had identical sequence to the EST contig, thus confirming a reading error in U79413. The updated sequence was submitted to GenBank [U79413]
The protein sequences of the bovine, human and mouse BPI-like genes were aligned using the SATCHMO (Simultaneous Alignment and Tree Construction using Hidden Markov mOdels)  with a window size of 13. The C-terminal domains of the two-domain proteins (i.e. long PLUNCs) were removed before aligning them to ensure appropriate alignment between the one- and two-domain proteins. The SATCHMO alignment of the protein sequences was converted into a nucleotide alignment using TRANALIGN program of EMBOSS . An unrooted tree was constructed using the maximum likelihood method in PHYML v2.4.4  with bootstrap support, at the nodes, computed for 1000 replications of the data. A general time-reversible model of DNA substitution (GTR) was used in the maximum likelihood and the initial tree used was BIONJ .
The rate of nonsynonymous (dN) and synonymous (dS) substitutions was calculated following the method of Yang and Nielsen  as implemented in the program yn 00 which is a part of PAML package (Phylogenetic Analysis by Maximum Likelihood ver 3.15) . Codeml, also part of the PAML package, was used to detect the positive selection for amino acid sequence divergence in the bovine BSP30 versus human and mouse PSP genes. The reduced alignment of the bovine BSP30 and human and mouse PSP genes were fitted with two maximum likelihood models, namely one ratio and two-ratio models. The one-ratio model assumed ω to be equal for all the branches in the reduced tree, while the two-ratio model allowed one ω value for the bovine branches and a possibly different value of ω for the Human and Mouse PSP branches. The test of differences between different models was carried out using a chi squared test. The evidence of positive selection was obtained where the two-ratio model was found to be significantly better than the one-ratio model, with ω estimated to be higher in the bovine lineage.
A sites model was also fitted to the reduced alignment of bovine BSP30 and human and mouse PSP genes to identify the amino acid residues under selection for divergence. The models implemented in codeml, namely, M1a, M2a, M7 and M8 were fitted for this purpose. The likelihood ratios from M1a and M2a and M7 and M8 were compared for the evidence of selection for divergence among sites. Bayesian probabilities  were calculated for each amino acid in the alignment, after removing ambiguity characters and those with Prob(ω >1) > 0.95 are reported.
For the genomic alignments, sequences were retrieved from hg17:chr20: 30,700,000–31,700,000, mm7:chr2: 153,400,000–154,400,000 and canFam2:chr24:24,900,000–25,900,000 and aligned with the assembled bovine region using OWEN . All sequences were assembled with the same parameters which consisted of an initial round using default parameters and then a second round with a lower threshold (1E-4) followed by manual removal of the off diagonal elements.
A range of bovine tissues were obtained from a Friesian-Holstein dairy cow at slaughter, snap frozen in liquid nitrogen and ground to a frozen powder in liquid nitrogen. RNA was isolated from the tissues using Trizol (Invitrogen) following the manufacturer's instructions. RNA was resolved on a formaldehyde-agarose gel, transferred to membrane and probed with 32P-labelled cDNA as previously described . The probes used were full length bovine BSP30C, SPLUNC3 and BPIL1 cDNA. The blots were washed at moderately high stringency (65°C in phosphate buffer ) and the signal was visualised by exposure to X-ray film.
Reverse transcriptase polymerase chain reaction (RT-PCR) was performed on RNA isolated from a similar range of bovine tissues to that described above. RT reactions were performed using 1 μg of RNA and MMLV reverse transcriptase in a 20 μl reaction following an established protocol . A 1 μl aliquot was subjected to PCR using the following primer sets: actin; CGCACCACTGGCATTGTCAT and TTCTCCTTGATGTCACGCAC, BPIL3; CCAGGGATGAAGCCTATCAA and TGTGAGGAGCCTTCAGCATA, BASE; GAAGGTCTCCAGCCTCTTCA and CTCAGGAATGAGCCTGCAAT, BSP30D; TGAGGCGGACCCAGAGAAGA and AATGCGTTACCAGGGACAATAC. The actin, BASE and BPIL3 reactions employed an annealing temperature of 55°C and proceeded for 30 cycles. The annealing temperature of the BSP30D reaction was 60°C. The amplified DNAs were resolved on agarose gels and visualised by staining with ethidium bromide.
The BSP30A amino acid sequence was submitted to the on-line protein threading program, PHYRE . The search returned a top structure prediction with an expect value of 2.2e-09, with a precision value of 100%. The thread was based on the crystal structure of BPI. The structure was viewed using the 3D molecule viewer module of the Vector NTI software package.
High throughput sequencing and assembly was done by the TIGR library construction, random sequencing, and closure teams. We wish to acknowledge the assistance of Brendan Haigh, Marita Broadhurst, and Grant Smolenski in facilitating the work as well as Ken Dodds for help with statistical analysis. We also wish to express our appreciation at being able to use relevant Bovine Genome Sequencing Project sequence prior to formal publication of the genome, and acknowledge in particular the work of Baylor HGSC. The research was supported through funding from the New Zealand Foundation for Research, Science and Technology.
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