The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2007, 179, 491 -504
Copyright © 2007 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Guethlein, L. A.
Right arrow Articles by Parham, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Guethlein, L. A.
Right arrow Articles by Parham, P.
Right arrowPubmed/NCBI databases
*Protein
*Substance via MeSH

Evolution of Killer Cell Ig-Like Receptor (KIR) Genes: Definition of an Orangutan KIR Haplotype Reveals Expansion of Lineage III KIR Associated with the Emergence of MHC-C1

Lisbeth A. Guethlein, Anastazia M. Older Aguilar, Laurent Abi-Rached and Peter Parham2

Department of Structural Biology, and Department of Microbiology and Immunology, School of Medicine, Stanford University, Stanford, CA 94305


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Orangutan (Pongo pygmaeus) MHC-C appears less evolved than human HLA-C: Popy-C is not fixed and its alleles encode only one (C1) of the two motifs for killer cell Ig-like receptor (KIR) ligands. To assess the structure and complexity of the orangutan KIR locus, the complete nucleotide sequence of an orangutan KIR haplotype was determined. The PopyKIR locus is flanked by LILR and FCAR and consists of seven genes and pseudogenes, two novel and five corresponding to known cDNA. Distinguishing all KIRs in this rapidly evolving KIR locus from the KIR3DX1 gene is an LTR33A/MLT1D element in intron 3. These two forms of KIR represent lineages that originated by duplication of a common ancestor. The conserved, framework regions of primate KIR loci comprise the 5' part of a lineage V KIR, the 3' part of a pseudogene, the complete 2DL4 gene, and the 3' part of a lineage II KIR. Although previously defined PopyKIR2DL4 alleles contain premature termination codons, the sequenced haplotype’s PopyKIR2DL4 allele encodes a full-length protein. A model for KIR evolution is proposed. Distinguishing the orangutan KIR haplotype from the proposed common ancestor of primate KIR haplotypes is an increased number to give three lineage III KIR genes in the centromeric part of the locus, the site for most human lineage III genes encoding HLA-C specific KIR. Thus, expansion of lineage III KIR is associated with emergence of MHC-C.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Killer cell Ig-like receptors (KIR)3 are a family of diverse and rapidly evolving MHC class I receptors expressed principally by NK cells (1), but also by subpopulations of {alpha}beta and {gamma}{delta} T cells (2, 3). KIR are type I transmembrane glycoproteins that have two or three extracellular Ig-like domains for binding MHC class I ligands, a stem region, and a signaling domain (the transmembrane region and cytoplasmic tail) for transducing either an inhibitory or activating signal. In humans, inhibitory KIR specific for polymorphic determinants of HLA-A, B, and C molecules function to make NK cells tolerant of self MHC class I and responsive to cells that lack a normal complement of MHC class I molecules (4, 5, 6, 7). Although the functions of the activating KIR are poorly defined, genetic correlations indicate they contribute to antiviral immunity (8, 9, 10), autoimmunity (11, 12, 13, 14, 15, 16, 17), and resistance to pre-eclampsia (18, 19).

KIR are encoded by a family of genes in the leukocyte receptor complex on chromosome 19q13.4. They are flanked by FCAR, the gene encoding the FcR for IgA, and the leukocyte Ig-like receptor (LILR) gene family. Human KIR haplotypes are highly variable because of differences in gene number, gene content, and allelic polymorphism (20, 21, 22, 23, 24, 25, 26). Conserved features of the human KIR gene family are the KIR3DL3 and KIR3DL2 genes, which define the centromeric and telomeric ends of the locus, respectively, and KIR3DP1 and KIR2DL4 that are centrally located. These conserved genes define three framework regions and two intervals of variable gene content (25, 26). The inhibitory HLA-C receptors are encoded by genes in the centromeric part of the locus, whereas the inhibitory HLA-A and B receptors are encoded by genes in the telomeric part of the locus. Recently, the novel and divergent KIR3DX1 (KIR3DL0) gene of unknown function has been located to the central part of the LILR gene family (27).

Characterization of KIR cDNA clones, combined with genomic typing of panels of individuals, has shown the KIR gene family varies considerably between primate species (28, 29, 30, 31, 32), as do the MHC class I genes (33). Notably, the two mouse KIR genes are situated on the X chromosome, not the LRC, and they do not encode NK cell receptors for MHC class I (34, 35). That role is fulfilled by a distinct gene family, Ly49, which encodes type 2 transmembrane glycoproteins having ligand-binding domains like those of C-type lectins (36, 37, 38, 39). Of the human MHC class I genes, the most recently evolved is HLA-C, for which orthologs have been found only in chimpanzee, bonobo, gorilla, and orangutan. Despite its recent origin, HLA-C now encodes the dominant ligands for inhibitory KIR (40). Whereas a minority of HLA-A and HLA-B allotypes are ligands for KIR, all HLA-C allotypes can serve this function. They form two groups: ones with asparagine at position 80, (called C1) which are ligands for KIR2DL2/3, whereas ones with lysine 80 (called C2) are ligands for KIR2DL1 (41, 42, 43). Almost all humans have KIR2DL1 and KIR2DL2/3, and can therefore use HLA-C mediated inhibition to regulate NK cell activity, whereas that is not the case for HLA-A and HLA-B.

In studying the evolution of MHC-C-mediated regulation of NK cells, the orangutan (Pongo pygmaeus) is a potentially informative species. In contrast to humans, where HLA-C is fixed and encodes both C1 and C2 allotypes, the orangutan Popy-C locus is present on ~50% of MHC haplotypes and all its allotypes are of the C1 group (28, 44). Previous study of orangutan KIR showed this limitation to Popy-C was reflected in the KIR. In humans, C1 specificity of KIR2D is determined by the presence of lysine at position 44, whereas C2 specificity is determined by methionine 44 (43). In the orangutan, we identified several KIR2D with lysine 44, but none with methionine 44 (28). In addition, several orangutan KIR2D with glutamic acid 44 were characterized. These observations suggested a model in which interactions between KIR and MHC-C in the orangutan are at a half-evolved state by comparison to the human. To investigate the genetics underlying this difference, we have determined the complete sequence of an orangutan KIR haplotype.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Isolation and characterization of cosmid clones

An orangutan cosmid library, MPMGc141, at the Deutsches Ressourcenzentrum für Genomforschung (Berlin, Germany), was screened with a probe containing PopyKIR3DLA, PopyKIR2DSB, and PopyKIR2DL4A cDNA (28). Of 31 clones obtained, 27 contained inserts and were analyzed by Southern blot with the probe used to screen the library. Twelve hybridizing clones were further studied to determine regions of overlap and to assign the clones to the two KIR haplotypes. This was accomplished by dot blot and sequence specific oligonucleotide hybridization to determine gene content, restriction mapping to look for regions with shared restriction patterns, and limited sequencing of subclones obtained from regions of identical restriction pattern.

For dot blots, 50 ng of cosmid DNA were mixed with 100 µl of 0.4 M NaOH, 10 mM EDTA followed by incubation at room temperature for 10 min. The DNA was applied to a prewetted filter in a vacuum manifold and the liquid was removed by vacuum. The membrane was washed with 5x SSPE (diluted from 30x SSPE: 4.5 M NaCl, 0.3 M NaH-2PO4, 30 mM Na2EDTA, final pH 7.4), cross-linked, and placed in prehybridization solution (6x SSPE, 0.2% polyvinylpyrrolidone, 0.2% Ficoll, 0.2% BSA, 0.5% SDS, and 100 µg/ml denatured salmon sperm DNA). The filters were incubated at 42°C for a minimum of 1 h, after which radiolabeled oligonucleotide probes were added. Filters were hybridized for 1 h, removed, and washed twice in 2x SSPE, 0.1% SDS at room temperature for 10 min. The membranes were exposed to x-ray film for 1 h to estimate the amount of DNA present on the membrane. Following exposure to film, the membranes were washed twice at high stringency in 6x SSPE, 1% SDS for 10 min. The wash temperature was determined from the nucleotide composition of the oligonucleotide probe used (Table I). Following the high-stringency wash, the membranes were again exposed to x-ray film.


View this table:
[in this window]
[in a new window]

  		Table I. Probes used for sequence specific oligonucleotide hybridization

 
Restriction maps were made using the BamHI, EcoRI, HindIII, PstI, XbaI, and XhoI restriction enzymes. Cosmids were first digested with SfiI to isolate the insert, which was then digested at several enzyme concentrations to produce partially digested fragments. The digested DNA was subjected to agarose gel electrophoresis and Southern blotting. The blots were probed with T7 or SP6 to identify fragments containing an insert end. The resulting restriction maps were compared with the complete cosmid digests to see whether all fragments were represented. Potentially overlapping cosmids were identified from these maps. Two cosmids had identical restriction maps.

As the cosmid library was made from DNA obtained from one orangutan, the cosmids were expected to represent two KIR haplotypes. To distinguish the haplotypes, we identified regions of different cosmids that had identical restriction maps. Separate aliquots of cosmid DNA were digested with EcoRI and BamHI, and the fragments were subcloned into pBluescriptKS+ (Stratagene). Limited end sequencing of identically sized fragments subcloned from different cosmids was used to determine whether the fragments were identical or different in sequence. The results allowed us to identify whether regions with an identical restriction map corresponded to the same or different haplotypes. A set of six overlapping cosmids, representing one complete KIR haplotype, was selected for sequencing. The sequence analysis confirmed the assignment of cosmids to the two haplotypes; the overlapping cosmids used to assemble the complete KIR haplotype had identical sequences in all the regions of overlap. In addition, the patterns of KIR-hybridizing bands obtained from Southern blotting of the selected cosmid clones were consistent with those obtained from similar analysis of genomic DNA from a panel of nine orangutans (see Fig. 1A). The six cosmids that are not parts of the sequenced haplotype represent the second KIR haplotype but do not completely cover it; dot blot analysis revealed the absence of KIR3DL3, the telomeric framework gene, from all six cosmids.


Figure 1
View larger version (31K):
[in this window]
[in a new window]

  		FIGURE 1. Organization and constituent genes of an orangutan KIR haplotype. A, Bands detected in Southern blots of different orangutan genomic DNAs (left panel) and cosmid DNAs digested with EcoRI and hybridized with a mixed KIR cDNA probe. Hybridizing bands are indicated by black boxes, the relative strength of hybridization by + or ++, the latter likely representing hybridization to more than one band of similar size. The six cosmids (1–6) used to obtain the KIR haplotype sequence gave five patterns of bands. Comp gives the band pattern of the complete KIR haplotype obtained from the set of six cosmids. It corresponds to orangutan KIR genotype A. The 1.6-kb band in cosmid 5 (gray box, +*) is located at the end of the cosmid and represents a partial fragment of what would be observed in the genomic digest. Cosmid identifiers are: 1, MPMGc141I20518Q2; 2, MPMGc141B1414Q2; 3, MPMGc141B1814Q2; 4, MPMGc141C06594Q2; 5, MPMGc141G11597Q2; and 6, MPMGc141P12584Q2. B, Map of the PopyKIR haplotype studied. The genes are shown by thin horizontal lines, their exons by vertical lines below. Thick horizontal lines show the cosmids sequenced. Cosmids 2 and 3 are identical and chimeric; the non-KIR sequences were removed before assembling the final sequence. C, A NJ tree comparing full-length human and chimpanzee LILRA to partial orangutan LILRA sequences. Trees with identical topology were obtained when sequences encoding extracellular or intracellular domains were analyzed separately. Like its human (LILRP2) and chimpanzee (PatrLILRP2) orthologs, PopyLILRP2 is a pseudogene with mutations that perturb splice sites and the coding region. Upstream of PopyLILRP2 in cosmid 1 are the two most 3' exons of PopyLILRP1. Like its human (LILRP1) and chimpanzee (PatrLILRP1) orthologs, PopyLILRPI is a pseudogene, it has splice site mutations flanking the penultimate exon. D, Schematic representation of the LRC and the KIR genes in primates, pig, rat, and mouse. Note that the mouse KIR genes are not associated with the LRC, but with the X chromosome. E, A NJ tree comparing the coding region sequences of the PopyKIR haplotype genes (shaded dark gray) with previously determined PopyKIR cDNA sequences. Branch length differences (*) arose because the cDNA sequences were incomplete. The coding regions of the PopyKIR3DLD1 and PopyKIR2DLB genes are identical to cDNA sequences obtained from different individual orangutans. Genes not represented by the cDNA sequences are PopyKIR3DL3 and PopyKIRDP, which correspond to human KIR3DL3 and KIR3DP1, respectively. The PopyKIR2DSE gene differs from the PopyKIR2DSC2 cDNA by a nonsynonymous substitution that changes residue 182 from cysteine to arginine. The gene sequence named PopyKIR2DSFn (n for null) differs from PopyKIR2DSD cDNA by four nucleotide substitutions (two synonymous and two nonsynonymous) and a nucleotide deletion in exon 5 that leads to premature termination. The PopyKIR2DL4C gene sequence is a novel allele of PopyKIR2DL4 that lacks the disabling mutations that characterize PopyKIR2DL4A and PopyKIR2DL4B. At 15 nt positions PopyKIR2DL4C differs from either PopyKIR2DL4A (one synonymous and two nonsynonymous) or PopyKIR2DL4B (three synonymous, two nonsynonymous and one deletion) or both (four synonymous and two nonsynonymous).

 
Nucleotide sequencing

The KIR haplotype sequence was determined by sequencing both strands of the cosmid subclones described above. Analysis of the EcoRI and BamHI subclones confirmed many of the junctions between adjacent subclones. Sequencing was performed on an ABI377 or Beckman Coulter CEQ instrument using the reagents and protocols recommended by the manufacturer. Subclones were first end sequenced. Smaller subclones (<2.5 kb) were sequenced by primer walking; medium length subclones (2.5–10 kb) were sequenced by deletion mutagenesis using the Erase-A-Base kit (Promega); longer subclones (>10 kb) were shotgun sequenced using the TOPO Shotgun Subcloning kit (Invitrogen Life Technologies). When an overlapping subclone to connect two sequenced subclones could not be found, primers were designed to PCR amplify a segment spanning the junction between the subclones. The amplification products were cloned and multiple clones sequenced to confirm the junction. Sequences were assembled using the Staden Package (45) (http://staden.sourceforge.net/). The finished sequence had quality scores >30. The PopyKIR haplotype sequence is deposited in GenBank with accession number EF014479.

Computational analyses

Phylogenetic analysis. The data set comprised the orangutan KIR genes obtained here and previously reported genomic sequences for human (AY320039, AC006293, AC011501), chimpanzee (BX842589), and rhesus macaque (BX842590, BX842591) KIR. Sequences of the individual loci were aligned using CLUSTAL X (46) and manually corrected in BIOEDIT (www.mbio.ncsu.edu/BioEdit/bioedit.html). The alignment was then divided into regions, generally following intron-exon boundaries. An exception was intron 6, which was further subdivided into three parts; the first of these (intron 6a) starts at the beginning of the intron and ends at the beginning of the deletion common to MmKIR3DL1 and MmKIR3DL10 (~750 bp), the second (intron 6b) begins here and ends at the beginning of the LINE insertion common to KIR3DL2 and PtKIR3DL1/2 (~2.9 kb), and the third (intron 6c) starts after the long interspersed nuclear element (LINE) insertion and ends at the end of the intron (~600 bp). These alignments were used for neighbor-joining (NJ) and parsimony analyses. NJ analysis was performed using MEGA version 3.1 (47) (www.megasoftware.net/) with 1000 replicates, pairwise deletion, midpoint rooting, and the Tamura-Nei method. PAUP*4.0b10 (48) (http://paup.csit.fsu.edu/) and the tree bisection-reconnection branch-swapping algorithm were used for parsimony analyses with 1000 replicates and a heuristic search. As comparison of the resulting trees revealed no differences, only NJ trees are presented in the figures.

Selection analysis. Selection analysis was performed on the sequences encoding the D1 and D2 domains for the seven orangutan lineage III KIR. Estimation of rate of nonsynonymous substitution (dN)/rate of synonymous substitution (dS) ({omega}) ratios was conducted by maximum likelihood using PAML version 3.14 (49) (http://abacus.gene.ucl.ac.uk/software/paml.html). A NJ analysis was first performed (as described above) and the likelihood of this tree topology was estimated for four site-specific models in which the selective pressure varied among different sites but the site-specific pattern was identical across all lineages. A likelihood ratio test was then conducted to compare a null model that does not allow {omega}>1 in the distribution with an alternative model that does. Likelihood ratio tests were performed for M1a (nearly neutral)/M2a (selection) and M7 (beta)/M8 (beta and {omega}). A Bayes Empirical Bayes approach was then used to identify codons belonging to the {omega}>1 site class. In all these analyses, the F3 x 4 model of codon frequencies was used.

Divergence time estimation. The divergence time for the two orangutan lineage III sublineages was estimated using the Bayesian relaxed molecular clock approach with the MULTIDISTRIBUTE program package (50, 51) (http://statgen.ncsu.edu/thorne/multidivtime.html). Genomic sequences were used for this analysis (from exons 2 to 4). All the nonrecombinant orangutan lineage III KIR were included, as well as KIR2DS4 and PtKIR2DS4 (calibration point), Mm KIR1D and KIR2DL4 (outgroup). The computer program ESTBRANCHES was used to estimate the branch lengths of the constrained topologies and the corresponding variance-covariance matrices. The F84+{gamma} model was used with maximum-likelihood parameters estimated previously by PAML. MULTIDIVTIME then used the variance-covariance matrices produced by ESTBRANCHES to run a Markov chain Monte Carlo analysis for estimating mean posterior divergence times on nodes with associated SD and 95% credibility interval. The Markov chain was sampled 10,000 times every 100 cycles and the burn-in stage was set to 100,000 cycles. Priors were set according to the guidelines defined in the MULTIDIVTIME manual. The root of the ingroup tree corresponds to the hominoid-Old World monkey separation and was set to 27 ± 3 million years ago (mya) to cover the range 21–33 mya as described previously (52). The separation of KIR2DS4 and Pt KIR2DS4 was used as the internal calibration point. It was constrained to be between 6 and 8 mya corresponding to the human-chimpanzee split. The lower limit corresponds to the lower estimate for the age of Sahelanthropus tchadensis, the oldest hominid fossil known to date (53); while 8 mya corresponds to the upper 95% credibility interval for the human chimpanzee divergence as established in a recent analysis (54).

Analysis of repetitive elements. The KIR haplotype sequences used for the analysis of repetitive elements were: human (AY320039, AC006283, AC011501, and AC009892), chimpanzee (BX842589), rhesus macaque (BX842591), orangutan (this analysis), pig (CR450381), rat (NW_047555), and mouse (AL672068 and NT_039385). In addition, the following cattle sequences obtained from Build 2.1 of the Bos taurus genome assembly were used for the intron 3 analysis (NW_937716, -NW_938268, NW_938467, NW_938583, NW_939141, NW_941773, NW_941846, NW_942117, NW_943617, NW_946585, and NW_980135). The sequences were submitted to the RepeatMasker server (RepeatMasker Open-3.0; www.repeatmasker.org) and the Censor server (55). Five separate regions were analyzed for the primate KIR haplotypes, the LILR to KIR interval, the region between the pseudogene and 2DL4, the KIR to FCAR interval, intragenic regions, and intergenic regions. Comparison of the primate KIR flanking regions was made with the pig LILR to KIR and KIR to FCAR regions, the rat Gp49B to KIR and KIR to FCAR regions, and the mouse PIR to NCR1 and KIR flanking regions. An alignment of primate, rodent, pig, and cattle sequences including exon 3, intron 3, and exon 4 was constructed. This data set was used to ascertain the repetitive element content of intron 3 as well as for phylogenetic analyses.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Genomic context of the orangutan KIR locus

The complete sequence of an orangutan KIR haplotype was obtained from six overlapping cosmid clones. It contains seven PopyKIR genes arrayed in the head-to-tail configuration seen in other primate species (25, 56). Upstream of the KIR genes is the LILR locus, downstream the FCAR locus (Fig. 1B). The part of the PopyLILR locus defined here is orthologous to the syntenic region of the human and chimpanzee LILR loci (94.6 and 93.7% sequence identity, respectively). It contains all of the PopyLILRP2 pseudogene (also called ILT10) and part of the PopyLILRP1 pseudogene (ILT9) (Fig. 1C). The part of the PopyFCAR gene defined here has 95.7% sequence similarity with the corresponding parts of the human and chimpanzee FCAR genes and 92.5% sequence similarity with the rhesus macaque FCAR gene. Thus, the organization of the PopyKIR locus and its genomic context within the leukocyte receptor complex (LRC) is like that observed in other primate species.

Comparison was also made with the KIR loci of three nonprimate species: pig, rat, and mouse (Fig. 1D). For the pig, which has a single KIR gene (57), the order of the loci is the same as in primates: LILR-KIR-FCAR. Although this order is conserved in the rat, the KIR gene and its accompanying KIR gene fragment are differently arrayed in a tail-to-tail configuration (extracted from NW_047555). Furthermore, the regions that flank the rat KIR genes are longer in the rat (55 kb upstream and 25 kb downstream) compared with the pig (11 and 8 kb) and primates (3–15 and 7–10 kb). More divergent is the mouse, where the LRC lacks both KIR and FCAR genes. The latter appears absent from the mouse genome and the two KIR genes are located on the X chromosome (34) in a head-to-head configuration (extracted from AL672068).

Gene content of the orangutan KIR haplotype

Of the seven genes in the orangutan KIR haplotype, two are novel and five correspond to previously characterized cDNA clones (Fig. 1E) (28). One of the novel genes, PopyKIR3DL3, is the orangutan equivalent of human KIR3DL3, the framework gene of lineage V that is adjacent to the LILR locus (Fig. 1B). The other novel gene, PopyKIRDP, is a pseudogene that lies upstream of PopyKIR2DL4 (lineage I), the position also occupied by a pseudogene in human, chimpanzee, and rhesus KIR haplotypes. The interval between PopyKIR3DL3 and PopyKIRDP contains three lineage III genes (PopyKIR2DSE, PopyKIR2DSFn, and KIR2DLB), an organization like that in the corresponding region of the group A human KIR haplotype. Downstream of PopyKIR2DL4, there is only one additional gene in the orangutan haplotype. PopyKIR3DLD1 is a lineage II gene that flanks the FCAR locus and corresponds to the lineage II framework gene present in other primate species. Overall, the organization of the orangutan KIR haplotype resembles that of the human KIR A haplotype (25) and the chimpanzee KIR haplotype described by Sambrook et al. (56) (Fig. 2).


Figure 2
View larger version (26K):
[in this window]
[in a new window]

  		FIGURE 2. Structure and gene organization of KIR haplotypes from four primate species. Shown is a schematic representation of KIR haplotypes from human (25 ), chimpanzee (56 ), orangutan (this study), and rhesus macaque (56 ) for which complete nucleotide sequences have been determined. Under "Framework" are shown the gene and gene fragments that are conserved among the four species. Genes are colored according to the lineages described by Rajalingam et al. (32 ) with the modification described here of including the rhesus lineage IV genes (Mm3DL1 and 3DL10) in lineage II. The maps have been drawn to align the framework genes found in all species and are not drawn to scale.

 
Repetitive elements in the KIR locus

Repetitive elements in the flanking sequences. The regions that flank the KIR locus contain many repetitive elements. Comparison of the 5' flanking region, which extends from the final LILR exon to the first KIR exon, showed that no repetitive element is conserved between primate and nonprimate species (pig, mouse, and rat) (Fig. 3A). Similar comparison for the 3' flanking region, which extends from the final KIR exon to the first FCAR exon, identified a 332-bp fragment of a LINE element that is common to pig and primates and is interrupted by other insertions (Fig. 3B). Whereas this shared L1MA9 fragment flanks the pig FCAR gene, in primates it is at the other end of the region, away from FCAR and close to the KIR genes. That only one small fragment has been preserved from the many repetitive elements likely present in the common ancestor of the pig and primate KIR loci, points to the unusually dynamic history of the KIR locus.


Figure 3
View larger version (25K):
[in this window]
[in a new window]

  		FIGURE 3. Comparison of repetitive elements found in the regions flanking the KIR locus. Shown is a schematic representation of the repetitive elements found in the regions flanking the KIR locus. Exons are colored green, LINE red, SINE dark blue, retroviral elements light blue, simple repeats yellow, and others orange. A specific 40-mer repeat in the KIR-FCAR region is indicated in purple. Boxes to the right indicate repeats on the plus strand (relative to the coding direction of the KIR); boxes on the left indicate repeats on the minus strand. In the primate sequences, identical elements are aligned. Discontinuities represent insertion/deletion events. A, Repetitive elements in the centromeric flanking region, bounded by the final LILR and the upstream KIR. B, Repetitive elements in the telomeric flanking region bounded by KIR and FCAR. The limits of the L1MA9 element fragment shared by pig and primates are shown by the dashed lines. A 44-bp segment of this element (positions 6179–6223) is repeated five times in the rhesus macaque.

 
Among the primates, the 5' flanking region is similar in length and organization in orangutan (14.8 kb), chimpanzee (11.2 kb), and human (14.8 kb) but it diverges in the rhesus macaque (2.7 kb) (Fig. 3A). Comparison of repetitive elements indicates that in the common ancestor of the four primate species, this region resembled that now seen in the orangutan, with the addition of a 577-bp segment that is present in the other three species but absent from the orangutan. Subsequent evolution appears largely characterized by deletions: at least three in the rhesus lineage, one in the orangutan lineage, and two in the human/chimpanzee lineage. By comparison to the 5' flanking region, the 3' flanking region is relatively conserved in primates. Its length is 7.1 kb in human, 7.3 kb in orangutan, 7.5 kb in rhesus, and 9.9 kb in chimpanzee, the latter’s increase in size being due to expansion of a central repeat region (Fig. 3B).

Repetitive elements in the intergenic sequences. Apart from the region between the pseudogene and KIR2DL4, the segments that separate two KIR genes can be divided into three groups based upon the repetitive elements they contain (Fig. 4). Common to all these intergenic regions is a MER2B element and a LINE fragment (L1M5). Group 1 comprises sequences associated with the most telomeric KIR genes (lineage II KIR) (Fig. 4, group 1), and in them the LINE fragment (shown red in Fig. 4) is longer by ~140 bp than in the group 2 and 3 sequences. These two groups have an Alu insertion that is responsible for the further truncation of the LINE fragment. In group 2, the Alu is full length, whereas in group 3 it has been truncated as the result of a subsequent deletion event. Also specific to group 3 is the insertion of an additional Alu element within the MER2B element. There is no orangutan representative in group 3. These relationships indicate that group 1 represents the oldest form of the intergenic sequence and that the group 2 and then group 3 forms evolved from it by successive insertion and deletion events. Analysis of the sequences upstream of the lineage V and KIR2DL4 genes showed that ~350 and 600 bp, respectively, corresponding to the 3' end of the intergenic sequence, is present.


Figure 4
View larger version (75K):
[in this window]
[in a new window]

  		FIGURE 4. Three distinct groups of intergenic sequences identified in the primate KIR region. On the left is the NJ tree generated from the intergenic sequences. Each sequence is identified by its upstream and downstream gene and for the human sequences; the haplotype of origin is indicated by A (AC011501) or B (AY320039). The group 1 sequences are associated with the terminal lineage II gene of each haplotype and thus only one gene is listed. Gene names are colored by the lineage of the gene (I, orange; II, light blue; III, green; V, red; truncated pseudogenes, black). To the right is a schematic representation of the repeat elements found in the intergenic region. MER2B elements are shown in gold, Alu elements in dark blue, the mammalian interspersed repeat element in purple, and the LINE in red. The LINE is inserted in the complementary direction relative to the KIR coding direction. In the group 1 sequences, it comprises bases 4978-4567 of the L1M5 sequence; in the group 2 and 3, sequences it comprises bases 4978-4699 of L1M5.

 
Five intergenic sequences exhibit departures from the general features described above; they differ in unique ways, all but one involving insertion/deletion events. A mammalian interspersed repeat element is inserted between MER2B and the LINE in the region between PopyKIR2DLB and PopyKIRDP. The MER2B element 3' of PopyKIR3DLD1 is partly deleted. The region between PtNewI and PtNewII has a deletion that extends from the Alu within MER2B past the second Alu element. The interval bounded by Mm1D and Mm has an Alu inserted between the MER2B and LINE elements, and is also truncated relative to the other intergenic sequences, due to a deletion extending from 5' of exon 1 through to intron 3 of the pseudogene. Finally, the interval between MmNewI and the Mm1D contains both the MER2B with Alu insertion and the full-length Alu at the 3' end of the interval. Phylogenetic analysis using the 3' fragment common to all intergenic regions confirmed that this was not a recombinant sequence, but instead showed that the Alu insertion into the MER2B element preceded the deletion event within the group 3 intergenic sequences (data not shown).

Repetitive elements in the intragenic sequences. Apart from the recently reported and divergent KIR3DX1 (27), we find that intron 3 of all primate KIR contain a MLT1D element inserted into a LTR33A element. This comparison also confirmed that primate lineage II and III KIR genes are distinguished from other KIR genes by the insertion of an AluSq into the MLT1D/LTR33A structure (56, 58). Extending the analysis to nonprimate species, showed the pig, rat, and mouse KIR genes all have an MLT1D/LTR33A element in intron 3, albeit truncated in rat and mouse. In contrast, analysis of the cattle draft genomic sequences containing exons 3 and 4 revealed that only the cattle KIR related to BtKIR2DL1 (4 of the 18 sequences) contained the MLT1D/LTR33A insertion. The remaining cattle KIR lacked this element and had intronic sequences more closely related to those of KIR3DX1, which from phylogenetic and genomic analyses appears to represent a second KIR lineage that is encoded by an LRC gene away from the region flanked by the LILR and FCAR genes (27). Thus, the complex of MLT1D and LTR33A within intron 3 was likely formed by an insertion in the lineage of KIR flanked by LILR and FCAR, occurring after its separation from the KIR3DX1 lineage.

Analysis of the intronic repeat structure encompassed the entire gene. Previous reports have demonstrated the maintenance of the intronic repeats both within lineages as well as those common to all KIR (56, 58). Our analysis shows that all but the lineage II orangutan KIR maintain the repeat structure common to each of the lineages described previously (56) (data not shown). The lineage II differences are confined to introns 5 and 6. Intron 6 of PopyKIR3DLD lacks the LINE present in human and chimpanzee lineage II KIR. Mm3DL1 and Mm3DL10 also lack this LINE, which was therefore inserted after separation of the human and chimpanzee lineage from the orangutan lineage. Intron 5 of human and chimpanzee lineage II KIR is characterized by two LINE fragments and four Alu elements. Of these the orangutan lineage II KIR lacks the Alu element closest to exon 5, which is also a feature of rhesus lineage II KIR. Thus, insertion of this element into human/chimpanzee lineage II KIR occurred after separation from the orangutan lineage. The orangutan does have the insertion of an AluSp in intron 5 that is common to the hominoid sequences and absent from the rhesus sequences, indicating that this insertion predates the speciation event.

Refined definition for the framework regions of the primate KIR locus

Only the 5' part of KIR3DL3 forms the centromeric framework. Located at the centromeric end of the KIR locus, PopyKIR3DL3 corresponds to human KIR3DL3, chimpanzee PtKIRNewI, and rhesus macaque MmKIRNewI. Distinguishing PopyKIR3DL3 from KIR3DL3 and PtKIRNewI is the presence in PopyKIR3DL3 of an exon 6, which encodes a stem in the predicted protein product, a feature it shares with MmKIRnewI. Domain-by-domain phylogenetic analysis showed that PopyKIR3DL3 groups strongly with KIR3DL3, PtKIRNewI, and MmKIRnewI in exons 1–5 and the intervening introns, forming the lineage V KIR (Fig. 5A). From intron 5 through to the 3' end of the sequence, this lineage affinity is lost and PopyKIR3DL3 groups strongly with the PopyKIR2D sequences, a pattern showing that PopyKIR3DL3 is the product of recombination (Fig. 5, B–D). This result is confirmed by the association of a group 2 intergenic segment PopyKIR3DL3 (Fig. 4) in contrast to the association of group 3 segments with the lineage V KIR of other primate species.


Figure 5
View larger version (45K):
[in this window]
[in a new window]

  		FIGURE 5. The genes of lineage V have recombined in all species to encode proteins with distinct cytoplasmic tails. Shown are phylogenetic trees obtained by comparison of different regions of primate KIR genes. The lineage V genes are highlighted. A, In the region spanning exons 1 to 5 and introns 1–4, the lineage V KIR group together with strong support. B, Intron 5 of PopyKIR3DL3 groups with PopyKIR of lineage III, a grouping that is maintained throughout the 3' exons and introns, and not with other lineage V KIR. C, Chimpanzee and human KIR of lineage V share a common deletion beginning upstream of exon 6 (the exon encoding the stem of the molecule) and ending ~400 bp into intron 6, which does not allow for phylogenetic reconstruction within this interval. The nonorangutan lineage V genes remained grouped together in intron 6b (data not shown). Within in the latter part of intron 6 (designated intron 6c), the remaining lineage V KIR group within the larger lineage III group. D, In the region including exons 7–9 and introns 7 and 8, the lineage V KIR do not group together but are dispersed within the tree and tend to associate with other KIR from the same species.

 
The domain-by-domain phylogenetic analysis indicates that recombination is a general feature of lineage V genes. For KIR3DL3, PtKIRNewI, and MmKIRnewI, the recombinations occurred within intron 6, whereas for PopyKIR3DL3 the recombination breakpoint is in intron 5 (Fig. 5C). Each recombination involves a different donor gene, with the result that the lineage V KIR in each species has a distinctive signaling domain (transmembrane region and cytoplasmic tail) (Fig. 5D). From this interspecies comparison, the framework at the centromeric end of the KIR locus is more precisely defined as the 5' part of the lineage V gene (from exons 1 through to 5) rather than the entire gene.

KIR2DL4 and part of a pseudogene form the central framework. Adjacent to the cluster of orangutan lineage III KIR lies the PopyKIRDP pseudogene, which is at the same position in the KIR locus as human 3DP1, as well as chimpanzee (PtKIR3DP1) and rhesus (MmKIRDP) pseudogenes. The pseudogene sequence ends soon after the end of exon 5 and in all species a region characterized by a number of repetitive elements is found between the end of the pseudogene and the beginning of 2DL4. Phylogenetic analysis showed that each of these pseudogenes is a chimera. In the 5' part, PopyKIRDP associates strongly with the orangutan lineage III KIR, whereas human 3DP1 associates with 2DL5 of lineage I, the chimpanzee pseudogene associates with 2DP1 and the rhesus pseudogene, which lacks sequences corresponding to exons 1, 2, and 3, associates with other rhesus sequences (data not shown). The three hominoid pseudogenes group together in phylogenetic trees based on exon 5 sequences (Fig. 6A), although the grouping is weakly supported.


Figure 6
View larger version (18K):
[in this window]
[in a new window]

  		FIGURE 6. Similarity of exon 5 of the KIR pseudogene upstream from KIR2DL4 in hominoids. A, Phylogenetic tree for exon 5. The KIR pseudogenes are shaded dark gray. The region containing repetitive elements follows exon 5. B, Organization of repetitive elements in the region bounded by the pseudogene and KIR2DL4 in the primates. The schematic representation is colored as described in Fig. 3.

 
The region bounded by the pseudogene and 2DL4 appears, so far, to be unique to primates. We found no evidence for a similar repeat structure in either the upstream or downstream sequences flanking the single pig KIR or the rodent KIR genes. Within the primate sequences the region is well-conserved. In the orangutan, this region is identical in organization to that in human and chimpanzee, with the exception of a 1.3-kb region found in rhesus and orangutan and deleted in humans and chimpanzees (Fig. 6B). Unique to the rhesus macaque is the extension of an L2 element, the insertion of an AluY element, the absence of an AluSg element found in the hominoid sequences, and the insertion of a long terminal repeat element.

Domain-by-domain phylogenetic analysis shows that PopyKIR2DL4 groups strongly with human, chimpanzee, and rhesus 2DL4 sequences in all domains. Thus, there is no evidence for recombination with other KIR genes. The PopyKIR2DL4 gene defined represents a different allele (PopyKIR2DL4C) from the two previously characterized cDNA clones (PopyKIR2DL4A and PopyKIR2DL4B), which have mutations preventing expression of a full-length protein. PopyKIR2DL4A has a point mutation in exon 5 that results in premature termination; PopyKIR2DL4B has a single nucleotide deletion that causes a frameshift and premature termination. PopyKIR2DL4C has two intact ITIM motifs in the cytoplasmic tail as well as a charged residue in the transmembrane domain. In contrast, human and chimpanzee 2DL4 have only one ITIM motif and the charged residue in the transmembrane domain.

Taken together, these results show that the central framework of the hominoid KIR locus consists of exon 5 of the pseudogene, the KIR2DL4 gene and the region of unique sequence that joins them. Although exon 5 of MmDP does not group phylogenetically with exon 5 of the hominoid pseudogenes, the identity of the breakpoint within intron 5 and the homology of the repeat region supports a common origin for the primate pseudogenes in this region. Subsequent recombination with another KIR gene could explain the divergence of exon 5 of MmDP.

The telomeric framework is formed by the 3' portion of a lineage II gene. Situated between PopyKIR2DL4 and the FCAR gene is an orangutan lineage II KIR gene that corresponds to the previously characterized PopyKIR3DLD1 cDNA clone (28). In most domains, it is homologous to all other hominoid lineage II KIR. The exceptions to this are the previously described recombination events that group human KIR3DL1 with lineage III KIR from within intron 6 to the 3' end of the gene (32) and place KIR3DL2 outside of the lineage II group in intron 1. In addition, analysis of exon 3 shows that although KIR3DL2 groups with the other lineage II KIR, its branching pattern indicates a greater divergence than expected (data not shown). As discussed above, PopyKIR3DLD1 lacks two of the repetitive elements that characterize chimpanzee and human lineage II KIR, features shared with MmKIR3DL1 and MmKIR3DL10. One is a LINE element insertion in intron 6, the other is an Alu repeat in intron 5.

With the addition of PopyKIR3DLD1 to the KIR data set, the results of phylogenetic analysis show that MmKIR3DL1 and MmKIR3DL10, which were previously considered to be a separate lineage (lineage IV) (32), should be included in the lineage II KIR (Fig. 7). This grouping is supported by the sequence of a region encompassing the 5' end of the gene through to intron 6, excepting the 5' UTR, exon 1, and exon 5 where the deeper branching nodes were not well-resolved. Weak support (values <50 for the bootstrap analysis) was also observed for exon 4 and the parts of intron 6 (6a and 6c) that are retained in the MmKIR3DL1 and MmKIR3DL10 genes: the intron 6b region being absent. From exon 7 through to the 3' end of the sequence, all macaque KIR group together and are separated from the hominoid KIR. The analysis of intergenic repetitive elements (Fig. 4) suggests that this divergence is not a consequence of recombination, but of selection upon exons encoding the signaling domain of the rhesus macaque KIR. Deletion of the intron 6b region, which includes the MER70B/MSTB1 elements present in other hominoid KIR genes, appears to have been an event specific to the macaque genes of this lineage.


Figure 7
View larger version (11K):
[in this window]
[in a new window]

  		FIGURE 7. Rhesus macaque KIR3D are related to hominoid lineage II KIR. Shown are the results of domain-by-domain phylogenetic analysis for the lineage II KIR. The regions analyzed are indicated at the top, but not drawn to scale: exons being represented by black boxes with white lettering. The results obtained for each lineage II KIR examined are shown below. Dark gray shading shows grouping with other lineage II KIR supported >50% by bootstrap analysis; light gray shading shows grouping with <50% support by bootstrap analysis. Stippled shading indicates regions replaced by recombination. Empty boxes indicate analyses that had many unresolved branchings. Introns and exons at the end of the sequence were analyzed separately and together to provide more power to the analysis. Only the results of the grouped analysis are shown. #, The insertion of a LINE into intron 6 of the progenitor of the chimpanzee and human lineage II KIR. This LINE is absent from all other KIR. Furthermore, the absence of an Alu element found in intron 5 of all other KIR, denoted by *, is a feature shared by the macaque and orangutan lineage II KIR. The two rhesus KIR lack the intron 6b region as indicated by the gap in the diagram.

 
Inclusion of the macaque KIR3DL sequences within lineage II is consistent with the genomic placement of MmKIR3DL1 adjacent to the macaque FCAR gene, a location occupied by a lineage II KIR in all hominoid sequences examined to date. Definition of precise endpoints for the framework is complicated by the deletions within the rhesus macaque sequences as well as the divergence of the tail-encoding exons of the rhesus macaque. Minimally, the framework comprises the region extending from intron 3 through intron 6. In the apes, the region may be considered as extending from intron 3 to the 3' end of the gene.

Orangutan lineage III KIR comprise two sublineages

In between PopyKIR3DL3 and PopyKIRDP are three lineage III KIR genes. These comprise one 2DL gene and two 2DS genes. The 2DL gene corresponds to the PopyKIR2DLB cDNA. The PopyKIR2DSE and PopyKIR2DSFn genes are related to the PopyKIR2DSC and PopyKIR2DSD cDNA, respectively (Fig. 1A). PopyKIRDSFn has a single base pair deletion in exon 5 resulting in a frameshift and premature termination indicating that it is a nonfunctional or null (n) allele. All three orangutan lineage III genes contain a pseudoexon 3, as seen in all lineage III KIR2D genes of other species. Like other pseudoexon 3 sequences, there is a 3-bp deletion relative to expressed exon 3 sequences. The pseudoexon has an intact 5' splice site in two of the three PopyKIR2D genes, the third gene (PopyKIR2DSFn) having an AG to AA change resulting in disruption of the splice site. In all three genes, the 3' splice site is lost through substitution of TT for GT.

The inhibitory PopyKIR2DLB and activating PopyKIR2DSE encode similar extracellular domains but different cytoplasmic tails. From the beginning of the gene through to intron 6, their sequences differ by only 2%, after which the sequence difference increases to 6% in the region encoding the signaling domain. In this region, the short-tailed PopyKIR2DSE and PopyKIR2DSFn differ by only 0.6% of their sequence, but are divergent elsewhere. Like PopyKIR2DLB and PopyKIR2DSE, pairs of human lineage III KIR with similar extracellular domains have opposing signaling function: 2DS1 with 2DL1, 2DS2 with 2DL2.

Several lineage III PopyKIR genes identified by cDNA analysis are not a part of the sequenced haplotype. To obtain further knowledge of their sequences, we characterized the region encompassing exons 2–4 of the PopyKIR2DSA, PopyKIR2DSB, PopyKIR2DSC, and PopyKIR2DLA genes. Phylogenetic analyses of the combined genomic and cDNA sequence data identified two sublineages for the sequences spanning exons 3–5. PopyKIR2DSA and PopyKIR2DSB comprise one sublineage, PopyKIR2DLA, PopyKIR2DSC and PopyKIR2DSFn the other. PopyKIR2DLB, PopyKIR2DSD, and PopyKIR2DSE represent interlineage recombinants, having intron 2-intron 4 of the PopyKIR2DLA sublineage (sublineage I) and exon 5 of the PopyKIR2DSA sublineage (sublineage II). Using the sequences spanning exons 2–4, the divergence time of the two sublineages was calculated to be 10.3 ± 1.25 mya, indicating that the two sublineages arose after separation of the orangutan ancestors from human/chimpanzee ancestors,10–18 mya (59, 60). This time is also later than the 13.5 mya estimated for the origin of the short-tailed hominoid KIR (52). Thus, the short-tailed hominoid KIR ancestor was formed before orangutan speciation and subsequently underwent orangutan-specific diversification, a model supported by phylogenetic analysis showing PopyKIR2D branches at a position orthologous to all the human and chimpanzee KIR with no affinity for any subgroup of them.

There are 29 positions of variation in the D1 and D2 domains of lineage III PopyKIR. Eighteen of these positions define the two lineages and 7 are substitutions unique to a single PopyKIR. At positions 36 and 44, there are three motifs which distinguish sublineage 1 (N36, K44), sublineage 2 (Y36, K44), and the recombinants (H36, E44) (Fig. 8A). The D1 and D2 domains of the PopyKIR2D were examined for evidence of natural selection. Recombinant sequences were removed for the analysis encompassing both domains and replaced when the domains were analyzed separately. Residues 143 and 148 were shown to be positively selected in both analyses, while selective pressure on residue 36 was only indicated in the analysis that included the recombinant sequences (Table II). As the specificity determining residue 44 is identical in the nonrecombinant sequences, there was no evidence for selection in the two-domain analysis. When the domains were analyzed separately, the value obtained for residue 44 still failed to approach significance.


Figure 8
View larger version (78K):
[in this window]
[in a new window]

  		FIGURE 8. Orangutan lineage II KIR are divided into two sublineages. A, All positions of difference in the D1 and D2 domains of the orangutan and human lineage II KIR. KIR2DL2 was used as the reference sequence, identity is indicated with a dash. Positions that divide the orangutan KIR into two sublineages are coded yellow for sublineage 1 and green for sublineage 2. In the D1 domain, the human sequences are more similar to sublineage 2 and in the D2 domain they are more similar to sublineage 1. Positions with orangutan specific residues are shaded pink. The specificity determining residue 44 is indicated in red; the positions that were shown to be positively selected are shown in blue. Differences unique to a single sequence are shaded gray. B, The structure of KIR2DL2 bound to HLA-Cw3 is shown (1EFX; Ref. 61 ). Upper panel, The specificity determining residue 44 (yellow) and the positively selected residues (pink). Center panel, The sublineage determining residues (yellow). Bottom panel, The orangutan-specific residues (pink).

 

View this table:
[in this window]
[in a new window]

  		Table II. Analysis of positive selection on PopyKIR sequences

 
Modeling PopyKIR2D on the crystallographic structure of human KIR2D bound to HLA-C (61) showed that the selected residues are located away from the putative binding site for MHC class I, but on loops between the B and C (residue 36) or C and C' (residues 143 and 148) beta strands (Fig. 8B, upper panel). The residues that distinguish the two sublineages are located on the loops of the D1 domain located opposite to the binding site and on the face of the D2 domain implicated in interdomain contacts (Fig. 8B, middle panel). The sublineage differences might therefore affect the function of PopyKIR2D by altering the angle between the two domains (61, 62) as well as altering the contact with other molecules. Finally, two of the orangutan-specific resides (P71 and T72) are located near the specificity determining residue 44 and may affect binding ability, two (G144 and G145) are located on a loop that is likely involved in interdomain interactions, and the remaining are located in loops of the D2 domain that are located opposite to the binding site and may interact with other molecules (Fig. 8B, lower panel).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Evolution of the mammalian KIR gene family

Synthesis of our results with the published literature suggests a model for the evolution of the KIR gene family. At an early stage in mammalian evolution, a progenitor KIR3D gene duplicated to produce ancestors for the two modern KIR lineages. The KIR3DX1 lineage, which is represented by a single gene in primates (27) but has expanded in cattle, and the lineage of KIR containing a LTR33A/MLT1D element in intron 3, an insertion which took place after the initial duplication, but before subsequent expansion of the lineage. The fate of this second lineage has varied. In pigs, it remained a single gene (57), while in primates it evolved into an elaborate family of genes placed head to tail (Fig. 2). Although the two rat KIR genes are syntenic to pig and primate KIR, their orientation is tail to tail (extracted from NW_047555), whereas the two mouse KIR genes have moved to the X chromosome (34) where they have head-to-head orientation (extracted from AL672068).

The progenitor of the LTR33A/MLT1D-containing primate KIR was a KIR3D having a group 1 intergenic region on its 3' side and the shared 3' part of the group 2 and 3 intergenic region on its 5' side (Fig. 9, haplotype 1). Duplication of this gene produced two tandem genes in head-to-tail orientation and separated by a group 2 region which could have originated during the duplication, by truncation of the LINE element (Fig. 9, haplotype 2). This duplication either captured the AluSq common to the group 2 and group 3 intergenic sequences or alternatively, the element was inserted before successive duplications. The next duplication event resulted in the truncated pseudogene and capture of the repeat region now found between the KIR pseudogene and the lineage Ia KIR (Fig. 9, haplotype 3). Subsequent duplication and recombination events resulted in the formation of the lineage III progenitor in the lineage V-pseudogene interval (Fig. 9, haplotype 4). Also during this period, exon 4 was deleted from the lineage I KIR progenitor and an Alu insertion formed the first group 3 intergenic region (designated 3* in Fig. 8). In this manner, a primordial primate KIR locus containing all three framework regions and all major primate KIR lineages could have evolved (Fig. 9, haplotype 4). An additional duplication of the lineage III KIR and deletion within the associated group 3 intergenic interval formed the precursor haplotype present before the speciation leading to the rhesus occurred (Fig. 9, haplotype 5). After the speciation event, species-specific duplication and deletion events occurred resulting in the defined rhesus haplotype (56) (Fig. 9, haplotype Rhesus).


Figure 9
View larger version (49K):
[in this window]
[in a new window]

  		FIGURE 9. Intermediate steps in primate KIR haplotype evolution. A model for the evolution of the primate KIR haplotypes is shown. The gray boxes enclose the known primate KIR haplotypes (Rhesus, Orang, Chimp, HumA-human A haplotype, HumB-human B haplotype). The remaining haplotypes (numbered 1–7) are proposed intermediates in the evolution of the complex, the first five having been present in species ancestral to all primates. Intermediates 6 and 7 between the rhesus and orangutan haplotypes arose after the speciation event leading to the rhesus and before ape speciation events. The genes are aligned to the longest haplotype, the human B haplotype (far right). Genes are color coded by lineage as in Fig. 3, the pseudogene adjacent to the lineage I gene is coded dark gray, ancestral genes that represent more than one lineage are coded black, and the lineages descended from them are indicated (I-V, all lineages; I/V, lineages I and V; II/III, lineages II and III). R is the precursor to the repeat region that is part of the central framework. The evolution of the complex began with a single KIR gene (far left) bounded on one side by the group 1 intergenic region (blue) and on the other by sequence homologous to the 3' end of the group 2 and 3 intergenic sequences (light green) as well as sequences homologous to the repeat region now found between the pseudogene and KIR2DL4 (haplotype 1, all placental mammals, 135.5 mya). The progenitor gene duplicated resulting in the formation of the group 2 intron. Following this, another duplication occurred resulting in the formation of the truncated pseudogene and capture of the central repeat region (haplotype 3, primate specific, maximum 81 mya). Additional duplication events formed the progenitor of the lineage III KIR and insertion of the Alu in the intergenic region resulted in the formation of the group 3 intergenic progenitor (designated as 3* and shown in light orange). Subsequent deletion within this region formed the group 3 sequence (pink) (haplotype 5, before rhesus speciation, 27 mya). The rhesus haplotype was formed from this progenitor sequence. Subsequent to the speciation event separating the rhesus macaque from the hominoids, additional duplication and recombination occurred within the lineage III containing region. A minimum of two ancestral genes was present before the speciation event leading to the orangutan, one of which was a long tail KIR and the other a short tail KIR (haplotype 7, before orangutan speciation, 13 mya). Duplication and recombination has focused on the interval bounded by the lineage V KIR and the pseudogene in the orangutan and chimpanzee. In the human haplotypes, there has been additional duplication and recombination within the 2DL4 to lineage II KIR interval.

 
Following this speciation event, additional duplication of the lineage III KIR occurred (Fig. 9, haplotype 6). In addition, genes encoding the short-tailed KIR were formed during this interval (Fig. 9, haplotype 7). The defined haplotypes for the orangutan, chimpanzee (56), and human (AY320039, AC006293, AC011501, and Ref. 25) can all be derived from this progenitor haplotype through duplication, recombination, and diversification (Fig. 9, haplotypes Orang, Chimp, HumA, and HumB).

Expansion of lineage III KIR in the orangutan

The defined orangutan KIR haplotype is similar to that of the hypothetical primordial primate KIR haplotype (haplotype 4 Fig. 9). The main difference is that the single lineage III KIR gene in the ancestral haplotype has duplicated and diverged to give three lineage III KIR genes in the orangutan haplotype. This region in human KIR haplotypes contains genes encoding the inhibitory HLA-C receptors: KIR2DL1, 2, and 3. Thus, the expansion of lineage III KIR has occurred in the same time frame as the emergence of MHC-C.

Previous cDNA analysis identified Popy2DLA and Popy2DLB as the only inhibitory PopyKIR2DL. The sequenced haplotype contains Popy2DLB but not Popy2DLA, raising the possibility that they are alleles. Popy2DLA and Popy2DLB differ at 14 positions (3 in D1, 6 in D2, 5 in the stem, transmembrane, and cytoplasmic regions), included in these is residue 44, which is lysine in KIR2DLA and glutamic acid in KIR2DLB. In human KIR2DL of lineage III, lysine 44 is characteristic of C1 receptors, whereas glutamic acid 44 has not been observed. Thus, the sequenced haplotype does not encode PopyKIR2DLA, the best candidate for an inhibitory Popy-C receptor (preliminary analysis shows that a PopyKIR2DLA-IgFc fusion protein binds to MHC-C with C1 specificity; data not shown). Of the haplotype’s two other lineage III genes, Popy2DSE encodes an activating receptor with glutamate 44 and Popy2DSFn is impaired due to a nucleotide deletion. One possibility is that the sequenced haplotype does indeed lack a gene encoding an inhibitory C1-specific KIR, which would be analogous to the orangutan MHC haplotypes that lack the Popy-C gene. Alternatively, the PopyKIR2DLB receptor could be MHC-C specific, using glutamate at position 44 to interact with Popy-C allotypes.

Previous analysis has shown that the duplication which resulted in the formation of the activating KIR occurred before the speciation event leading to the orangutan. Our analysis showed there to be two distinct lineage III KIR sublineages in the orangutan. Divergence time estimation showed that these two groups were formed early in the orangutan lineage, but after the speciation event leading to the orangutan lineage. The mechanism was likely homogenization of the extracellular domain encoding exons of the lineage III KIR by recombination followed by divergence resulting in the two sublineages recognizable today. Analysis of the orangutan lineage III KIR provided evidence for selection acting on residues involved in interdomain interaction. These residues may be important in determining the hinge angle and thus the ligand-binding platform of the KIR molecule. Visual inspection of the sequence alignment also shows a cluster of substitutions occurring within the sequence that encodes the region corresponding to the face of the D2 domain that interacts with the D1 domain. In the orangutan, the lysine at position 44 is common to both sublineages, indicating the glutamic acid found in the recombinant sequences is more recently arisen.

KIR2DL4 is the only KIR gene conserved in higher primates

Analysis of an orangutan KIR haplotype and its comparison to haplotype sequences from other species has further defined the three conserved framework regions of the primate KIR locus. The centrally situated KIR2DL4 (lineage I) is the only gene preserved in its entirety in the orangutan, chimpanzee, human, and rhesus macaque. By contrast, only the 5' part of the lineage V gene at the 5' end of the KIR locus and the 3' part of the lineage II gene at the 3' end are conserved. Although previous analysis of cDNA identified only defective PopyKIR2DL4A and B alleles (28), the PopyKIR2DL4C allele of the sequenced haplotype encodes a full-length protein that is likely functional. Human KIR2DL4 is an HLA-G receptor (63) implicated in regulating NK cell functions during reproduction. Of potential importance is that orangutan and rhesus 2DL4 have two ITIM motifs, whereas human and chimpanzee 2DL4 have only one. Further evidence for functional differences between these species is that rhesus MHC-G is unexpressed, possibly supplanted by the related MHC-AG (64, 65), and that secreted HLA-G isoforms appear specific to the human species (66) and able to stimulate NK cells through endocytosis and signaling from intracellular vesicles (67). Thus, while KIR2DL4 is the most conserved KIR gene in the higher primates, its alleles and functions are continuing to evolve. There is no evidence, so far, for a 2DL4 gene in nonprimate species. Notably, the single pig KIR gene, which is syntenic to the primate KIR locus (57), has no special relationship with primate KIR2DL4. This finding is consistent with the model shown in Fig. 9, in which the KIR2DL4 precursor arises after two rounds of duplication of the progenitor KIR3D gene. The second duplication is coincident with the "capture" of the associated repeat region, which is conserved among the primate species studied, but absent in all others, thus placing this event at a time postdating the speciation event leading to the primate lineage.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants AI031168 and AI024258 (to P.P.) and a Ruth L. Kirschstein National Research Service Award (F30) from National Institutes of Health (to A.M.O.A.). Back

2 Address correspondence and reprint requests to Dr. Peter Parham, Department of Structural Biology, Stanford University, Fairchild D-159, 299 Campus Drive West, Stanford, CA 94305. E-mail address: peropa{at}stanford.edu Back

3 Abbreviations used in this paper: KIR, killer cell Ig-like receptor; NJ, neighbor joining; mya, million years ago; LRC, leukocyte receptor complex; LINE, long interspersed nuclear element; dN, rate of nonsynonymous substitution; dS, rate of synonymous substitution. Back

Received for publication January 3, 2007. Accepted for publication April 14, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Moretta, L., C. Bottino, D. Pende, R. Castriconi, M. C. Mingari, A. Moretta. 2006. Surface NK receptors and their ligands on tumor cells. Semin. Immunol. 18: 151-158. [Medline]
  2. Ferrini, S., A. Cambiaggi, R. Meazza, S. Sforzini, S. Marciano, M. C. Mingari, L. Moretta. 1994. T cell clones expressing the natural killer cell-related p58 receptor molecule display heterogeneity in phenotypic properties and p58 function. Eur. J. Immunol. 24: 2294-2298. [Medline]
  3. Uhrberg, M., N. M. Valiante, N. T. Young, L. L. Lanier, J. H. Phillips, P. Parham. 2001. The repertoire of killer cell Ig-like receptor and CD94:NKG2A receptors in T cells: clones sharing identical {alpha}beta TCR rearrangement express highly diverse killer cell Ig-like receptor patterns. J. Immunol. 166: 3923-3932. [Abstract/Free Full Text]
  4. Colonna, M., J. Samaridis. 1995. Cloning of immunoglobulin-superfamily members associated with HLA-C and HLA-B recognition by human natural killer cells. Science 268: 405-408. [Abstract/Free Full Text]
  5. D’Andrea, A., C. Chang, K. Franz-Bacon, T. McClanahan, J. H. Phillips, L. L. Lanier. 1995. Molecular cloning of NKB1: a natural killer cell receptor for HLA-B allotypes. J. Immunol. 155: 2306-2310. [Abstract]
  6. Pende, D., R. Biassoni, C. Cantoni, S. Verdiani, M. Falco, C. di Donato, L. Accame, C. Bottino, A. Moretta, L. Moretta. 1996. The natural killer cell receptor specific for HLA-A allotypes: a novel member of the p58/p70 family of inhibitory receptors that is characterized by three immunoglobulin-like domains and is expressed as a 140-kD disulphide-linked dimer. J. Exp. Med. 184: 505-518. [Abstract/Free Full Text]
  7. Wagtmann, N., S. Rajagopalan, C. C. Winter, M. Peruzzi, E. O. Long. 1995. Killer cell inhibitory receptors specific for HLA-C and HLA-B identified by direct binding and by functional transfer. Immunity 3: 801-809. [Medline]
  8. Artavanis-Tsakonas, K., K. Eleme, K. L. McQueen, N. W. Cheng, P. Parham, D. M. Davis, E. M. Riley. 2003. Activation of a subset of human NK cells upon contact with Plasmodium falciparum-infected erythrocytes. J. Immunol. 171: 5396-5405. [Abstract/Free Full Text]
  9. Khakoo, S. I., C. L. Thio, M. P. Martin, C. R. Brooks, X. Gao, J. Astemborski, J. Cheng, J. J. Goedert, D. Vlahov, M. Hilgartner, et al 2004. HLA and NK cell inhibitory receptor genes in resolving hepatitis C virus infection. Science 305: 872-874. [Abstract/Free Full Text]
  10. Martin, M. P., X. Gao, J. H. Lee, G. W. Nelson, R. Detels, J. J. Goedert, S. Buchbinder, K. Hoots, D. Vlahov, J. Trowsdale, et al 2002. Epistatic interaction between KIR3DS1 and HLA-B delays the progression to AIDS. Nat. Genet. 31: 429-434. [Medline]
  11. Yen, J. H., B. E. Moore, T. Nakajima, D. Scholl, D. J. Schaid, C. M. Weyand, J. J. Goronzy. 2001. Major histocompatibility complex class I-recognizing receptors are disease risk genes in rheumatoid arthritis. J. Exp. Med. 193: 1159-1167. [Abstract/Free Full Text]
  12. van der Slik, A. R., B. P. Koeleman, W. Verduijn, G. J. Bruining, B. O. Roep, M. J. Giphart. 2003. KIR in type 1 diabetes: disparate distribution of activating and inhibitory natural killer cell receptors in patients versus HLA-matched control subjects. Diabetes 52: 2639-2642. [Abstract/Free Full Text]
  13. Suzuki, Y., Y. Hamamoto, Y. Ogasawara, K. Ishikawa, Y. Yoshikawa, T. Sasazuki, M. Muto. 2004. Genetic polymorphisms of killer cell immunoglobulin-like receptors are associated with susceptibility to psoriasis vulgaris. J. Invest. Dermatol. 122: 1133-1136. [Medline]
  14. Nelson, G. W., M. P. Martin, D. Gladman, J. Wade, J. Trowsdale, M. Carrington. 2004. Heterozygote advantage in autoimmune disease: hierarchy of protection/susceptibility conferred by HLA and killer Ig-like receptor combinations in psoriatic arthritis. J. Immunol. 173: 4273-4276. [Abstract/Free Full Text]
  15. Momot, T., S. Koch, N. Hunzelmann, T. Krieg, K. Ulbricht, R. E. Schmidt, T. Witte. 2004. Association of killer cell immunoglobulin-like receptors with scleroderma. Arthritis Rheum. 50: 1561-1565. [Medline]
  16. Luszczek, W., M. Manczak, M. Cislo, P. Nockowski, A. Wisniewski, M. Jasek, P. Kusnierczyk. 2004. Gene for the activating natural killer cell receptor, KIR2DS1, is associated with susceptibility to psoriasis vulgaris. Hum. Immunol. 65: 758-766. [Medline]
  17. Martin, M. P., G. Nelson, J. H. Lee, F. Pellett, X. Gao, J. Wade, M. J. Wilson, J. Trowsdale, D. Gladman, M. Carrington. 2002. Susceptibility to psoriatic arthritis: influence of activating killer Ig-like receptor genes in the absence of specific HLA-C alleles. J. Immunol. 169: 2818-2822. [Abstract/Free Full Text]
  18. Hiby, S. E., J. J. Walker, K. M. O’Shaughnessy, C. W. Redman, M. Carrington, J. Trowsdale, A. Moffett. 2004. Combinations of maternal KIR and fetal HLA-C genes influence the risk of preeclampsia and reproductive success. J. Exp. Med. 200: 957-965. [Abstract/Free Full Text]
  19. Varla-Leftherioti, M., M. Spyropoulou-Vlachou, T. Keramitsoglou, M. Papadimitropoulos, C. Tsekoura, O. Graphou, C. Papadopoulou, M. Gerondi, C. Stavropoulos-Giokas. 2005. Lack of the appropriate natural killer cell inhibitory receptors in women with spontaneous abortion. Hum. Immunol. 66: 65-71. [Medline]
  20. Hsu, K. C., X. R. Liu, A. Selvakumar, E. Mickelson, R. J. O’Reilly, B. Dupont. 2002. Killer Ig-like receptor haplotype analysis by gene content: evidence for genomic diversity with a minimum of six basic framework haplotypes, each with multiple subsets. J. Immunol. 169: 5118-5129. [Abstract/Free Full Text]
  21. Martin, A. M., J. K. Kulski, S. Gaudieri, C. S. Witt, E. M. Freitas, J. Trowsdale, F. T. Christiansen. 2004. Comparative genomic analysis, diversity and evolution of two KIR haplotypes A and B. Gene 335: 121-131. [Medline]
  22. Norman, P. J., C. V. Carrington, M. Byng, L. D. Maxwell, M. D. Curran, H. A. Stephens, D. Chandanayingyong, D. H. Verity, K. Hameed, D. D. Ramdath, R. W. Vaughan. 2002. Natural killer cell immunoglobulin-like receptor (KIR) locus profiles in African and South Asian populations. Genes Immun. 3: 86-95. [Medline]
  23. Shilling, H. G., L. A. Guethlein, N. W. Cheng, C. M. Gardiner, R. Rodriguez, D. Tyan, P. Parham. 2002. Allelic polymorphism synergizes with variable gene content to individualize human KIR genotype. J. Immunol. 168: 2307-2315. [Abstract/Free Full Text]
  24. Uhrberg, M., N. M. Valiante, B. P. Shum, H. G. Shilling, K. Lienert-Weidenbach, B. Corliss, D. Tyan, L. L. Lanier, P. Parham. 1997. Human diversity in killer cell inhibitory receptor genes. Immunity 7: 753-763. [Medline]
  25. Wilson, M. J., M. Torkar, A. Haude, S. Milne, T. Jones, D. Sheer, S. Beck, J. Trowsdale. 2000. Plasticity in the organization and sequences of human KIR/ILT gene families. Proc. Natl. Acad. Sci. USA 97: 4778-4783. [Abstract/Free Full Text]
  26. Yawata, M., N. Yawata, L. Abi-Rached, P. Parham. 2002. Variation within the human killer cell immunoglobulin-like receptor (KIR) gene family. Crit. Rev. Immunol. 22: 463-482. [Medline]
  27. Sambrook, J. G., A. Bashirova, H. Andersen, M. Piatak, G. S. Vernikos, P. Coggill, J. D. Lifson, M. Carrington, S. Beck. 2006. Identification of the ancestral killer immunoglobulin-like receptor gene in primates. BMC Genomics 7: 209[Medline]
  28. Guethlein, L. A., L. R. Flodin, E. J. Adams, P. Parham. 2002. NK cell receptors of the orangutan (Pongo pygmaeus): a pivotal species for tracking the coevolution of killer cell Ig-like receptors with MHC-C. J. Immunol. 169: 220-229. [Abstract/Free Full Text]
  29. Hershberger, K. L., J. Kurian, B. T. Korber, N. L. Letvin. 2005. Killer cell immunoglobulin-like receptors (KIR) of the African-origin sabaeus monkey: evidence for recombination events in the evolution of KIR. Eur. J. Immunol. 35: 922-935. [Medline]
  30. Hershberger, K. L., R. Shyam, A. Miura, N. L. Letvin. 2001. Diversity of the killer cell Ig-like receptors of rhesus monkeys. J. Immunol. 166: 4380-4390. [Abstract/Free Full Text]
  31. Khakoo, S. I., R. Rajalingam, B. P. Shum, K. Weidenbach, L. Flodin, D. G. Muir, F. Canavez, S. L. Cooper, N. M. Valiante, L. L. Lanier, P. Parham. 2000. Rapid evolution of NK cell receptor systems demonstrated by comparison of chimpanzees and humans. Immunity 12: 687-698. [Medline]
  32. Rajalingam, R., P. Parham, L. Abi-Rached. 2004. Domain shuffling has been the main mechanism forming new hominoid killer cell Ig-like receptors. J. Immunol. 172: 356-369. [Abstract/Free Full Text]
  33. Adams, E. J., P. Parham. 2001. Species-specific evolution of MHC class I genes in the higher primates. Immunol. Rev. 183: 41-64. [Medline]
  34. Hoelsbrekken, S. E., O. Nylenna, P. C. Saether, I. O. Slettedal, J. C. Ryan, S. Fossum, E. Dissen. 2003. Cutting edge: molecular cloning of a killer cell Ig-like receptor in the mouse and rat. J. Immunol. 170: 2259-2263. [Abstract/Free Full Text]
  35. Welch, A. Y., M. Kasahara, L. M. Spain. 2003. Identification of the mouse killer immunoglobulin-like receptor-like (Kirl) gene family mapping to chromosome X. Immunogenetics 54: 782-790. [Medline]
  36. Anderson, S. K., J. R. Ortaldo, D. W. McVicar. 2001. The ever-expanding Ly49 gene family: repertoire and signaling. Immunol. Rev. 181: 79-89. [Medline]
  37. George, T. C., L. H. Mason, J. R. Ortaldo, V. Kumar, M. Bennett. 1999. Positive recognition of MHC class I molecules by the Ly49D receptor of murine NK cells. J. Immunol. 162: 2035-2043. [Abstract/Free Full Text]
  38. Held, W., J. Roland, D. H. Raulet. 1995. Allelic exclusion of Ly49-family genes encoding class I MHC-specific receptors on NK cells. Nature 376: 355-358. [Medline]
  39. Yokoyama, W. M., B. F. Plougastel. 2003. Immune functions encoded by the natural killer gene complex. Nat. Rev. Immunol. 3: 304-316. [Medline]
  40. Valiante, N. M., M. Uhrberg, H. G. Shilling, K. Lienert-Weidenbach, K. L. Arnett, A. D’Andrea, J. H. Phillips, L. L. Lanier, P. Parham. 1997. Functionally and structurally distinct NK cell receptor repertoires in the peripheral blood of two human donors. Immunity 7: 739-751. [Medline]
  41. Boyington, J. C., P. D. Sun. 2002. A structural perspective on MHC class I recognition by killer cell immunoglobulin-like receptors. Mol. Immunol. 38: 1007-1021. [Medline]
  42. Winter, C. C., J. E. Gumperz, P. Parham, E. O. Long, N. Wagtmann. 1998. Direct binding and functional transfer of NK cell inhibitory receptors reveal novel patterns of HLA-C allotype recognition. J. Immunol. 161: 571-577. [Abstract/Free Full Text]
  43. Winter, C. C., E. O. Long. 1997. A single amino acid in the p58 killer cell inhibitory receptor controls the ability of natural killer cells to discriminate between the two groups of HLA-C allotypes. J. Immunol. 158: 4026-4028. [Abstract]
  44. Adams, E. J., G. Thomson, P. Parham. 1999. Evidence for an HLA-C-like locus in the orangutan Pongo pygmaeus. Immunogenetics 49: 865-871. [Medline]
  45. Staden, R., K. F. Beal, J. K. Bonfield. 2000. The Staden package, 1998. Methods Mol. Biol. 132: 115-130. [Medline]
  46. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, D. G. Higgins. 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25: 4876-4882. [Abstract/Free Full Text]
  47. Kumar, S., K. Tamura, M. Nei. 2004. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform. 5: 150-163. [Abstract/Free Full Text]
  48. Swofford, D. L.. 2003. PAUP*: phylogenetic analysis using parsimony (*and other methods), version 4 Sinauer Associates, Sunderland.
  49. Yang, Z.. 1997. PAML: a program package for phylogenetic analysis by maximum likelihood. Comput. Appl. Biosci. 13: 555-556. [Free Full Text]
  50. Thorne, J. L., H. Kishino. 2002. Divergence time and evolutionary rate estimation with multilocus data. Syst. Biol. 51: 689-702. [Medline]
  51. Thorne, J. L., H. Kishino, I. S. Painter. 1998. Estimating the rate of evolution of the rate of molecular evolution. Mol. Biol. Evol. 15: 1647-1657. [Abstract]
  52. Abi-Rached, L., P. Parham. 2005. Natural selection drives recurrent formation of activating killer cell immunoglobulin-like receptor and Ly49 from inhibitory homologues. J. Exp. Med. 201: 1319-1332. [Abstract/Free Full Text]
  53. Brunet, M., F. Guy, D. Pilbeam, H. T. Mackaye, A. Likius, D. Ahounta, A. Beauvilain, C. Blondel, H. Bocherens, J. R. Boisserie, et al 2002. A new hominid from the Upper Miocene of Chad, Central Africa. Nature 418: 145-151.
  54. Kumar, S., A. Filipski, V. Swarna, A. Walker, S. B. Hedges. 2005. Placing confidence limits on the molecular age of the human-chimpanzee divergence. Proc. Natl. Acad. Sci. USA 102: 18842-18847. [Abstract/Free Full Text]
  55. Jurka, J., V. V. Kapitonov, A. Pavlicek, P. Klonowski, O. Kohany, J. Walichiewicz. 2005. Repbase update, a database of eukaryotic repetitive elements. Cytogenet. Genome Res. 110: 462-467. [Medline]
  56. Sambrook, J. G., A. Bashirova, S. Palmer, S. Sims, J. Trowsdale, L. Abi-Rached, P. Parham, M. Carrington, S. Beck. 2005. Single haplotype analysis demonstrates rapid evolution of the killer immunoglobulin-like receptor (KIR) loci in primates. Genome Res. 15: 25-35. [Abstract/Free Full Text]
  57. Sambrook, J. G., H. Sehra, P. Coggill, S. Humphray, S. Palmer, S. Sims, H. H. Takamatsu, T. Wileman, A. L. Archibald, S. Beck. 2006. Identification of a single killer immunoglobulin-like receptor (KIR) gene in the porcine leukocyte receptor complex on chromosome 6q. Immunogenetics 58: 481-486. [Medline]
  58. Martin, A. M., E. M. Freitas, C. S. Witt, F. T. Christiansen. 2000. The genomic organization and evolution of the natural killer immunoglobulin-like receptor (KIR) gene cluster. Immunogenetics 51: 268-280. [Medline]
  59. Chaimanee, Y., D. Jolly, M. Benammi, P. Tafforeau, D. Duzer, I. Moussa, J. J. Jaeger. 2003. A Middle Miocene hominoid from Thailand and orangutan origins. Nature 422: 61-65.
  60. Chaimanee, Y., V. Suteethorn, P. Jintasakul, C. Vidthayanon, B. Marandat, J. J. Jaeger. 2004. A new orang-utan relative from the Late Miocene of Thailand. Nature 427: 439-441. [Medline]
  61. Boyington, J. C., S. A. Motyka, P. Schuck, A. G. Brooks, P. D. Sun. 2000. Crystal structure of an NK cell immunoglobulin-like receptor in complex with its class I MHC ligand. Nature 405: 537-543. [Medline]
  62. Fan, Q. R., E. O. Long, D. C. Wiley. 2001. Crystal structure of the human natural killer cell inhibitory receptor KIR2DL1-HLA-Cw4 complex. Nat. Immunol. 2: 452-460. [Medline]
  63. Rajagopalan, S., E. O. Long. 1999. A human histocompatibility leukocyte antigen (HLA)-G-specific receptor expressed on all natural killer cells. J. Exp. Med. 189: 1093-1100. [Abstract/Free Full Text]
  64. Arnaiz-Villena, A., P. Morales, E. Gomez-Casado, M. J. Castro, P. Varela, R. Rojo-Amigo, J. Martinez-Laso. 1999. Evolution of MHC-G in primates: a different kind of molecule for each group of species. J. Reprod. Immunol. 43: 111-125. [Medline]
  65. Langat, D. K., J. S. Hunt. 2002. Do nonhuman primates comprise appropriate experimental models for studying the function of human leukocyte antigen-G?. Biol. Reprod. 67: 1367-1374. [Abstract/Free Full Text]
  66. Castro, M. J., P. Morales, J. Martinez-Laso, L. Allende, R. Rojo-Amigo, M. Gonzalez-Hevilla, P. Varela, J. Moscoso, M. Garcia-Berciano, A. Arnaiz-Villena. 2000. Lack of MHC-G4 and soluble (G5, G6) isoforms in the higher primates, Pongidae. Hum. Immunol. 61: 1164-1168. [Medline]
  67. Rajagopalan, S., Y. T. Bryceson, S. P. Kuppusamy, D. E. Geraghty, A. van der Meer, I. Joosten, E. O. Long. 2006. Activation of NK cells by an endocytosed receptor for soluble HLA-G. PLoS Biol. 4: e9[Medline]



This article has been cited by other articles:


Home page
 Genome ResHome page
P. J. Norman, L. Abi-Rached, K. Gendzekhadze, J. A. Hammond, A. K. Moesta, D. Sharma, T. Graef, K. L. McQueen, L. A. Guethlein, C. V.F. Carrington, et al.
Meiotic recombination generates rich diversity in NK cell receptor genes, alleles, and haplotypes
Genome Res., May 1, 2009; 19(5): 757 - 769.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. K. Moesta, L. Abi-Rached, P. J. Norman, and P. Parham
Chimpanzees Use More Varied Receptors and Ligands Than Humans for Inhibitory Killer Cell Ig-Like Receptor Recognition of the MHC-C1 and MHC-C2 Epitopes
J. Immunol., March 15, 2009; 182(6): 3628 - 3637.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. K. Moesta, P. J. Norman, M. Yawata, N. Yawata, M. Gleimer, and P. Parham
Synergistic Polymorphism at Two Positions Distal to the Ligand-Binding Site Makes KIR2DL2 a Stronger Receptor for HLA-C Than KIR2DL3
J. Immunol., March 15, 2008; 180(6): 3969 - 3979.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Guethlein, L. A.
Right arrow Articles by Parham, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Guethlein, L. A.
Right arrow Articles by Parham, P.
Right arrowPubmed/NCBI databases
*Protein
*Substance via MeSH

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS