Two Orangutan Species Have Evolved Different KIR Alleles and Haplotypes

Abstract

The immune and reproductive functions of human NK cells are regulated by interactions of the C1 and C2 epitopes of HLA-C with C1-specific and C2-specific lineage III killer cell Ig-like receptors (KIR). This rapidly evolving and diverse system of ligands and receptors is restricted to humans and great apes. In this context, the orangutan has particular relevance because it represents an evolutionary intermediate, one having the C1 epitope and corresponding KIR but lacking the C2 epitope. Through a combination of direct sequencing, KIR genotyping, and data mining from the Great Ape Genome Project, we characterized the KIR alleles and haplotypes for panels of 10 Bornean orangutans and 19 Sumatran orangutans. The orangutan KIR haplotypes have between 5 and 10 KIR genes. The seven orangutan lineage III KIR genes all locate to the centromeric region of the KIR locus, whereas their human counterparts also populate the telomeric region. One lineage III KIR gene is Bornean specific, one is Sumatran specific, and five are shared. Of 12 KIR gene-content haplotypes, 5 are Bornean specific, 5 are Sumatran specific, and 2 are shared. The haplotypes have different combinations of genes encoding activating and inhibitory C1 receptors that can be of higher or lower affinity. All haplotypes encode an inhibitory C1 receptor, but only some haplotypes encode an activating C1 receptor. Of 130 KIR alleles, 55 are Bornean specific, 65 are Sumatran specific, and 10 are shared.

Figure 1. KIR gene content of five orangutan BAC clones

Using orangutan KIR cDNA as probes, five clones (CH253-75E4, -76D24,-134A21, -272D24 and -418C19) were isolated from BAC library CH253 constructed from genomic DNA obtained from Segundo, a male orangutan and son of Susie. A) Shows the PCR primers used to determine presence or absence of the ILT10, KIR3DL3, KIR3D, and FCAR genes in each BAC clone. B) Under ‘PCR’ are shown the results of the PCR analysis. Dark gray shaded boxes denote a positive PCR amplification and presence of the gene in the BAC clone. Under ‘Sequencing’ are shown the results of a sequencing analysis that assessed the presence of lineage II KIR3D sequences. A gray-shaded box denotes presence of 3D-1, 3D-2 or 3D-3 sequence. Also listed under ‘Sequencing’ are the locations of the end sequences of the BAC clone compared to the human genome reference sequence. Under ‘Haplotype’ is given the haplotype to which each BAC clone corresponds: M denotes the maternal haplotype inherited from Susie and P the paternal haplotype. With completion of the KIR haplotype sequences, 3D-1 was seen to correspond to KIR3DS1*001, 3D-2 corresponded to KIR3DS1*002, and 3D-3 corresponded to KIR3DL1*001 on the M haplotype and to KIR3DL1*002 on the P haplotype. C) Shows the primer sequences, cycling parameters, expected product sizes and location of the primers used for genotyping of orangutan KIR.

Figure 2. Organization of genes and other elements in three orangutan KIR haplotypes

A) Shows the three orangutan KIR haplotypes (Pongo-H1 to H3) for which we determined complete nucleotide sequences. Like the human KIR locus, the orangutan KIR locus is flanked by LILR genes on one side, and by FCAR, NCR1 and NLRP7 genes on the other. Gene boxes are colored according to the lineage of the KIR gene: green for lineage I, yellow for lineage II inhibitory receptors, orange for lineage II activating receptors, blue for lineage III and purple for lineage V. This color code is also spelled out in the gene boxes of the Pongo-H1 haplotype. For the lineage III KIR genes, the boxes for genes encoding activating receptors have a lighter color than the boxes for those encoding inhibitory receptors. The boxes for genes flanking the KIR locus are shaded black. In haplotypes Pongo-H1 and Pongo-H2, the small, orange box flanking the LILRP1 gene denotes an inserted sequence that is unique to orangutan. The characteristics of this insertion are shown in B). B) The repetitive element and gene content of the ‘IN’ region were determined using RepeatMasker analysis of the region and BLAST searching of GenBank. Repeat elements are color-coded and the name of the most similar human element is shown. Arrows indicate the 5′ to 3′ direction of the element. In red is ponPygV1R-ps1924, a vomeronasal 1 receptor pseudogene that was found as part of the Orangutan Genome Sequencing project.

Figure 3. High-resolution orangutan KIR genotypes

Shown are KIR genotypes for 19 Sumatran (S) orangutans [A and B] and 10 Bornean (B) orangutans [C and D]. In addition, three Sumatran-Bornean hybrid orangutans (H) and one orangutan (CP81) of unknown provenance (U) were analyzed [E]. Also included are KIR types for the H1, H2 and H3 haplotype sequences [F]. The genotypes were determined either by typing [A, C, and D] or sequencing [B, E, and F]. A dark gray box denotes presence of a gene. Where known, allele names are given in orange characters for Sumatran orangutans (Poab) and blue characters for Bornean orangutans (Popy). Ambiguous typing is indicated by “/”.Presence of two allotypes is indicated by “+”. KIR2DL10/S10 typing discriminated activating and inhibitory forms, which are designated S and L, respectively. KIR2DL4 typing failed to resolve some allotypes; 002g includes *002, *003, *006, *00701, *00704, *008, *010, and 005g includes *004 and *005. ‘N’ in an allotype name indicates presence of a substitution predicted to abrogate expression. Under ‘Species’ S for Sumatran (Pongo abelli), B for Bornean (Pongo pygmaeus) and H for individuals who are hybrids of the two species ‘U’ denotes individuals for whom the species is unknown. Under 2DS15, ‘nd’ indicates that typing was ‘not done’ for the panel as the presence of the gene was discovered only through assembly of the sequence data from the GAGP dataset. Its absence in the Sumatran orangutans of the GAGP dataset was confirmed. On the right under ‘Haplotypes’ are given the two haplotypes that combine to form the individual's genotype. Of the orangutans analyzed here, 21 were from our panel (A, C and D), 2 were represented by the BAC libraries, and 10 are part of the GAGP dataset (22).

Figure 4. High-resolution orangutan KIR haplotypes

A) Eleven groups of KIR haplotypes (H1- H11) are distinguished by KIR gene content. Presence of a gene is indicated by a shaded box and absence by a white box. Framework genes are distinguished by dark gray boxes. Genes encoding functional lineage III receptors are indicated by blue shading, with light blue for activating KIR and dark blue for inhibitory KIR. All other genes that are present are represented by light gray shaded boxes. KIR3DL3 was present in all individuals and not included in this table as it was not resolved at the allele level in all haplotypes. Within a group the haplotypes are distinguished by their allele content. Allele names are given inside the gene boxes. Species designations and limitations in the typing are as described in the legend to Figure 3. The two columns to the right of the gene boxes give the occurrence of each allele-content KIR haplotype and each gene-content KIR haplotype in the dataset. All the orangutan KIR haplotypes are species specific. B) Haplotypes were grouped by the number of functional activating and inhibitory MHC-C receptors that they encoded and are displayed in the ascending order by number of receptors. The percentage of each category of haplotype is indicated in the two columns on the right of the table.

Figure 5. KIR2DS15 is a species-specific KIR of the Bornean orangutan

A) Shows a phylogenetic tree constructed from the coding sequences of 122 of the 130 known alleles for the orangutan KIR genes (alleles with only single exons sequenced were removed). KIR indicated by orange circles are from Sumatran orangutans, those indicated by blue circles are from Bornean orangutans, and those indicated by yellow circles are present in both orangutan species. The segments of the outer circle group the KIR according to their lineage: I, II, III, and V. In bold and highlighted is KIR2DS15, a specific gene of the Bornean orangutan that was not previously known. Two groups of KIR2DL11 alleles (2DL11*001g and 2DL11*002g) differ in the sequence encoding the cytoplasmic tail. Recombinant 3DL1 alleles that contain a segment of sequence derived from lineage III KIR are designated lin III rec. Bootstrap values greater than 50 at major nodes are shown on the tree. B) Shows the amino acid differences that distinguish the orangutan lineage III KIR proteins. For each of the eight functional KIR, as well as the encoded product of the KIRDP pseudogene (which lacks exons encoding the stem and transmembrane regions), a consensus sequence was made from all allotypes. These sequences are aligned against the consensus of the eight functional KIR.

Figure 6. KIR2DS15 has features that suggest it has lost or altered MHC binding affinity

A) Shows the position of the T52, K54, N72 motif that characterizes KIR2DS15. The position of the residues are colored on the 2DL2 structure (1EFX). The specificity determining residue 44 is colored orange and the three residues of the TKN motif (52, 54, and 72) are colored blue. Three views of the molecule are shown, side, end, and top. B) Shows all combinations of residues found for positions 44, 52, 54, and 72 in a dataset comprised of all known KIR sequences from macaque, orangutan, gorilla, chimpanzee, and human. Presence of the motif is indicated by a dark gray box in the leftmost grid, under ‘N’ are the total number of occurrences, the third table shows the protein motifs found for the four residues. Highlighted in yellow are residues found in the TKN motif of KIR2DS15. The gray shading next to the table indicates identical protein motifs that are encoded by different sequences. The DNA sequences encoding the motifs are shown in the rightmost table.

Figure 7. Allelic diversity of orangutan KIR

A) Shows the frequency of individual KIR genes in the panel of haplotypes. Haplotypes identical by descent are counted only once. Bars in the histogram are colored to indicate the dataset that was used to calculate the frequency, red (Sumatran WGS), dark blue (Bornean WGS), light orange (complete Sumatran), and light blue (complete Bornean). The WGS datasets are the haplotypes determined from the GAGP dataset and include all KIR genes. The complete dataset includes all haplotypes determined by typing or BAC sequencing, however, presence of KIR2DS15 and DP were not tested for all individuals so no value is reported. The shading of the bar for KIR2DS15 indicates that it is present in at least 50% of the haplotypes, but may be a component of all Bornean haplotypes. The ‘**’ for KIR2DS15 and KIRDP indicate that these were not part of the typing protocol used and their status in the orangutan panel is unknown. Only the results from the WGS individuals are shown for these genes. B) Shows the pairwise difference within and between species for alleles at each locus. The distribution of values is indicated by the bar and whisker graphs showing median, first quartile, and min/max values. Individual measures are indicated by dots. Orange is the within Sumatran calculation, blue is within Bornean, and gray between Sumatran and Bornean. C) Shows a table of the average pDist values (mean) and the standard deviation (S. D.) for each group of comparisons. D) Shows the number of alleles and allotypes for each gene broken down into those that are species-specific and those that are shared between species.