The expanded cattle KIR genes are orthologous to the conserved single-copy KIR3DX1 gene of primates
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- Guethlein, L.A., Abi-Rached, L., Hammond, J.A. et al. Immunogenetics (2007) 59: 517. doi:10.1007/s00251-007-0214-x
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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.
The killer cell immunoglobulin-like receptors (KIR) of primates are diverse, polymorphic major histocompatibility complex class I receptors expressed by subpopulations of natural killer (NK) cells and T cells (Uhrberg et al. 2001). The family of genes encoding these variable and rapidly evolving KIR is located in the leukocyte receptor complex (LRC), flanked on one side by the leukocyte immunoglobulin-like receptor (LILR) gene family, and on the other side by FCAR, the gene encoding the immunoglobulin A receptor (FcαRI) of myeloid cells (Wende et al. 1999; Wilson et al. 2000). An additional KIR gene, named KIR3DX1, was recently discovered in a location distinct from the other KIR. It is in the middle of the LILR gene family, between the two duplicated blocks of LILR genes and bounded by the LAIR2 and LILRA2 genes (Sambrook et al. 2006b). KIR3DX1 is conserved as a single-copy gene in four of the five primate species examined with an apparent species-specific duplication in the fifth. From sequencing KIR3DX1 cDNA from single human and chimpanzee individuals, Sambrook et al. 2006b identified limited polymorphism: with a single synonymous substitution between the two human alleles and five non-synonymous substitutions between the two chimpanzee alleles. KIR3DX1 is unique in not having the LTR33A/MLT1D element in intron 3 that is present in all other primate KIR (Martin et al. 2000).
KIR have been reported for several non-primate species. An analysis of horse cDNA provides evidence for a single EqcaKIR3DL gene (Takahashi et al. 2004). Genomic analysis showed that the pig has a single KIR gene (SuscKIR2DL1), placed between LILR and FCAR in the homologous position occupied by the expanded lineage of primate KIR (Sambrook et al. 2006a). SuscKIR2DL1 is of the same lineage as the expanded primate KIR and contains the LTR33A/MLT1D repeat in intron 3. This element also characterizes the rat KIR (Hoelsbrekken et al. 2003), which is present in the homologous location between LILR and FCAR, as well as the two mouse KIR, which are not in the corresponding location due to their transposition to the X chromosome (Hoelsbrekken et al. 2003; Welch et al. 2003). A search of GenBank revealed a single cat KIR gene that is flanked by LILR and FCAR and also contains the intron 3 element. This composite element comprises an LTR33A fragment interrupted by the insertion of a MLT1D element. Its presence in a wide range of mammalian KIR genes suggests that the insertion event occurred early in KIR evolution and that the inserted element is a useful lineage marker.
Outside of the primates, the only species reported to have an expanded KIR gene family is cattle, for which four functional KIR genes and one pseudogene were described from analysis of cDNA (McQueen et al. 2002; Storset et al. 2003). In addition, McQueen et al. looked for the presence of the intron 3 MLT1D/LTR33A repeat in the bovine KIR by polymerase chain reaction amplification of genomic DNA followed by cloning and sequencing of the introns 3. They described two clones that contained the MLT1D/LTR33A element but were unable to assign them to a locus. To assess the relationships of the cattle KIR to the two lineages of primate KIR, we searched the database from the cattle genome project (Bovine genome build 2.1 at http://www.ncbi.nlm.nih.gov/) for further KIR sequences. We identified 22 KIR-containing contigs: Nine contained one or more full-length KIR genes along with flanking sequences, 13 contained partial KIR gene sequences.
Figure 2a shows the results of the analysis for exon 3. In addition to showing the divergence of the two main KIR lineages, the tree defines four sub-lineages within the second lineage of cattle KIR. These relationships were similarly observed in the tree of exon 4 sequences (data not shown). The analyses of exons 1 + 2 and exon 6 also revealed the two main cattle KIR lineages but not the separation into sub-lineages, likely due to the small size of these exons. Analysis of exons 7–9, encoding the transmembrane and cytoplasmic tail of the KIR, also showed the lineages corresponding to the BotaKIR2DL1-related sequences vs all others (Fig. 2b). When the BotaKIR2DL1-related KIR were excluded from the analysis of exons 7–9, the resulting tree of the other lineage of cattle KIR showed two sub-lineages corresponding to the KIR having short and long cytoplasmic tails (Fig. 2c).
Phylogenetic trees of exon 5 of the BotaKIR, encoding the D2 domain, lacked the divergent lineages characteristic of the other exons. Comparing the BotaKIR, exon 5 sequences with exon 5 sequences of other mammalian KIR shows that the cattle sequences form a monophyletic group with very strong support. Disrupting this group, to cluster BotaKIR2DL1 with the other 3DL-lineage sequences and the remaining cattle KIR with the 3DX1-lineage sequences results in a tree topology with a markedly reduced parsimony score (Templeton test, α = 0.001). This supports the hypothesis that all of the cattle KIR exon 5 sequences share a unique common ancestor, likely due to a recombination that replaced the exon 5 sequence of one lineage with that of the other. Further analysis, including sequences derived from other species, will be necessary to resolve the direction of the recombination.
Groups 3–13 in Table 1 correspond to the 3DX1 lineage cattle KIR genes. As with the primate KIR3DX1, the introns 3 of these genes do not contain the LTR33A/MLT1D element. Thus, the variable lineage of functional KIR in cattle is the result of diversification of the 3DX1 lineage. This contrasts with the situation in primates, where the other KIR lineage expanded and KIR3DX1 remained a conserved single-copy gene.
Analysis of the gene order in those contigs containing multiple KIR genes provided preliminary mapping information. Contig NW_943617 contains the 3′ portion of BotaKIR3DL1 linked to the 5′ portion of BotaKIR2DL1 with an intergenic interval of approximately 2 kb, a distance similar to that observed in the primate KIR region (Wende et al. 1999; Wilson et al. 2000). BotaKIR3DL1p is linked to the BotaKIR2DL1-related gene fragment with a similar 2-kb intergenic interval in three separate contigs (NW_938467, NW_938583, and NW_942117). Two additional linkages were observed that had contiguous sequence for the intergenic region, group 12 to group 5 (BotaKIR3DS1) in NW_937716 and group 7 to group 9 in NW_941846. The intergenic interval of these latter two pairs was approximately 7 kb, much longer than that between BotaKIR3DL1 and BotaKIR2DL1.
Recombination appears to have changed the gene order in the cattle and primate LRCs. The linkage of BotaKIR3DL1 to BotaKIR2DL1 is different from the map positions observed for the two divergent lineages in primates, where KIR3DX1 is separated from the 3DL-lineage KIR containing the LTR33A/MLT1D element by genes of the LILR family (Sambrook et al. 2006b), a distance of approximately 180 kb. NW_001003629 provides evidence that cattle LILR are situated on the other side of the KIR genes compared to primates and other species.
So far, cattle have stood out as the only non-primate species in which the KIR genes have expanded through gene duplication and diversification. In this paper, we have shown that that cattle KIR comprise two lineages. The lineage that is related to the highly diversified primate KIR has limited diversity. In contrast, the highly diversified cattle KIR are related to primate KIR3DX1, a conserved single-copy gene for which the function is unknown.
The research described in this paper was supported by NIH grant AI024258 to P.P.