Evolutionary patterns of killer cell Ig-like receptor genes in Old World monkeys

Killer cell Ig-like receptors (KIRs) modulate the cytotoxic effects of Natural Killer cells. KIR genes are encoded in the Leucocyte Receptor Complex and are characterized by their high haplotypic diversity and polymorphism. The KIR system has been studied in only three species of Old World monkeys, the rhesus macaque, the cynomolgus macaque, and the sabaeus monkey, displaying a complexity rivaling that of hominids (human and apes). Here we analyzed bacterial artificial chromosome draft sequences spanning the KIR haplotype of three other Old World monkeys, the vervet monkey (Chlorocebus aethiops), the olive baboon (Papio anubis) and the colobus monkey (Colobus guereza). A total of 25 KIR gene models were identified in these species, predicted to encode receptors with 1, 2, and 3 extracellular Ig domains, all of them with long cytoplasmic domains having two putative ITIMs, although three had a positively charged residue in the transmembrane domain. Sequence and phylogenetic analyses showed that most Old World monkeys shared five classes of KIR loci: i) KIR2DL5/3DL20 in the most centromeric region, followed by ii) the single Ig domain-encoding locus KIR1D, iii) the pseudogene KIR2DP, iv) the conserved KIR2DL4, and v) the highly diversified KIR3DL/H loci in the telomeric half of the cluster. An exception to this pattern was the KIR haplotype of the colobus monkey that lacked the KIR1D, KIR2DP, and KIR2DL4 loci of the central region of the cluster. Thus, Old World monkeys display a broad spectrum of KIR haplotype variation that has been generated upon an ancestral haplotype architecture by gene duplication, gene deletion, and non-homologous recombination.     

Bimodal evolution of the killer cell Ig-like receptor (KIR) family in New World primates

The immunoglobulin-like receptor (KIR) gene family in New World primates (Platyrrhini) has been characterized only in the owl monkey (Aotus sp.). To gain a better understanding of the KIR system in Platyrrhini, we analyzed a KIR haplotype in Ateles geoffroyi, and sequenced KIR complementary DNAs (cDNAs) from other three Atelidae species, Ateles hybridus, Ateles belzebuth, and Lagothrix lagotricha. Atelidae expressed a variable set of activating and inhibitory KIRs that diversified independently from their Catarrhini counterparts. They had a unique mechanism to generate activating receptors from inhibitory ones, involving a single nucleotide deletion in exon 7 and a change in the donor splice site of intron 7. The A. geoffroyi haplotype contained at least six gene models including a pseudogene, two coding inhibitory receptors, and three coding activating receptors. The centromeric region was in a tail-to-tail orientation with respect to the telomeric region. The owl monkey KIR haplotype shared this organization, and in phylogenetic trees, the centromeric genes clustered together with those of A. geoffroyi, whereas their telomeric genes clustered independently. KIR cDNAs from the other Atelidae species conformed to this pattern. Signatures of positive selection were found in residues predicted to interact with the major histocompatibility complex. Such signatures, however, primarily explained variability between paralogous genes but not between alleles in a locus. Atelidae, therefore, has expanded the KIR family in a bimodal fashion, where an inverted centromeric region has remained relatively conserved and the telomeric region has diversified by a rapid process of gene duplication and divergence, likely favored by positive selection for ligand binding.     

Identification of the mouse killer immunoglobulin-like receptor-like (<Emphasis Type="Italic">Kirl</Emphasis>) gene family mapping to Chromosome X

Natural killer (NK) inhibitory receptors, which recognize major histocompatability complex (MHC) proteins in humans, are known as killer Ig-like receptors (KIRs) and are encoded by a multi-gene immunoglobulin (Ig) superfamily. In a screen for genes differentially expressed in the mouse thymus, we discovered the first close rodent homologue of the NK receptor KIR family, which we named KIR- Like (Kirl). KIRL1 shares 40% amino acid identity with primate KIR family members, with the majority of the homology contained within the Ig-like ectodomains. KIRL1 is more similar to the KIRs than to any other known member of the Ig domain-containing leukocyte receptor superfamily. This highly significant homology suggests that the KIR family did not arise independently in primates, as has been previously suggested, but rather evolved from a primordial gene already present in the common rodent/primate ancestor. KIRL1 lacks the cytoplasmic protein motifs that mediate inhibition in KIRs (immunoregulatory tyrosine inhibiting motif, ITIM); KIRL1 also lacks the transmembrane activation signature (a conserved K residue involved in association with the immunoregulatory tyrosine activating motif-containing DAP12 molecule) found in some KIRs. Nevertheless, we hypothesize that Kirl1 is functional, for the following reasons: (1) Kirl1 mRNA is expressed at high levels in immature thymocytes; (2) Kirl1 is regulated during thymocyte development; (3) KIRL1 protein is detected in thymus. We also show that the mouse genome contains a closely related, transcribed gene, which we name Kirl2. Kirl2 encodes a KIR-like molecule with three Ig-like domains and also lacks tyrosine-based immunoregulatory motifs in its cytoplasmic region.     

Diversity of the killer cell Ig-like receptors of rhesus monkeys

Because the killer cell Ig-like receptors (KIRs) have only been characterized in humans and chimpanzees, we do not have a full understanding of their evolutionary history. Therefore, cDNAs encoding the KIR molecules of five rhesus monkeys were characterized, and were found to differ from the KIR molecules identified in humans and chimpanzees. Whereas only one KIR2DL4 molecule is detected in humans and chimpanzees, two distinct KIR2DL4 homologues were identified in the monkeys. Although the two human KIR3DL molecules are limited in their polymorphism, the KIR3DL homologues in the monkeys were highly polymorphic. Up to five KIR3DL homologues were identified in each monkey that was studied, and eleven distinct KIR3DL molecules were detected in the five rhesus monkeys. Two novel families of KIR molecules were identified in the rhesus monkeys, KIR3DH and KIR1D. The KIR3DH molecules have three Ig domains, transmembrane domains homologous to KIR2DL4 molecules that contain an arginine, and short cytoplasmic domains. With these features, the KIR3DH molecules resemble the activating forms of the human KIR molecules. The KIR1D molecule encodes only one complete Ig domain before a frame-shift in the second Ig domain occurs, leading to early termination of the molecule. Multiple splice variants of KIR1D exist that encode at least one Ig domain, as well as transmembrane and cytoplasmic domains. The extensive diversity of the rhesus monkey KIR3DL homologues and the novel KIR3DH and KIR1D molecules suggests that the KIR family of molecules has evolved rapidly during the evolution of primates.     

Complexity in cattle <Emphasis Type="Italic">KIR</Emphasis> genes: transcription and genome analysis

Killer immunoglobulin (Ig)-like receptors (KIRs) are the major functional natural killer (NK) cell receptors in human. The presence of KIR genes has only recently been demonstrated in other (non-primate) species, and their expression, genomic arrangement, and function in these species have yet to be investigated. In this study, we describe the KIR gene family in cattle. KIR sequences were amplified from cDNA derived from four animals. Seventeen new sequences were identified in total. Some are alleles of two previously described genes, and the remainder are representative of at least four additional genes. These cDNA data, together with analysis of the cattle genome sequence, confirm that, as in humans, cattle have multiple inhibitory and activating KIR genes, with variable haplotype composition, and putative framework genes. In contrast to human, the majority of the cattle KIR genes encode three Ig-domain KIRs; most of the inhibitory genes encode only one immunoreceptor tyrosine-based inhibitory motif (ITIM), and the activating genes encode molecules with arginine rather than the more usual lysine in the transmembrane domain. A divergent gene, 2DL1, encodes a two Ig-domain KIR with an unusual D0-D2 structure, and a distinct signaling domain with two ITIMs. Similarity to pig and human two Ig-domain (D0-D2) KIRs suggest these may be more related to an ancestral gene than the other cattle KIR genes. Cattle have multiple NKG2A-related genes and at least one Ly49 gene; thus, the data presented here suggest that they have the potential to express more major histocompatibility complex-binding NK receptors than other species.     

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-C

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.     

Nature of allelic sequence polymorphism at the KIR3DL3 locus

KIR3DL3 is a framework gene of the Leukocyte Receptor Complex, present in all individuals and haplotypes analysed to date. We describe 17 novel KIR3DL3 alleles, including seven single nucleotide polymorphic (SNP) positions within the coding region. Sequence variation within introns included a VNTR within intron 1. As KIR3DL3 mRNA is known to be expressed in decidual NK cells, we investigated the impact of KIR3DL3 allelic variation on pre-eclampsia. No statistical difference in allele frequency or polymorphism was observed between pre-eclampsia patient and control cohorts. Linkage disequilibrium (LD) analysis of exonic SNPs suggested that recombination may be a mechanism of generating sequence diversity within KIR3DL3. A potential recombination hotspot was located within intron 5. A strong LD was detected between polymorphism in exon 6 of KIR3DL3 and the KIR gene −2DL3 or -2DS2 loci, which define the centromeric end of two main haplotypes (A and B) of the KIR cluster. Comparison of primate KIR sequences indicated that the Ig domains of KIR3DL3 are highly conserved between chimpanzee, gorilla and humans. Investigation of KIR3DL3 dN/dS ratios indicated a greater level of synonymous mutations consistent with purifying selection, although positive selection was detected acting on two sites within the stem region.Electronic supplementary material Supplementary material is available for this article at and is accessible for authorized users.     

Evolution of the human killer cell inhibitory receptor family

Phylogenetic analysis of different domains of human natural killer cell inhibitory receptors (KIR) implicated both intragenic duplication and deletion of exons and interlocus recombination in the evolution of these receptors. In phylogenies of the extracellular immunoglobulin (Ig) superfamily C2-set domains and of the pre-membrane (PM) domain, KIR receptors having two C2-set domains and those having three such domains tended to form separate clusters. However, the phylogenies of the transmembrane (TM) and cytoplasmic (CYT) domains showed quite different topologies, suggesting that major sites of interlocus recombination have been between exon 6 (encoding PM) and exon 7 (encoding TM) and between exon 7 and exons 8-9 (encoding CYT). Examination of the pattern of nucleotide substitution in the exons encoding Ig C2-set domains supported the hypothesis that positive Darwinian selection has acted to diversify the residues within these domains that are involved in contact with class I MHC molecules.     

The Structure of the Atypical Killer Cell Immunoglobulin-like Receptor,KIR2DL4

The engagement of natural killer cell immunoglobulin-like receptors (KIRs) with their target ligands, human leukocyte antigen (HLA) molecules, is a critical component of innate immunity. Structurally, KIRs typically have either two (D1-D2) or three (D0-D1-D2) extracellular immunoglobulin domains, with the D1 and D2 domain recognizing the α1 and α2 helices of HLA, respectively, whereas the D0 domain of the KIR3DLs binds a loop region flanking the α1 helix of the HLA molecule. KIR2DL4 is distinct from other KIRs (except KIR2DL5) in that it does not contain a D1 domain and instead has a D0-D2 arrangement. Functionally, KIR2DL4 is also atypical in that, unlike all other KIRs, KIR2DL4 has both activating and inhibitory signaling domains. Here, we determined the 2.8 Å crystal structure of the extracellular domains of KIR2DL4. Structurally, KIR2DL4 is reminiscent of other KIR2DL receptors, with the D0 and D2 adopting the C2-type immunoglobulin fold arranged with an acute elbow angle. However, KIR2DL4 self-associated via the D0 domain in a concentration-dependent manner and was observed as a tetramer in the crystal lattice by size exclusion chromatography, dynamic light scattering, analytical ultracentrifugation, and small angle x-ray scattering experiments. The assignment of residues in the D0 domain to forming the KIR2DL4 tetramer precludes an interaction with HLA akin to that observed for KIR3DL1. Accordingly, no interaction was observed to HLA by direct binding studies. Our data suggest that the unique functional properties of KIR2DL4 may be mediated by self-association of the receptor.     

KIR2DL5 can inhibit human NK cell activation via recruitment of Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2)

Human NK cells use class I MHC-binding inhibitory receptors, such as the killer cell Ig-like receptor (KIR) family, to discriminate between normal and abnormal cells. Some tumors and virus-infected cells down-regulate class I MHC and thereby become targets of NK cells. Substantial evidence indicates that the mechanism of KIR-mediated inhibition involves recruitment of the protein tyrosine phosphatases, Src homology 2-containing protein tyrosine phosphatase-1 (SHP-1) and SHP-2, to two phosphorylated cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). KIR2DL5 is a type II member of the KIR2D family with an atypical extracellular domain and an intracytoplasmic domain containing one typical ITIM and one atypical ITIM sequence. Although KIR2DL5 structure is expressed by approximately 50% of humans and is conserved among primate species, its function has not been determined. In the present study, we directly compared functional and biochemical properties of KIR2DL5, KIR3DL1 (a type I KIR with two ITIMs), and KIR2DL4 (the only other type II KIR, which has a single ITIM) in a human NK-like cell line. Our results show that KIR2DL5 is an inhibitory receptor that can recruit both SHP-1 and SHP-2, and its inhibitory capacity is more similar to that of the cytoplasmic domain of KIR2DL4 than KIR3DL1. Interestingly, inhibition of NK cell cytotoxicity by KIR2DL5 was blocked by dominant-negative SHP-2, but not dominant-negative SHP-1, whereas both dominant-negative phosphatases can block inhibition by KIR3DL1. Therefore, the cytoplasmic domains of type II KIRs (2DL4 and 2DL5) exhibit distinct inhibitory capacities when compared with type I KIRs (3DL1), due to alterations in the canonical ITIM sequences.     

The expanded cattle <Emphasis Type="Italic">KIR</Emphasis> genes are orthologous to the conserved single-copy <Emphasis Type="Italic">KIR3DX1</Emphasis> gene of primates

Cattle are the only non-primate species for which expansion of the killer cell immunoglobulin-like receptor (KIR) genes has been reported. We analyzed cattle KIR sequences to determine their relationship to the two divergent lineages of primate KIR: one comprising the KIR3DX1 gene of unknown function, the second comprising all other primate KIR genes, which encode variable major histocompatibility complex class I receptors. Phylogenetics and analysis of repetitive elements shows that cattle KIR subdivide into the same two lineages as primate KIR. Unlike the primates, the lineage of variable and likely functional cattle KIR corresponds to the KIR3DX1 lineage of primate KIR, whereas the variable lineage of primate KIR is represented in cattle by one KIR gene and a related gene fragment.     

Fine Mapping and in silico Isolation of the EUI1 Gene Controlling Upper Internode Elongation in Rice

Upper internode elongation in rice is an important agronomic trait. Well-known mutants with an elongated uppermost internode (eui) are important germplasms for developing unsheathed-panicle male-sterile lines in hybrid rice breeding. We finely mapped the eui1 gene and identified its candidate gene using in silico analysis based on previous research work and rice genomic sequence data. The rice eui1 gene was mapped to two overlapping BAC clones, OSJNBa0095J22 and OSJNBb0099O15, between the markers AC40 and AC46, that were 0.64 cM apart and spanned approximately 152 kb. A simple sequence repeat (SSR) marker AC41 that cosegregated with eui1 was located in an intron of a putative cytochrome P450-related gene. In silico analysis suggested that this encoded the cytochrome CYP714D1. Allelic sequencing confirmed that EUI1 corresponded to this P450 gene. A gamma ray-induced eui1 mutant carried a deletion in exon II of the EUI1 gene, and resulted in a frame-shift deletion that produced a truncated polypeptide. We conclude that the EUI1 gene controlling the upper internode elongation in rice is 9804 bp long, and comprises two exons and one intron. The length of the cDNA is 1931 bp containing a 1734 bp ORF, a 110 bp 5′-UTR and a 87 bp 3′-UTR. The ORF encodes an unknown 577 amino acid functional protein, that appears to be a member of the cytochrome P450 family. Hongli Ma, Shubiao Zhang: These authors contributed equally to this work     

Rapid expansion of killer cell immunoglobulin-like receptor genes in primates and their coevolution with MHC Class I genes

The gene family of killer cell immunoglobulin-like receptors (KIRs) in primates provides the first line of defense against virus infection and tumor transformation. Interacting with MHC class I molecules, KIRs can regulate the cytotoxic activity of natural killer (NK) cells and distinguish the tumor and virus infected cells from normal body cells. Phylogenetic analysis and comparison of domain structures identified three major groups of KIR genes (group I, II, and III genes). These groups of KIR genes, generated by a series of gene duplications, have acquired different MHC-binding specificity. Inference of ancestral KIR sequences suggested that the functional divergence of group I genes from group II genes occurred by positive selection at the MHC-binding sites after duplication. Our evolutionary study has shown that group I genes diverged from group II genes about 17 million years ago (Mya) apparently after separation of hominoids from Old World (OW) monkeys. Around the same time, gene duplication generating the class I MHC-C locus appears to have occurred. These findings suggest that KIR and MHC class I genes have coevolved as an interacting system. The KIR gene family has experienced a rapid expansion in primate species. The rate of expansion of this gene family seems to be one of the highest among all hominoid gene families. The KIR gene family is also subject to birth-and-death evolution.