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Keywords:

  • NK receptor;
  • KIR;
  • Non-human primate;
  • Recombination;
  • Molecular evolution

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Killer cell immunoglobulin (Ig)-like receptors (KIR) were characterized in the West African sabaeus monkey (Chlorocebus sabaeus) to elucidate the mechanism by which diversity evolves in this family of molecules. Complementary DNA encoding four forms of KIR molecules, including KIR3DL, KIR2DL4, KIR2DL5, and KIR3DH forms, were identified in two unrelated sabaeus monkeys. A novel hybrid form showing features found in both KIR2DL5 and KIR3DH was also identified. Both the KIR3DL and KIR3DH forms from the sabaeus monkey were considerably more polymorphic than any KIR form identified in great apes or humans. The polymorphic residues of the three Ig-like domains were frequently located in structural loops, indicating that point mutations have occurred in these regions. The three Ig-like domains of the KIR3D forms of six primate species were found to have different patterns of clustering in phylogenetic trees, suggesting that each Ig-like domain has a distinct phylogenetic history. This variation in relationships suggests that repeated recombination events have occurred between the Ig-like domains during the evolution of the KIR family in primates. Recombination between individual Ig-like domains, in addition to point mutations, provides a mechanism for generating the diversity of the KIR genes.

Abbreviations:
cDNA:

Complementary DNA

Cs:

Chlorocebus sabaeus

IgSF:

Ig superfamily

ITIM:

Immunoreceptor tyrosine-based inhibition motif

Mm:

Macaca mulatta

Pt:

Pan troglodytes

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Genetic polymorphism is central to the ability of the vertebrate immune system to recognize a diversity of microbial antigens. Members of the Ig superfamily (IgSF) of molecules subsume the role of providing this recognition capability. The evolution of this large family of proteins is believed to have been the result of gene duplication followed by diversification by mutation 1. Most of the Ig-like domains of the IgSF are encoded by a single exon, providing a discrete unit for expansion of the IgSF members by such genetic mechanisms as exon shuffling and unequal crossing over.

Killer cell Ig-like receptors (KIR) are members of the IgSF 2, 3. Expressed in humans on NK cells and a subset of T lymphocytes, they interact directly with classical MHC class I molecules 4. In humans, KIR molecules are encoded by a multigene family whose members are located in close proximity to one another on chromosome 19 5. Like other IgSF members, the KIR family is believed to have originated by duplication of a single gene, followed by additional duplication events to expand the gene number 6, 7. Unequal crossing over appears to have subsequently resulted in KIR gene deletions, expansions, and formation of hybrids 6, 8, 9. Studies of the KIR family of humans and several great ape species demonstrate the frequent appearance of novel sequences, as well as the absence of other KIR genes 6, 1012. In fact, few KIR genes are conserved between humans and common chimpanzees, the majority of genes having undergone species-specific divergence 10.

Our recent characterization of KIR sequences of Indian-origin rhesus monkeys (Macaca mulatta; Mm-KIR) revealed significant differences between the rhesus monkey KIR forms and those of humans and apes 13. Up to five highly polymorphic Mm-KIR3DL sequences were found in each rhesus monkey studied. While KIR3DS sequences were not detected, several sequences of the novel KIR3D form, Mm-KIR3DH, were identified. These polymorphic Mm-KIR3DH sequences encode a molecule with three Ig-like domains, but with truncated cytoplasmic tails due to the deletion of the first cytoplasmic exon (exon 8). The Mm-KIR3DH form resembles the activating forms of human KIR, with a transmembrane domain containing an arginine and a short cytoplasmic tail that does not contain inhibitory motifs.

To elucidate further the molecular evolution of the primate KIR molecules, we characterized the KIR forms of a second Old World monkey, the sabaeus monkey. The sabaeus monkey is a West African species (Chlorocebus sabaeus or Cercopithecus sabaeus) or subspecies (C. aethiops sabaeus) of a group of monkeys referred to as African green monkeys 14, 15. Although the taxonomy of this monkey continues to be debated, we will use sabaeus monkey as the common name and the most recent genus and species designation Chlorocebus sabaeus in this report 15, 16. Using PCR amplification of complementary DNA (cDNA) to characterize the KIR sequences, we identified the sabaeus monkey homologs for the KIR3DL, KIR2DL4, KIR2DL5, and Mm-KIR3DH forms in two animals. Phylogenetic analysis of the KIR3D forms suggests that repeated recombination events have occurred between the Ig-like domains during the evolution of these sequences.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Isolation of cDNA encoding sabaeus monkey KIR molecules

cDNA clones encoding sabaeus monkey KIR forms were obtained by RT-PCR using primers based on the conserved sequences of human or rhesus KIR forms. Clones were characterized from two unrelated sabaeus monkeys using RNA isolated from PBMC. All sabaeus monkey KIR sequences have been given the prefix “Cs” to indicate Chlorocebus sabaeus, followed by the KIR form (Fig. 1). The Cs-KIR1D molecule will be described in another report.

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Figure 1. KIR forms identified in two sabaeus monkeys. (A) Schematic representation of the structures of sabaeus monkey KIR molecules. The R denotes an arginine in the transmembrane domain. ITIM (I/V/L/SxYxxL/V) are indicated in the cytoplasmic domain. (B) An X indicates sequences detected in an individual animal. Only putative splice variant sequences were detected for the form marked with an asterisk (*). Cs-KIR3DL and Cs-KIR3DH sequences were classified into types based on amino acid sequences with differences greater than 2%.

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Identification of cDNA encoding Cs-KIR2DL4

A sabaeus monkey cDNA sequence encoding a KIR form with D0 and D2 domains, an arginine in the transmembrane domain, and a long cytoplasmic tail was identified using a KIR2DL4-specific forward primer (Fig. 2). This sequence is likely from an ortholog of the human and rhesus monkey KIR2DL4 genes and is therefore designated Cs-KIR2DL4. Only one type of Cs-KIR2DL4 sequence was identified, which was identical in both monkeys (Fig. 1B). Like the rhesus monkey KIR2DL4 sequences, Cs-KIR2DL4 has two immunoreceptor tyrosine-based inhibitory motifs (ITIM) in the cytoplasmic domain.

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Figure 2. Predicted amino acid sequence and structural domains of sabaeus monkey KIR2DL4. Dashes (-) indicate undetermined amino acids of Cs-KIR2DL4 prior to the forward PCR primer, tildes (∼) indicate amino acids encoded by the PCR primer used to amplify the cDNA, and periods (.) indicate identity with the Cs-KIR2DL4 sequence. Inverted triangles (▾) indicate cysteine residues that likely form intradomain disulfide bridges. Double bars indicate a predicted beta-bulge formed by a WSXS/PS or L/VSAPS motif. Potential N-linked glycosylation sites are boxed. Vertical bars indicate the location of the sequence corresponding to exon 7 in human KIR3DL genes. Plus signs (+) indicate the locations of characteristic features of Mm-KIR2DL4.2 13. An asterisk (*) marks the characteristic arginine residue of all KIR2DL4 sequences in the transmembrane domain. ITIM are indicated by bars above the motifs. The sequences of KIR2DL4 orthologs from rhesus monkeys, common chimpanzees, and humans are shown for comparison 10, 13, 38. The cysteines found in the ITIM-like motifs of the human and common chimpanzee KIR2DL4 sequences are indicated in reverse text.

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Identification of cDNA encoding Cs-KIR3DL

Sabaeus monkey cDNA sequences encoding a KIR form with three Ig-like domains and a long cytoplasmic tail that has two ITIM were identified (Fig. 3). This family of molecules is designated as Cs-KIR3DL. Four types of Cs-KIR3DL were identified in the PBMC of two unrelated sabaeus monkeys, with no type shared between the two animals (Fig. 1B). The amino acid differences between the various types are not localized to any particular region of the receptor sequences, but greater differences are seen in the Ig-like domains and the cytoplasmic region. The Cs-KIR3DL sequences possess 73–76% amino acid identity to human KIR3DL1 and KIR3DL2 and 86–91% amino acid identity to rhesus monkey KIR3DL types 13, 17. Like Mm-KIR3DL, Cs-KIR3DL sequences have a D0 domain that is three amino acids longer than those of human KIR3DL forms and also a transmembrane domain that is one amino acid shorter than those of the human KIR3DL molecules. On the basis of sequence comparison, none of the sabaeus monkey KIR3DL sequences can be designated as orthologous to any human, ape or rhesus monkey KIR3DL sequences.

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Figure 3. Predicted amino acid sequences and structural domains of sabaeus monkey KIR3DL and KIR3DH forms. Tildes (∼), periods (.), inverted triangles (▾), boxed sequences, double bars, asterisk (*), vertical bars, and single horizontal bars indicate the same features as in Fig. 2. Dashes (-) indicating the absence of amino acids are used to aid in alignment. The rhesus monkey KIR3DL and KIR3DH types, Mm-KIR3DL7 and Mm-KIR3DH1 are included for comparison 13. (A) Signal sequence and Ig-like domains. Putative beta-strands and loops of the Ig-like folds are indicated above the consensus sequences of the D1 and D2 domains 22. Loop residues that have been identified to make contact with MHC class I by analysis of human KIR2DL/HLA-C co-crystal structures are underlined in the consensus sequence 20, 21. The three-amino acid deletion in the D0 domain of Cs-KIR3DH4 is highlighted in yellow. The two regions with high rates of non-synonymous substitutions are highlighted in green. (B) Stem, transmembrane, and cytoplasmic domains. A filled circle (•) indicates the cysteine in the stem domain of KIR3DH sequences. The sequence corresponding to exon 8 is missing in the Cs-KIR3DH molecules.

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Identification of cDNA encoding Cs-KIR3DH

cDNA sequences were identified encoding a sabaeus monkey KIR form with three Ig-like domains and a short cytoplasmic tail (Fig. 3). These sequences share a number of features with the rhesus monkey KIR3DH form and were therefore designated Cs-KIR3DH. The transmembrane region of the Cs-KIR3DH form has an arginine and an extra amino acid in the transmembrane region not found in KIR3DL. These changes are characteristic of both the KIR2DL4 and Mm-KIR3DH forms. The stem region of the Cs-KIR3DH form contains a cysteine, which is also found in several of the Mm-KIR3DH types, but not in KIR2DL4. This cysteine is not in the same location as in the human KIR3DL2 molecule, but may allow formation of disulfide-linked dimers 18, 19. Like Mm-KIR3DH, Cs-KIR3DH sequences also lack the exon 8 region, which encodes a portion of the cytoplasmic domain. This 53-nucleotide deletion causes a frameshift and early termination of the protein. Consequently, the Cs-KIR3DH molecules are truncated and have no ITIM.

Four distinct types of Cs-KIR3DH were found, with Cs-KIR3DH1 expressed in the PBMC of both animals evaluated (Fig. 1B). The detection of three Cs-KIR3DH sequences in monkey U040 indicates that there is more than one KIR3DH locus in this animal. Cs-KIR3DH1 and Cs-KIR3DH2 have very similar Ig-like domains, but divergent transmembrane domains. Unlike the other Cs-KIR3DH and Cs-KIR3DL sequences, the D0 domain of Cs-KIR3DH4 lacks the same three amino acids as the human KIR3D sequences. The Cs-KIR3DH sequences have only 84–88% amino acid identity to rhesus monkey KIR3DH types and, based on sequence comparison, do not appear to be orthologous to these rhesus monkey KIR3DH sequences.

Variability in the Ig-like domains of Cs-KIR3D forms

The amino acid substitutions in the three Ig-like domains of Cs-KIR3DL and Cs-KIR3DH sequences suggest that the extracellular regions of these different sequences may recognize distinct MHC class I alleles. The primary sequences of the Cs-KIR3D forms can be overlaid into the crystal structure of the homologous D1 and D2 domains of the inhibitory human KIR2DL1 and KIR2DL2 molecules in complex with their HLA-C ligands 2022. In these crystal structures, six loops linking the beta-strands in the Ig-like domains contain the residues that interact with the HLA-C ligands. The variable residues of the Cs-KIR3D sequences frequently appear in regions homologous to these loops. In particular, the sequences homologous to loops 3, 5 and 6 contain a number of non-conservative residue substitutions, at positions where KIR2DL1 and/or KIR2DL2 have been shown to contact HLA-C (underlined in Fig. 3A).

When an analysis of nucleotide substitutions was performed on the sabaeus monkey KIR3D sequences, two regions containing a concentration of codons with high rates of non-silent substitutions were identified (Fig. 4A). Sharp local increases in the number of non-synonymous changes were detected in the loop 3 region of the D1 domain and in the loop 5 region of the D2 domain. In the first region, seven of the amino acids located in positions 186–196 are under strong positive selection (Figs. 3A, 4A). Residue 186 is frequently an asparagine, forming the typical N-linked glycosylation signal NXS/T, and may be glycosylated. Fewer non-synonymous mutations were observed in the loop 3 region of rhesus monkey KIR3D sequences, suggesting that the contribution of the interaction of loop 3 with the ligand may differ in these two species. The other region under strong selective pressure is located at positions 250–252 in loop 5 of the D2 domain (Figs. 3A, 4A). By tracking substitutions through the phylogenetic trees, non-synonymous substitutions within a primate species in KIR genes could be found to be propagated either through recombination or through base substitution (data not shown, see results below).

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Figure 4. Location of the polymorphic positions in the Ig-like domains of the Cs-KIR3D forms. (A) Analysis of non-synonymous and synonymous substitutions in the three Ig-like domains of the sabaeus monkey, human, and rhesus monkey KIR3D sequences, with the substitutions plotted cumulatively for each codon, moving from left to right along the protein. This plot allows one to visualize regional mutation hot spots within a set of protein sequences (very steep regions) and highly conserved regions (very flat regions). The seven amino acids shown here in capital letters using the single amino acid code, NStTsDlAGtY (positions 186–196), of the D1 domain and the amino acids QNS (positions 250–252) of the D2 domain are under strong positive selection. There is a potential for approximately 25% of all substitutions to be silent; therefore, if there were no constraints on mutations and if they occurred at random (a dN/dS ratio of 1), non-synonymous substitutions would accumulate three times faster than synonymous ones. For this reason, the slope for non-synonymous substitutions is steeper on the plots. Thirteen rhesus monkey sequences, Mm-KIR3DL 1–11, 17, and 18, and Mm-KIR3DH1–4 and the three human reference sequences for KIR3DL1 (NM_013289.1), KIR3DL2 (NM_006737.2), and KIR3DS1 (NM_014514.1) were used in this analysis 13, 17. (B) Ribbon diagram of the D1 and D2 domains of human KIR2DL1 indicating (in purple) the amino acid positions of Cs-KIR3DL3 that vary from the consensus sequence of Fig. 3A. The side chains shown are those of the human KIR2DL1 sequence 22. (C) Ribbon diagram indicating the amino acid positions in the D1 and D2 domains of Cs-KIR3DH3 that vary from the consensus sequence of Fig. 3A.

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Comparisons of representative KIR3D sequences from humans, common chimpanzees, pygmy chimpanzees, orangutans, rhesus monkeys and sabaeus monkeys revealed that the longest stretches of amino acids that are perfectly preserved across all KIR3D sequences are two regions of the D2 domain (data not shown) 2, 6, 10, 11, 13, 17. These two regions of five amino acids, positions 245–249 (LSCSS) and positions 294–296 (YRCFG), are found in the B and F beta-strands of the D2 domain, respectively. These regions include the cysteine residues that form the intradomain disulfide bridges and therefore may be structurally constrained 22.

The allospecificity of human KIR2DL is determined by dimorphisms of residue 44 in loop 2 of the D1 domain of these molecules, which directly interacts with residue 80 of HLA-C 23, 24. The conservation at the analogous position to residue 44 (position 163) in the D1 domain of all four Cs-KIR3DL sequences and three of the Cs-KIR3DH sequences indicates that allospecific recognition may not occur at this location in Cs-KIR3D forms. However, the Cs-KIR3D sequences do show variation in additional loops predicted from the human KIR2DL crystal structures that are not involved in the interaction with the HLA-C ligands. Amino acids of Cs-KIR3DL3 that vary from the consensus sequence shown in Fig. 3 include three amino acids in the loop located in the D2 domain between the beta-strands C’ and E. These amino acid changes are indicated on the crystal structure of human KIR2DL1 (Fig. 4B). In addition to amino acid differences in the putative ligand contact loops (loops 2, 4–6), Cs-KIR3DH3 varies from the consensus in loops formed between the B and C beta-strands and the F and G beta-strands of the D1 domain, and the C’-E loop of the D2 domain (Fig. 4C). These additional loops may also contribute to ligand interactions of Cs-KIR3D molecules. The C’-E loop is particularly intriguing because variability of this loop is also seen in human KIR sequences 25. Moreover, a significant rotation of this loop was found in the crystal structure of the activating receptor KIR2DS2, despite sequence conservation 26.

Additionally, a substantial number of residue substitutions were observed in the D0 domains of the Cs-KIR3D sequences, which may contribute to ligand recognition. In fact, studies of deletion mutants of human KIR3DL1 have demonstrated that all three intact Ig-like domains are required for binding to its HLA-B ligand 27. Of the Cs-KIR3D forms, the D0 domain of Cs-KIR3DH4 is unique, and, like the human KIR3D forms, it has three fewer amino acids in the region predicted to form the C beta-strand. This deletion of three amino acids results in the loss of two predicted N-linked glycosylation sites in this domain (Fig. 3A). A two-amino acid deletion in the predicted C' beta-strand was previously shown to enhance interaction of Pan troglodytes (Pt)-KIR3DL1/2 with HLA-B ligands 28. The insertion/deletion of the three amino acids into/from the D0 domain and the alteration in glycosylation of Cs-KIR3D molecules may also affect ligand interaction.

Identification of cDNA encoding a Cs-KIR2DL5 splice variant and the novel form Cs-KIR3DH/vL5

cDNA sequences were identified that encode two unique KIR forms, Cs-KIR2DL5sv1 and Cs-KIR3DH/vL5 (Fig. 5). These forms possess a D0 domain that is strikingly divergent from Cs-KIR3DL, Cs-KIR2DL4, and Cs-KIR3DH, but which has 98–100% identity to the D0 domain of rhesus monkey KIR2DL5 (Mm-KIR2DL5) types. Like Cs-KIR2DL4, this D0 domain is three amino acids shorter than that of Cs-KIR3DL and most Cs-KIR3DH types. These forms also possess an extra amino acid in the transmembrane domain that is found in Cs-KIR2DL4 and Cs-KIR3DH, but not in Cs-KIR3DL.

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Figure 5. Predicted amino acid sequences and structural domains of sabaeus monkey KIR2DL5sv1 and KIR3DH/svL5. Tildes (∼), periods (.), inverted triangles (▾), dashes (-), boxed sequences, double bars, putative beta-strands, loops of the Ig-like folds, MHC class I contact residues from human KIR2DL/HLA-C co-crystal structures, filled circle (•), asterisk (*), vertical bars, and single horizontal bars are indicated as in Fig. 3. The rhesus monkey sequence Mm-KIR2DL5.2 and sabaeus monkey sequences Cs-KIR3DH3 and Cs-KIR3DL2 are included for comparison 13.

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The first form is designated Cs-KIR2DL5sv1 because of the similarity of its sequence to that of Mm-KIR2DL5 and its apparent lack of both D1 and D2 domains. It may represent a splice variant of an as yet unidentified full-length Cs-KIR2DL5 sequence encoding a D0–D2 KIR form. In fact, a human splice variant of KIR2DL5 without a D2 domain has been previously reported 29. The transmembrane domain of Cs-KIR2DL5sv1 is identical to Mm-KIR2DL5.2, one of the two Mm-KIR2DL5 types identified in rhesus monkeys. However, Cs-KIR2DL5sv1 has a cytoplasmic region with greater identity to the sabaeus monkey KIR3DL sequences than Mm-KIR2DL5 sequences. It appears that a recombination event occurred at the second cytoplasmic exon (exon 9) with a Cs-KIR3DL sequence. The cytoplasmic tail of the Cs-KIR2DL5sv1 molecule is of the same length as that of Cs-KIR3DL and is 31 amino acids shorter than that of Mm-KIR2DL5.

The second novel KIR form is designated Cs-KIR3DH/vL5 because of the similarity of its sequence to that of Mm-KIR2DL5 and Cs-KIR2DL5sv1 in the D0 region, and because of its homology to Cs-KIR3DH in the D2, stem, transmembrane and cytoplasmic regions. Numerous amino acid changes not shared by any other sabaeus monkey KIR molecule are found in the D1 domain of Cs-KIR3DH/vL5, most notably in the loop 2 and loop 3 regions. In particular, residue 160, which is analogous to the loop 2 residue 44 of the human KIR2DL forms, is the nonpolar amino acid isoleucine, not a polar amino acid like that found in all Cs-KIR3DL and Cs-KIR3DH molecules. Like in Cs-KIR3DH sequences, there is a cysteine in the stem region and an arginine in the transmembrane region of Cs-KIR3DH/vL5. Also as seen in Cs-KIR3DH sequences, Cs-KIR3DH/vL5 lacks the sequence corresponding to exon 8 and, accordingly, should terminate early without a substantial cytoplasmic domain.

Phylogenetic analysis of KIR3D sequences

To examine the evolutionary history of the sabaeus monkey KIR3D molecules, Cs-KIR3DL and Cs-KIR3DH sequences were compared to KIR3D sequences previously obtained from humans and four other primate species: common chimpanzees, pygmy chimpanzees, orangutans, and rhesus monkeys 2, 6, 10, 11, 13, 17. Initially, extensive analysis using Simplot and bootscanning was performed 30. Multiple crossover points were apparent between the different exons of the Ig-like domains, suggesting recombination was occurring between these exons (Fig. 6). Because the genomic sequence of the KIR3D genes is not available for any of the nonhuman primate species, the precise boundary of the recombination breakpoints within the introns was not determined. While phylogenetic analyses of primate KIR3D forms have previously been performed, they have generally been done using the full coding sequences, or with all three of the Ig-like domains analyzed together 10, 11, 31. In order to examine more precisely the evolutionary relationships of the primate KIR3D sequences, phylogenetic trees were constructed using the maximum-likelihood method with bootstrapping, treating each of the three Ig-like domains as a distinct entity (Fig. 7). Branching patterns consistent with those shown in Fig. 7 were also observed using neighbor-joining trees (data not shown).

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Figure 6. Bootscan analysis provides evidence of recombination events. (A) Bootscan analysis of the Mm-KIR3DL5 sequence as the query against Mm-KIR3DL2, Mm-KIR3DL18, and Pt-KIR3DL6 sequences was performed with a sliding-window size of 200 nucleotides, a step size of 20 base pairs, and 100 bootstrap replicates. (B) Bootscan analysis using the Cs-KIR3DH3 sequence as the query against Cs-KIR3DL3, Mm-KIR3DL7, and Mm-KIR3DL2 sequences.

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Figure 7. Phylogenetic relationships between the three Ig-like domains of the KIR3D forms. Maximum-likelihood trees were constructed for each of the KIR3D Ig-like domains. Bootstrap values were determined for 100 replications of the maximum-likelihood tree, and values >50% are shown. The scale bar represents 10 mutations per 100 nucleotide sequence positions. The KIR3D sequences were examined from the following species: human (no prefix), common chimpanzee (Pt-), pygmy chimpanzee (Pp-), orangutan (Popy-), rhesus monkey (Mm-), and sabaeus monkey (Cs-) 6, 10, 11, 13, 17. The human KIR reference sequences for KIR3DL1 (NM_013289.1), KIR3DL2 (NM_006737.2), and KIR3DS1 (NM_014514.1) were used. The outgroup was a sabaeus and rhesus monkey KIR3D consensus sequence. Examples of rearranging associations of Ig-like domains between KIR3D sequences from the rhesus or sabaeus monkey sequences are highlighted in color to assist in tracking. Trees are drawn vertically to better illustrate potential recombination events.

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In each of the trees, the human, common chimpanzee, pygmy chimpanzee, and orangutan KIR3D sequences form one distinct lineage, and the rhesus and sabaeus monkey sequences form another. Within these lineages, there was not a clear intraspecies association of sequences, nor was there an association of KIR3D forms based on potential receptor signaling. Most strikingly, many of the KIR3D sequences form relationships with different sequences in each tree, indicating that each Ig-like domain has a different phylogenetic history. Like the simplots and bootscans, the shifting of associations between KIR3D sequences determined for Ig-like domains suggests that recombination between Ig-like domains occurred during the evolution of these KIR sequences. For many of the sabaeus and rhesus monkey KIR3D sequences, only low bootstrap support was obtained for the phylogenetic relationships. In contrast, the relationship between Cs-KIR3DH1 and Cs-KIR3DH2 shows at least 90% support in all three Ig-like domains, consistent with their high amino acid identity.

We were able to detect examples of rearrangement between Ig-like domains of KIR3D sequences from an individual primate species for both the rhesus and sabaeus monkey. The rhesus monkey KIR3DL5 (Mm-KIR3DL5) sequence clusters with Mm-KIR3DL18 in the D0 domain with a bootstrap support of 82%. Yet, in the D1 and D2 Ig-like domains, the Mm-KIR3DL5 sequence forms a cluster with the Mm-KIR3DL1, 2, 3, and 4 sequences. These associations are supported by bootstrap values of 94% and 100% in the D1 and D2 domains, respectively. These alterations in relationship were confirmed using bootscanning, and the crossover point was detected in the region of the exon boundary between the D1 and D2 domains (Fig. 6A). Importantly, the Mm-KIR3DL5 sequence was identified from a different rhesus monkey than Mm-KIR3DL1–4 and Mm-KIR3DL18 sequences, indicating that these findings do not represent a PCR-generated artifact. Although the relationships do not have high bootstrap support, a similar example is seen with the sabaeus monkey Cs-KIR3DL2 and Cs-KIR3DL3 sequences. These sabaeus KIR3DL sequences associate in both the D0 and D2 domains, but not the D1 domain, indicating that the D1 domains of these sequences are more divergent (Fig. 7).

In an example of rearrangement of individual Ig-like domains that illustrates recombination between KIRDH and KIRDL genes, the Mm-KIR3DLl0 sequence associates with Mm-KIR3DL9 in the D2 domain with 100% bootstrap support, and a trend for a relationship between these molecules is shown in the D1 domain tree. However, in the D0 Ig-like domain, Mm-KIR3DL10 is most closely related to a cluster of rhesus monkey KIR sequences that includes Mm-KIR3DL (5, 7, and 18) and Mm-KIR3DH (1 and 2). This D0 cluster containing both KIR3DH and KIR3DL sequences has a bootstrap value of 77%. The differing relationships of these KIR3DL and KIR3DH sequences indicates that shuffling of Ig-like domains has occurred during the evolution of these two forms of immune receptors. These recombinations between Ig-like domains occurred in addition to the distinct recombination events at the transmembrane exon that most likely generated the KIR3DH form.

An additional example of changing associations between KIR3DL and KIR3DH forms involves sequences found in the two different monkey species (Figs. 6B, 7). The sabaeus monkey KIR3DH3 sequence (Cs-KIR3DH3) is associated with Cs-KIR3DL3 in the D0 domain, but forms a cluster with the rhesus monkey sequences Mm-KIR3DL7 and Mm-KIR3DL18 in the D1 domain. Although these associations did not have high bootstrap values using maximum-likelihood analysis, there is 88% bootstrap support for the clustering of these sabaeus and rhesus monkey sequences in the D1 domain using the neighbor-joining method (data not shown). The change in association of Cs-KIR3DH3 with Cs-KIR3DL3 in the D0 domain to Mm-KIR3DL7 in the D1 domain was confirmed by bootscanning (Fig. 6B). Interspecies phylogenetic clustering suggests that an Ig-like domain that existed prior to speciation gave rise to the Ig-like domains currently identified in these two primate lineages. While the Ig-like domain was then maintained in both species, further recombination events occurred to alter the relationship with the other two Ig-like domains, as well as the type of KIR3D form in which this Ig-like domain is found.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Knowledge of the sabaeus monkey KIR forms that have been identified in this study sheds considerable light on the evolutionary history of the primate KIR forms. There is considerable divergence of the KIR sequences of humans from those of common chimpanzees since the evolutionary divergence of these species approximately 6 million years ago 10. Even greater differences in KIR forms exist between sequences identified in two Old World monkey species (Cercopithecoidea) and those of Hominoidea superfamily members. This is consistent with our knowledge that the human and Cercopithecoidea lineages diverged approximately 25 million years ago 15, 32. The ancestral lineages of the sabaeus and rhesus monkeys are estimated to have diverged approximately 9 million years ago 15. Examination of the KIR families from these two monkey species reveals that the KIR forms of the sabaeus monkey are more similar to those of the rhesus monkey than any of the other species that have been characterized 6, 1012, 19, 33.

The detection of the KIR3DH form in the sabaeus monkey suggests a shared evolutionary history of the KIR family in Old World monkey species. This receptor was previously described in the rhesus monkey, but not in apes. It has features of both KIR3DL and KIR2DL4 molecules and seems to have originated from an intergenic recombination between these two KIR loci. It appears that convergent evolution has generated different KIR forms with short cytoplasmic tails. Since there is no evidence for the typical activating or short-tailed receptors in the sabaeus monkey, Cs-KIR3DH may act as an activating form of KIR in this species.

Gene duplication and repeated recombination of Ig-like domains provide mechanisms for generating both species-specific diversity and the intraspecies polymorphism of KIR molecules. That the lineage of KIR3D sequences in Old World monkey species is distinct from the KIR3D forms of apes and humans indicates that many of these gene duplication events have occurred in the last 25 million years (Fig. 7) 17. The evolutionary mechanism of diversification by exon shuffling is implicit in KIR sequence data from apes previously reported by Parham and his colleagues. An orangutan KIR3DL sequence provided evidence for clear inter-locus recombination involving a single exon encoding an Ig-like domain 11. Moreover, the recombination of KIR3D sequences within the Ig-like domains that occurred following the divergence of the common and pygmy chimpanzees appeared to follow exon boundaries 6, and this is confirmed here (Fig. 7). In fact, a previous analysis of KIR sequences for possible recombination events showed a strong correlation between the detected breakpoint locations and protein domain junctions 12. Our phylogenetic analysis of the individual Ig-like domains of primate KIR3D sequences revealed that recombination events, such as exon shuffling, between KIR Ig-like domains appear to have been common during the evolution of this receptor family (Fig. 7).

The rapidly evolving classical MHC class I molecules are also members of the IgSF. Considerable effort has been focused on understanding the generation of the allelic diversity of the highly polymorphic MHC class I molecules. While recombination can occur, it seems that selection of mutations within the peptide binding region of the molecules most likely drives the polymorphism of this family of genes 34. As illustrated in Fig. 7, each of the three exons encoding the Ig-like domains of the KIR3D genes appears to have undergone multiple recombination events. Thus, the extent of the recombination between the KIR3D molecules is greater than that seen with MHC class I molecules.

The Cs-KIR3DH/vL5 sequence most likely originated from an intergenic recombination event between Cs-KIR2DL5 and Cs-KIR3DH genes. The D0 domain of Cs-KIR3DH/vL5 is nearly identical to that of Cs-KIR2DL5sv1, but this sequence has an arginine-containing transmembrane domain and an early terminated cytoplasmic domain like Cs-KIR3DH sequences. Cs-KIR3DH/vL5 has a highly divergent D1 domain, while its D2 domain shares homology to that of Cs-KIR3DL2. Further recombination events and point mutations could be responsible for the unique amino acid changes observed in this sequence. The recombination between various KIR forms blurs the distinctions between KIR alleles and KIR loci.

The discovery of hybrid forms, such as Cs-KIR3DH and Cs-KIR3DH/vL5, emphasizes the apparent plasticity of the KIR gene family. However, the sabaeus monkey also has certain KIR forms that have been found in all species studied thus far. These include the genes encoding KIR with three Ig-like domains and the genes encoding KIR2DL4. The presence of sequences homologous to these genes in the sabaeus monkey also supports the idea that KIR2DL4 and KIR3DL compose framework loci 6. These loci can provide the background upon which the haplotypic diversity of the KIR family is created via intergenic recombination, duplication, and deletion. Polymorphic diversity is a prominent feature of molecules such as Cs-KIR3DL and Cs-KIR3DH and could have been generated by both point mutations and recombination. The array of receptors found in the sabaeus monkey suggests that KIR diversity is an evolutionary response to the variation of the MHC class I system, the presumed ligand for at least several of the KIR forms. The co-evolution of the KIR and MHC class I families, most probably in response to pathogens, appears to have selected for KIR forms and sequences unique to particular Old World monkey species 10, 13.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

Cloning of KIR cDNA

EDTA-anti-coagulated peripheral blood samples were obtained from two unrelated sabaeus monkeys, numbers U040 and U097. PBMC were isolated by centrifugation over Ficoll-Hypaque (Ficopaque; Pharmacia, Piscataway, NJ). Total RNA was extracted from approximately 3×106 PBMC using the RNeasy Mini kit with a DNase treatment step (Qiagen, Chatsworth, CA). First-strand cDNA was generated using avian myeloblastosis virus reverse transcriptase and an oligo-dT primer (Promega, Madison, WI).

KIR sequences homologous to human KIR3DL and rhesus monkey KIR3DH were amplified by PCR from both monkeys using the human KIR-specific Ig3Up and Ig3Down primers 18. The novel form Cs-KIR3DH/vL5 was also amplified from monkey U097 with this primer pair. Reactions (50 µl) included 2 µl cDNA, 1× PCR Buffer II, 2 mM MgCl2, 200 µM of each of the four dNTP, 50 pmol of each of the two primers, and 2.5 U AmpliTaq Gold (Perkin-Elmer, Foster City, CA). Samples were incubated for 10 min at 94°C to activate the AmpliTaq Gold. This was followed by 30–35 cycles of denaturation at 94°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 90 s, and a final extension at 72°C for 10 min.

Cs-KIR3DL and KIR sequences homologous to rhesus monkey KIR2DL5 (putative splice variant of Cs-KIR2DL5) were amplified from monkey U040 using the human KIR-specific primers F23 and R1441 2. The PCR conditions employed were the same as described above, except that the annealing temperature was 54°C. Cs-KIR3DH sequences were also amplified from both sabaeus monkeys using the reverse primer Cs3DHR (5′-CGTGGGCARGAGACAGTGA-3′), which takes advantage of a 4-nucleotide deletion in the KIR3DH sequences of both rhesus and sabaeus monkeys, in conjunction with the F23 primer at an annealing temperature of 57°C.

The sabaeus monkey KIR sequence homologous to KIR2DL4 was amplified from both monkeys using the primers KIR2DL4F1 and Ig3Down and an annealing temperature of 58°C. The KIR2DL4-specific forward primer KIR2DL4F1 (5′-GTCAGGACAAGCCCTTCTGC-3′) was designed based on the conserved sequences of other primate KIR2DL4 homologs, taking advantage of a 20-nucleotide conserved stretch of the D0 domain of these sequences that is divergent from other KIR sequences.

PCR products were analyzed on 1% agarose gels, and larger and smaller products were separately excised. Purified PCR products (MinElute Gel Extraction kit, Qiagen) were ligated into the p-GEM-T Easy vector (Promega) for sequencing (Applied Biosystems, Foster City, CA). Complete DNA sequences of each clone were assembled using SeqMan (DNASTAR, Madison, WI). Approximately 60–150 clones were completely sequenced for each primer pair. The sequences were submitted to GenBank and assigned the following accession numbers: AY648235 (Cs-KIR2DL4), AY483137–AY483140 (Cs-KIR3DL), AY483141–AY483143 and AY648234 (Cs-KIR3DH), AY648238 (Cs-KIR2DL5sv), and AY648239 (Cs-KIR3DH/L5). The structure of the extracellular domains of human KIR2DL1 (PDB accession 1NKR) was modeled using Swiss-PDB viewer (http://us.expasy.org/spdbv/).

Phylogenetic analysis

Non-synonymous and synonymous substitutions were calculated using SNAP (http://hiv-web.lanl.gov) 35. The synonymous and non-synonymous changes were tracked without correction (pS and pN) for each codon 36.

Simplots and bootscanning were performed using the Ig-like domains of the sabaeus and rhesus monkey KIR3D sequences (http://sray.med.som.jhmi.edu/RaySoft/SimPlot/) 30. For this analysis, neighbor joining with Kimura's two-parameter model and a Ts/Tv ratio of 2.0 for the distances was used for the phylogenic reconstruction. Bootscan analysis was performed with a sliding-window size of 200 nucleotides, a step size of 20 base pairs, and 100 bootstrap replicates.

Maximum-likelihood trees for each of the KIR3D Ig-like domains were generated using the optimum model selected based on the Akaike Information Criteria as implemented in MODELTEST 37, used in conjunction with PAUP program (http://paup.csit.fsu.edu/, PAUP version 4.0 by David Swofford, Sinauer Associates, Inc., Sunderland, MA; http://bioag.byu.edu/zoology/crandall_lab/dposada.html). Different models were selected for the D0, D1 and D2 domains: HKY + gamma, alpha =0.4613; TVMef + gamma, alpha =0.8032; and TrN + gamma, alpha =0.4324, respectively. Bootstrap values were determined for 100 replications of the maximum-likelihood tree to determine confidence in each node of the tree.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements

This work was supported by a grant from the National Institutes of Health to N.L.L. K.L.H. was supported by a National Research Service training grant. We thank Dr. V. Hirsch for providing the blood samples from the sabaeus monkeys. Nucleotide sequence data reported are available in the GenBank database under the accession numbers: AY483137–AY483143 and AY648234–AY648239.

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