Characterisation of Bovine Leukocyte Ig-like Receptors
Louise Hogan1,2*., Sabin Bhuju3,5., Des C. Jones4, Ken Laing1, John Trowsdale4, Philip Butcher1,
Mahavir Singh3,5, Martin Vordermeier2, Rachel L. Allen1
1Centre for Infection, Division of Clinical Sciences, St George’s, University of London, Cranmer Terrace, London, United Kingdom, 2TB Research Group, Animal Health and
Veterinary Laboratories Agency (AHVLA), Weybridge, New Haw, United Kingdom, 3Department of Gene Regulation and Differentiation, Helmholtz-Zentrum fu ¨r
Infektionsforschung, Braunschweig, Germany, 4Immunology Division, Department of Pathology, University of Cambridge, Cambridge, United Kingdom, 5LIONEX
Diagnostics & Therapeutics, Braunschweig, Germany
Leukocyte Immunoglobulin-like receptors (LILR) are innate immune receptors involved in regulating both innate and
adaptive immune functions. LILR show more interspecies conservation than the closely related Killer Ig-like receptors, and
homologues have been identified in rodents, primates, seals and chickens. The murine equivalents, paired Ig-like receptors
(PIR), contain two additional immunoglobulin domains, but show strong sequence and functional similarities to human
LILR. The bovine genome was recently sequenced, with preliminary annotations indicating that LILR were present in this
species. We therefore sought to identify and characterize novel LILR within the Bos taurus genome, compare these
phylogenetically with LILR from other species and determine whether they were expressed in vivo. Twenty six potential
bovine LILR were initially identified using BLAST and BLAT software. Phylogenetic analysis constructed using the neighbour-
joining method, incorporating pairwise deletion and confidence limits estimated from 1000 replicates using bootstrapping,
indicated that 16 of these represent novel bovine LILR. Protein structures defined using protein BLAST predict that the
bovine LILR family comprises seven putative inhibitory, four activating and five soluble receptors. Preliminary expression
analysis was performed by mapping the predicted sequences with raw data from total transcript sequence generated using
Genome Analyzer IIx (Illumina) to provide evidence that all 16 of these receptors are expressed in vivo. The bovine receptor
family appears to contain receptors which resemble the six domain rodent PIR as well as the four domain LILR found in
Citation: Hogan L, Bhuju S, Jones DC, Laing K, Trowsdale J, et al. (2012) Characterisation of Bovine Leukocyte Ig-like Receptors. PLoS ONE 7(4): e34291.
Editor: Nikolas Nikolaidis, California State University Fullerton, United States of America
Received December 6, 2011; Accepted February 25, 2012; Published April 2, 2012
Copyright: ? 2012 Hogan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This project was funded by St George’s University of London and the Animal Health and Veterinary Laboratories Agency. The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The co-author Rachel Allen is a PLoS ONE Editorial Board member. Two of the authors are employed by a commercial company (LIONEX
Diagnostics & Therapeutics). This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
Members of the Leukocyte Immunoglobulin-like receptor
(LILR) family are potent modulators of immune function.
Through their expression on monocytic cells, LILR can regulate
Toll Like Receptor (TLR) activity [1,2] and antigen presenting
phenotype [3,4,5,6]. This ability to regulate both innate and
adaptive immune functions of antigen presenting cells indicates
that LILR play a pivotal role in shaping immune responses.
Accordingly, LILR activity has been shown to influence the
immune response during bacterial and viral infection, determining
the outcome of disease [2,7,8].
The human LILR family consists of eleven functional genes and
two pseudogenes [9,10,11] encoded within the leukocyte receptor
complex (LRC) on chromosome 19q13.4 [10,12]. LILR belong to
the Ig superfamily, and each possess between 2–4 extracellular C2-
type Ig domains  and a type I transmembrane domain .
The IgC2 domains within the human LILR family are referred to
The 11 receptors of the human LILR family have been
subgrouped on the basis of their signalling ability and their
ligand specificity. Receptors classified as inhibitory (LILRB1-5)
have a cytoplasmic tail containing 2–4 immunoreceptor tyrosine-
based inhibitory domains (ITIMs). Activating LILR (LILRA1, 2,
4–6) lack any signalling motif of their own, but instead possess a
charged arginine residue which enables association with ITAM
encompassing adaptor proteins such as FceRIc . LILRA3,
whose coding sequence contains no transmembrane or signalling
domains, is expressed as a soluble molecule and is absent from
some haplotypes [11,16]. Despite their designation as activating,
receptors which signal through ITAM domains are capable of
exerting inhibitory effects on cell function [15,17,18].
LILR orthologues are found in rodents where they are known as
the paired immunoglobulin-like receptors (PIR). The PIR family
contains multiple activating receptors (PIR-A) but only one
inhibitory receptor, PIR-B [19,20,21]. A further murine homo-
logue, LILRB4 (previously known as gp41B1), also exists
[19,20,21,22,23,24]. PIR contain six Ig domains , but share
sequence similarity with the D1-D4 domains in LILR, as well as
expression profile, functional effects and ligand specificity for
MHC-I, to the extent that the murine receptors are also capable of
recognising human MHC-I [25,26].
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The preservation of two structurally similar receptor groups,
LILR and PIR, indicates that these receptors and their immune
functions may be highly conserved through evolution. This is in
contrast to the closely-related KIR, for which a structurally
unrelated family of receptors known as Ly49 has evolved in some
species to perform the same functional role . To date, LILR
homologues and LILR like sequences have been identified in
humans [28,29,30], rodents , chimpanzees , gray seals
 and chickens [31,32,33]. We sought to investigate the
presence of LILR genes and their expression in the domesticated
cow (Bos Taurus). Due to the high level of LILR conservation
between species, we hypothesised that the structure and function
of bovine LILR will resemble that of human LILR. In support of
this, bovine KIR homologues have been identified, and their
structure and function, but not location, were found to be
homologous to that of human KIR [34,35,36].
The bovine genome has recently been sequenced and
published , and 35 predicted bovine LILR have been
annotated (Table 1). Of these 35 predicted bovine LILR, 17
were annotated on NCBI GenBank using the gene prediction
method GNOMON, and the remaining 18 receptors were
annotated on Ensembl, using Genebuild. Table 1 details the gene
ID, transcript ID, sequence length of each previously predicted
bovine LILR, and which human receptor they are thought to be
orthologous to. These sequences are mapped to the same region
of bovine chromosome 18 (chr18:62833056-63654437), with the
exception of two sequences mapped to position chr18:1073938-
Table 1. This table depicts the gene ID, transcript ID, sequence length and location, and which human receptor each predicted
bovine LILR is thought to be orthologous to.
Source Gene IDTranscript ID BpHuman LILR
ENSBTAG00000037830ENSBTAT00000056991 579 Novel
ENSBTAG00000038797 ENSBTAT00000057210636 Novel
ENSBTAG00000038828 ENSBTAT000000108781947 Novel
ENSBTAG00000038828 ENSBTAT000000252681899 Novel
ENSBTAG00000038828 ENSBTAT000000519251278 Novel
ENSBTAG00000005841 ENSBTAT000000260481794 Novel
LOC786365 XM_001250913.3 8704PIRA4
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Figure 1. Gene arrangement within the human and bovine LRC. This diagram presents data from NCBI on the locations of predicted and
confirmed genes found within the bovine LRC, located on 18q13.4, and compared to the annotated human LRC located on 19q13.4. This data is
correct at time of print, although is subject to change due to unallocated genomic scaffolds within the bovine genome. The basic arrangement of the
bovine LRC is almost the reverse of the human LRC, with a few exceptions. The KIR and FCaR can be found at the centromeric end of the bovine LRC,
whereas in the human LRC they are located at the telomeric end. The LILR are upstream of the KIR in the human LRC, compared to the bovine LRC,
where the BL are found downstream of predicted KIR. Furthermore, OSCAR, LENG1, CNOT3 and MBOAT7 are located at the telomeric end of the
bovine LRC, compared to the centromeric end of the human LRC.
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1097100 and seven Ensembl sequences which have been mapped
to three currently unknown contigs (Un.004). As many of these
sequences show considerable overlap, and information is limited
regarding their protein structure or relationship to human LILR,
we sought to further analyse these sequences and search the
bovine genome for other potential LILR. Here, we address
whether these predicted bovine LILR sequences have been
adequately annotated and further describe the identification,
classification and expression of 16 novel bovine LILR transcripts
in bovine immune cell subsets.
Bioinformatic Identification of Bovine LILR Homologues
Coding sequences (CDS) and the corresponding peptide
sequences were obtained from the Consensus CDS (CCDS)
database available through Ensembl (http://www.ncbi.nlm.nih.
gov/CCDS/) for all human LILR and KIR, bovine KIR, murine
PIR and key primate LILR. These sequences were then used to
search all current bovine genome assemblies (Project reference
AAFC03000000) through the Bos taurus nucleotide BLAST
programme available through NCBI (http://www.ncbi.nlm.nih.
gov/genome/seq/BlastGen/BlastGen.cgi?taxid=9913). As LILR
contain Ig domains, which are found in many other proteins, the
data obtained from BLAST were confirmed, and the chromo-
somal locations were clarified using the BLAST-Like Alignment
Tool (BLAT) (UCSC Genome Bioinformatics, USA, http://
identified as encoding potential bovine LILR was then analysed
using GeneScan (http://genes.mit.edu/GENSCAN.html, ) or
GeneMark (http://exon.gatech.edu/eukhmm.cgi ), to predict
the CDS and resulting peptides, and the predicted protein
domains and exon boundaries were obtained using protein
BLAST, available through NCBI (http://blast.ncbi.nlm.nih.gov/
Blast.cgi?PAGE=Proteins), and Ensembl respectively.
). The genomicDNA
Sequence comparisons of the predicted CDS were analysed
using Clustalw, a multiple sequence alignment tool, in which the
results are given as a percentage of identity to pairwise alignments
Phylogenetic analysis of the amino acid sequences for the
individual Ig domains was performed using MEGA5 software
, and constructed using the neighbour-joining method ,
incorporating pairwise deletion and confidence limits estimated
from 1000 replicates using bootstrapping . Pairwise deletion
was used as there is a high number of sequences to be analysed
and complete deletion would have resulted in a significant
reduction in the amount of each sequence available for analysis.
Prediction of Transmembrane Helices
Transmembrane regions were mapped using the TMHMM
Server v. 2.0 available at the Center for Biological Sequence
Analysis, Technical University of Denmark, and the arginine
residues and ITIMs were mapped to each peptide sequence, using
the established V/L/S/NxYxxL/V ITIM motif .
To determine whether mRNA transcripts corresponding to our
newly identified sequences are expressed, cDNA transcripts
generated from RNA isolated from cultured peripheral blood
cells of cows were mapped to the putative LILR sequences.
Purification of mRNA from total RNA, cleavage of mRNA, cDNA
synthesis and library preparation were performed according to
manufacturer’s standard protocols using the mRNA-Seq-8 Sample
Preparation Kit (Illumina). Cluster generation, primer hybridiza-
tion and sequencing reactions were performed following the
manufacturer’s recommended protocols . Reads with an
expected read length of 36-bases were generated from each library
using a Genome Analyzer IIx (Illumina).
To evaluate the best prediction server for each corresponding
bovine LILR, the chromosomal sequences chr18:62832447-
63370336, Un.004.625:2160-77036, and Un.004.1339:17781-
25700 extracted from the bovine genome were used as reference
for mapping. Mapping was performed using CLC Genomics
Workbench 4.7.2 with mismatch cost of 2 using ungapped
alignment. Positional mapping of each base for all mappable
reads was calculated using custom designed Perl script using the
SAM file. Each chromosomal segment containing predicted
sequences was inspected with Artemis  using the predicted
annotations obtained based on each prediction server and the
positional mapping of reads. The predicted exonic sequence was
further inspected and supported by the presence of regions with a
high density of mappable reads from the RNA-sequencing. The
most likely prediction was selected based on the combined
annotation and RNA-sequence analysis for each bovine LILR.
The RNA-sequence reads were re-mapped using the same
reference sequence but with a lower mismatch cost of 1. Regions
mapped with more than 99% coverage were considered to
represent exonic sequence expressed in peripheral blood cells of
Identification of Potential Bovine LILR Transcripts
Using human LILR sequences to search the bovine genome
with both BLAST and BLAT, we identified a total of 77 open
reading frames (ORF’s) on three contigs as containing potential
bovine LILR. Each of these ORF’s was then analysed by
GeneScan and GeneMark, to obtain the predicted CDS and
corresponding protein sequence, which were then aligned to
domains using protein BLAST to identify Ig domains. Of the 77
ORF sequences, 26 were found to contain Ig domains and were
selected for further analysis; 19 were located on chromosome 18
between 62,832,497–63,653,564, five sequences were located on
unknown contig 004.625 between 2210–76986, and finally, one
sequence was found to be located on a further unknown contig
004.1339 between 17831–25650 (data not shown). From this point
on, the newly identified transcripts will be referred to as bovine
LILR (BL) 1–26.
Identification of Different Receptor Families
We then sought to confirm that the sequences identified encode
BL rather than other closely related Ig receptors. To do this, we
compared the BL to LILR/PIR receptors from other species,
including human, murine, bovine, chimpanzee, orang-utan, gorilla
and rhesus macaque. Furthermore, as bovine equivalents of other
Ig receptors located within the human LRC have also been
mapped to this region of chromosome 18 in Bos taurus, we
extended our search of the annotated sequences to include all
LRC sequences mapped within the region of chr18:62832447-
63700000. The location of bovine Ig receptors currently mapped
to this region, such as the bovine KIR and OSCAR, indicate that
the organisation of the bovine LRC is considerably different to
that of the human LRC (figure 1) . As proteins encoded within
the LRC are structurally and functionally related, it becomes more
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difficult to isolate and distinguish between one particular set of
sequences and another. We therefore also included human and,
where available, bovine SIGLEC, OSCAR, KIR, FcGRT, FcaR,
LAIR, GpVI and NKp46 in our analysis.
The exonic regions encoding each Ig domain for all sequences
were analysed separately using phylogenetic analysis, which was
performed using neighbour-joining bootstrap consensus trees
inferred from 1000 replicates, pairwise deletion and amino acid
p distance. Because the IgC2 domains fold in a characteristic way
for each receptor, and contain a distinct binding site, it is possible
to group receptor domains on a functional basis and determine
which Ig superfamily receptor group they belong to. The
phylogenetic analyses of these domains are shown in figures 2a-d.
From the 26 putative LILR transcripts, 16 receptors are closely
associated with LILR transcripts from other species. The bovine
LILR appear to have evolved in a distinct manner compared to
that of other species, but bovine LILR do not appear to be as
divergent as murine PIR are to primate LILR. There are six
transcripts (BL1, BL2, BL17, BL18, BL19 and BL24) which
appear to be novel genes that closely resemble KIR transcripts,
although whether these genes are novel KIR sequences is an exact
distinction that can not be made from this data. BL3 resembles the
already annotated bovine LAIR-1 gene, however it is mapped to a
different region on chromosome 18. This could be due to recent
rearrangements in the bovine genome where an effort has been
made to position unallocated genomic scaffoldings. Two tran-
scripts, BL15 and BL16, did not cluster with any groups of
receptors in this analysis. And finally, BL5 Ig1 is more closely
associated with LILR than the other Ig receptor groups analysed,
however, it appears to be an outlier within the LILR group, and
the Ig2 domain is not closely related to any other group of
receptors. Therefore, further investigation is required to determine
exactly what type of receptor this transcript encodes.
Interestingly, in our analysis (data not shown) the previously
0000012925, ENSBTAT00000051925, ENSBTAT00000056991
and the novel Gorilla LILR ENSGGOT00000031548 are all
more closely grouped with the KIR family than LILR, and contain
1–3 Ig domains, which is more characteristic of the KIR genes.
Moreover, although the Ig2 domain of ENSBTAT00000052581 is
clustered within the LILR transcripts, the Ig1 and Ig3 domains
again are more closely associated with the KIR group. BL5
together with ENSBTAT0000001231, ENSBTAT00000050632,
ENSBTAT00000054607, ENSBTAT00000057210, ENSBTAT0
0000052297 and XM_2684429 are clustered separately from the
other receptor groups, despite all these transcripts (with the
exception of ENSBTAT00000052297) having an Ig1 domain that
matches the LILR group. These receptors only contain two Ig
domains, and may represent a separate receptor family, or a group
of very divergent LILR.
Comparison of Novel Sequences with Previously
We then sought to ascertain whether any of the sequences
identified in our search were identical to any of the previously
annotated sequences. Several comparisons were made to deter-
mine whether any duplications were present;, the chromosome
position for each sequence was mapped, whole sequences were
compared using clustalw, and individual Ig domains were
phylogenetically analysed as described above. Three of the
previously annotated sequences, XM_002695415.1, NM_00110
2357.1 and XR_083953.1, were excluded from the phylogenetic
analysis, as these sequences could not be mapped within the
specified chromosomal regions, and therefore their exonic regions
could not be fully determined. A chromosome map is shown in
figure 3, and the phylogenetic analysis is shown in Figure S1a-f
and summarized in table 2.
The chromosome map shown in figure 3 demonstrates that
several of the previously annotated bovine LILR overlap at least
partially with those found in our search. Although EN-
SBTAT00000026048, XM_002684433.1, XM_001788788.2 and
XM_002684429.1 overlap with BL26, BL10, BL7, BL8 and
XM_870741.4 respectively, clustalw analysis showed that none of
these overlapping sequences had a similarity of $95% (data not
shown). However, it is interesting to note that XM_002684429.1
and XM_870741.4 are mapped to the same position and have an
alignment score of 95%, and are therefore possibly two
alternatively spliced transcripts of the same gene.
In our combined analysis, the clustalw results (data not shown),
indicated that a total of 85 sequences were $95% similar to one or
more other sequences. Of these, 41 of the previously annotated
sequences were $95% similar to other annotated sequences, 32
previously annotated sequences were $95% similar to a newly
identified BL sequence, and 12 of the newly identified BL
sequences were $95% similar to another newly identified BL
sequence. These results were expected as LILR are very similar in
structure and function both in humans and between species, and
some receptors may differ only in their ligand binding sites. BL14
was found to be identical to both XM_001787176.2 and
ENSBTAT00000052297 along the entire sequence length,
however,itis worth noting
SBTAT00000052297 have short sequence lengths of 945aa, and
840aa respectively, which might account for the increased identity
matches. Furthermore, these sequences are located in three
different positions on the chromosome.
Each BL transcript contains up to 6 Ig domains. Phylogenetic
analysis (figure S1 a-f and table 2) demonstrated that between
our 16 BL transcripts there were a total of 17 Ig domains present
that were also identified amongst the previously annotated
transcripts. There were six of the newly identified sequences for
which all of the Ig domains could be matched to all the Ig
domains encoded in another annotated transcript, and in two
cases these new BL sequences were matched entirely to two
different annotated sequences. From the six new sequences, three
of the matches, BL10, BL11 and BL12, are located in different
non-overlapping positions on the chromosome. BL4 partially
overlaps XM_007256691.2, and therefore the Ig domains for
these two receptors may be identical, although in our search we
identified a different ORF. Finally, BL21 was shown to share
identical Ig domains with both ENSBTAT00000025268 and
ENSBTAT00000010878, which are two transcripts from the
same gene, and BL26 has identical Ig domains to EN-
SBTAT00000026048. These transcripts are located in the same
chromosomal positions, but, as the clustalw results indicated
Figure 2. Phylogenetic analysis of Ig domains from receptors located within the LRC of different species. Phylogenetic analysis of Ig
domains from receptors located within the LRC of different species: The available Ig domains from SIGLEC, OSCAR, KIR, FcGRT, FcaR, LAIR, GpVI and
NKp46 in species including human, murine, bovine, chimpanzee, orang-utan, gorilla and rhesus macaque were analyzed using phylogeny.
Phylogenetic trees were constructed using the neighbour-joining bootstrap consensus method inferred from 1000 replicates, pairwise deletion and
amino acid p distance. The graphs show Ig1–4 domains (a-d respectively). The receptor groups are labelled with LILR shown in red and PIR in blue.
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BL21 had a 75% and 74% identity to ENSBTAT00000025268
and ENSBTAT00000010878 respectively, and BL26 had an
85% identity to ENSBTAT00000026048. Consequently, we
concluded that our newly identified transcripts are significantly
different from those previously reported. However, it cannot be
ruled out that they are different transcripts of the same gene.
Therefore, from the results presented here, we suggest that all
transcripts identified in our search are novel, and have not been
Structure of Novel BL
The predicted structure for the bovine LILR are shown in
Figure 4. Based on the presence or absence of ITIMs we predict
that there are four activating BL and seven inhibitory BL, a similar
repertoire to the six activating and five inhibitory receptors found
in humans. We have also identified five receptors within the
bovine genome which, on the basis of this analysis, would be
predicted to be expressed only in a soluble form. In the human
system, LILRA3 is the only genomically encoded soluble receptor,
although a common splice site allows most human LILR to be
expressed in a soluble form . The signaling domains found in
the bovine LILR are more conserved than those found in human
LILR. The human receptors contain between 2–4 ITIMs, with
VxYxxL and SxYxxL being present on every receptor, whereas
the bovine receptors only contain one ITIM on each receptor,
either VxYxxV or VxYxxL. It is not clear whether this difference
in signaling domains is due to a loss in the bovine genome, or if an
extra signaling domain has evolved in primates. It is also possible
that human LILR could possess a higher capacity for cellular
regulation than bovine LILR, as the Y-2domains have been shown
to determine the binding of the cytosolic tyrosine kinases SHP-1
and SHP-2 [49,50,51].
Potential Ligand Binding
Following the solution of a crystal structure for LILRB1 with an
HLA-A2 ligand, it was predicted that a subset of LILR (known as
group 1), will bind to MHC-I, on the basis of sequence similarities
. To establish whether any of our newly identified bovine
receptors share ligand specificity, individual exons encoding Ig
domains in each sequence were phylogenetically analyzed.
The human group 1 LILR, which share MHC-I ligand
specificity, show similar clustering patterns for each of the Ig
domains (figure 5a-d) that contain their ligand binding site .
No similar clustering patterns were observed for bovine LILR.
Figure 3. Chromosomal locations for predicted bovine LILR.
Chromosomal locations for bovine LILR: Bovine LILR have been mapped
to compare the sequence locations of those found in our search
compared to the previously annotated sequences.
Table 2. Summary of phylogenetic analysis in figures 2a-f,
indicating where transcripts contained identical Ig domains.
New LILR Sequences
Transcript IDBL4 BL10BL11 BL12BL21 BL26
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This indicates that bovine LILR may recognise a broader range of
ligands than human LILR.
Two of the 16 BL transcripts, BL7 and BL10, were more closely
clustered with the PIR transcripts, than that of the human LILR.
These two transcripts both contain six Ig domains, similar to PIR.
Therefore, we repeated our phylogenetic analysis to include all six
Ig domains in the PIR-like receptors. The results, shown in
figure 6, suggest that Bos taurus may possess both LILR and PIR
transcripts. This raises the possibility that there is a common
ancestral gene for PIR and LILR which has divided to produce
two separate lineages, with cattle appearing to have expanded and
maintained both. In support of this, Martin, et al, have shown that
LILR and PIR possess a type I IgC2 domain which is thought to
be the most ancient IgC2 domain, from which other domains have
in fact evolved [32,54]. The hypothesis that both PIR and LILR
arise from a common ancestral gene is strengthened by the
observation that the gorilla LILRA5 homologue, appears to share
domains from both PIR and LILR; Ig1 and Ig2 closely resemble
that of LILR domains, whereas, the Ig3 and Ig4 closely resemble
that of PIR domains (Figures 2a-d and 6). Therefore, it is possible
that Bos taurus have maintained both receptor groups.
Expression of BL
Finally, we sought to determine whether any of our newly
identified bovine receptors were expressed in bovine PBMC’s. To
confirm expression of these genes, we evaluated if they were
transcribed in bovine PBMC stimulated with bovine tuberculin
PPD. The transcriptome of bovine PBMC was determined by
applying Next Generation Sequencing techniques using a Genome
Analyzer IIx (Illumina). The resulting sequence data was
compared to our predicted BL sequences. As depicted in table 3,
we found evidence that all our predicted BL are expressed in
PBMC’s, and showed that for six of the 16 predicted BL (BL7,
BL10, BL11, BL13, BL22, and BL26) more than 99% of the
predicted exonic sequence was mapped, thus validating our in
In this study, we sought to identify and characterise novel BL
using a bioinformatic approach. Our initial search yielded 26
transcripts as potential BL, which were then analysed using
phylogeny alongside other structurally similar receptor families
also belonging to the Ig-superfamily. By including all available
receptor families located on the LRC from as many species as
possible, we determined that 16 of these 26 transcripts were LILR
The bovine genome is now fully sequenced, and as a result
several predicted BL have been previously annotated on NCBI
and Ensembl. To ensure our sequences were not duplications of
any of these previously reported sequences, we analysed and
compared; exonic domains using phylogeny, whole sequences
using clustalw, and the mapped position of each sequence. The
exonic domains of six of our newly identified sequences were
shown to have a high similarity with previously annotated
sequences, however the clustalw results and chromosomal location
were significantly different. Therefore, we concluded that the 16
Figure 4. Predicted structures of bovine LILR compared to that of the known human LILR. Predicted structures of bovine LILR compared
to that of the known human LILR: To determine the potential structures of bovine LILR the Ig domains were identified using protein BLAST (NCBI),
transmembrane regions were mapped using the TMHMM Server v. 2.0 (Centre for Biological Sequence Analysis, Technical University of Denmark), and
the arginine residues and ITIMs were identified and located on each peptide sequence, using the established V/L/S/NxYxxL/V ITIM formula .
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Figure 5. Phylogenetic analysis of bovine and human Ig domains. Phylogenetic analysis of bovine and human Ig domains: The Ig domains of
predicted bovine and human LILR were analyzed by constructing neighbour-joining bootstrap consensus trees inferred from 1000 replicates, pairwise
deletion and amino acid p distance. The graphs show the comparison of Ig1–4 domains (a-d respectively).
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BL sequences described here are novel sequences, and have not
been previously characterised.
Our results indicate that the bovine genome encodes a total of
four activating BL, seven inhibitory BL and five soluble BL, with
the possibility of further common splice sites which allow the other
BL to be expressed in soluble form. This is a different repertoire of
receptors compared to those found in humans, as there is only one
human genomically encoded soluble receptor, LILRA3. Soluble
receptors, through their nature, are thought to have a higher
capacity for cellular regulation, and therefore the presence of five
soluble BL in cattle may indicate these receptors have an extended
regulatory role. It is worth noting that in humans deficiency of the
soluble LILRA3 is associated with Sjogren’s syndrome and
multiple sclerosis, indicating an important regulatory role for
soluble receptors [55,56,57].
As cattle possess more BL it was interesting to find that the
inhibitory BL possess fewer signaling domains than those of their
human counterparts. The human receptors contain between 2–4
canonical and permissive ITIMs, with VxYxxL and SxYxxL being
present on every receptor, whereas the bovine receptors only
contain one canonical ITIM on each receptor, either VxYxxV or
VxYxxL. This possibly provides a greater diversity of signaling
abilities in human LILR than that of BL.
The analysis of human LILR has demonstrated that group I
receptors, which all share ligand specificity for MHC I, will cluster
together as the exonic Ig domains are highly similar. We analyzed
the BL exonic domains to determine if any similar clustering
patterns could be identified, which may be indicative of shared
ligand specificity. However, in our anaylsis there were no patterns
of clustering detected, which may attest to the fact that the BL
have a broader range of ligand specificity.
To date, LILR have been reported as highly conserved receptors
within all mammalian species, with the exception of PIR in mice. BL
appear to have evolved independently to both the PIR and LILR,
whilst remaining more conserved than PIR. However, it is interesting
to note that the BL genes seem to have retained sequences that
resemble both LILR and PIR, which are thought to stem from a
from a common ancestral gene, KIR3DX . Approximately
two daughter genes, KIR3DL and KIR3DX. Whereas in primates
the KIR3DL gene expanded and gave rise to primate KIR, in cattle,
the KIR3DX gene is the ancestor for the bovine KIR genes, and in
humans this gene is now a non-functional pseudogene .
Finally, by analysing bovine PBMC stimulated with bovine
tuberculin PPD, we found evidence for the expression of all 16
newly identified BL, six of which we found .99% expressed.
Further investigation is required to determine exactly which
subsets of PBMC’s each receptor is expressed on, and to elucidate
ligand specificities. However, given the role of LILR in humans,
we predict that BL will play an important key role in bovine innate
In summary, we have identified 16 novel BL, which appear to
have evolved and maintained both LILR and PIR –like sequences
from a common ancestral gene. The BL repertoire includes four
activating receptors, six inhibitory receptors and five further
soluble receptors, six of which we have confirmed the expression of
in the peripheral blood cells of Bos taurus. The Genbank accession
numbers for each sequence are details in table S1. We suggest an
appropriate name for these receptors would be Bovine Ig-like
Receptors (BIR) (Table S1).
annotated bovine LILR Ig domains. Phylogenetic analysis of Ig
domains from predicted bovine LILR: The Ig domains from both the sequences
identified in our search and those previously annotated were analyzed by
constructing neighbour-joining bootstrap consensus trees inferred from 1000
replicates, pairwise deletion and amino acid p distance. The graphs show the
comparison of Ig1–6 domains (a-f respectively) and identical Ig domains are
highlighted in red.
Phylogenetic analysis of newly and previously
Suggested nomenclature for novel sequences.
The perl script used was kindly provided and written by Andreas Do ¨tsch,
Helmholtz Zentrum fu ¨r Infektionsforschung.
Conceived and designed the experiments: LH SB MV. Performed the
experiments: LH SB. Analyzed the data: LH SB DCJ KL JT. Contributed
reagents/materials/analysis tools: LH SB DCJ KL MS. Wrote the paper:
LH DCJ SB RA PB.
Figure 6. Phylogenetic analysis of murine and predicted bovine PIR Ig domains. Phylogenetic analysis of murine and predicted bovine PIR
Ig domains: The Ig domains from murine PIR and the predicted bovine PIR (BL7 and BL10) were analyzed using phylogeny. Phylogenetic trees were
constructed using the neighbour-joining bootstrap consensus method inferred from 1000 replicates, pairwise deletion and amino acid p distance.
The graph shows the clustering of the individual Ig domains.
Table 3. Percentage of the predicted BL sequence mapped
to the sequence derived by RNA-sequence analysis of Bos
taurus peripheral blood cells (PBMCs).
Bovine Leukocyte Ig-like Receptors
PLoS ONE | www.plosone.org11 April 2012 | Volume 7 | Issue 4 | e34291
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