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Departments of Structural Biology and Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Analysis and comparison of NK cell receptors for MHC class I first involved human and mouse, species for which there appears no orthology between the MHC class I genes. The results revealed remarkable difference in their NK cell receptors. The Ly49 lectin-like receptors that give mouse NK cells diverse receptors for polymorphic H-2 determinants (reviewed in Refs. 2 and 3) are represented by a single nonfunctional Ly49L gene in humans (4, 5). Conversely, the killer cell Ig-like receptors (KIR) that provide human NK cells with diverse receptors for polymorphic HLA class I determinants, are absent from mice. Common to human and mouse are the CD94:NKG2 form of lectin-like receptors that are specific for complexes of a nonclassical class I molecule (Qa1b in mouse, HLA-E in human) and hydrophobic peptides derived from the leader peptides of other MHC class I H chains (6, 7, 8).
To explore further this remarkable species diversity, we previously studied NKC and LRC receptors in the common chimpanzee (Pan troglodytes) (9, 10), a species which has orthologs for all the expressed human HLA class I genes (11). In comparison to human, the chimpanzee CD94 and NKG2 family members are structurally conserved as is the inhibitory class I specificity of the CD94:NKG2A receptor (9). Orthologs for each human CD94,NKG2 family member were identified, with the exception of NKG2C, for which there are two chimpanzee paralogs (10).
A quite different situation was observed for the KIR. A minority (three) of chimpanzee KIR are orthologous to human KIR, the majority having homologous relationships indicative of rapid evolution through gene duplication, deletion, and other mechanisms of recombination (9). Within the KIR specific for MHC-A and -B, species-specific divergence in structure correlated with differences in MHC class I specificity consistent with coevolution of MHC class I and KIR to maintain ligand-receptor interaction. In contrast, MHC-C-specific KIR with identical specificity in the two species had accumulated considerable structural divergence that could not simply be attributed to pressure for maintaining ligand-receptor interaction.
The study described here concentrates on another ape species,
the orangutan (Pongo pygmaeus). Being more divergent from
humans than the chimpanzee, the orangutan has an MHC in
which the class I genes have several defined differences
from their human counterparts (11). Whereas
Popy-E and -G are orthologous (12, 13), the Popy-A and -B genes may not be
orthologs of HLA-A and -B (14, 15).
Most important is that the Popy-C locus is only present on
50% of MHC haplotypes; and thus, a significant minority
of orangutans lack an MHC-C locus (14). To see
how these differences have impacted the evolution of NK cell receptors
in the higher primates, we have characterized orangutan NK cell
receptors.
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Materials and Methods |
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Orangutan peripheral blood samples were obtained from the Yerkes Regional Primate Center (Atlanta, GA). Nine orangutans were used for this study comprising seven males and two animals of unknown gender. This number included two parent:offspring pairs, Lokan:Loklok and Ayer:Jantan. Results from Lokan:Loklok are consistent and show segregation of a haplotype with a single 3DL gene and a haplotype with two 3DL genes. Neither of Ayer’s 3DL alleles was recovered from Jantan; however, only four clones were available for sequencing. One of the alleles carried by Jantan is present in two other genotypes with three 3DL sequences, so it is possible that Jantan also has three 3DL sequences, one of which was not recovered in the course of this study.
RNA and cDNA preparation
PBMC were isolated on Ficoll-Hypaque gradients (Amersham Pharmacia Biotech, Piscataway, NJ). For one individual (Allen), cultured cells were used as the source of RNA. PBMC (5 x 106) were cultured with irradiated RPMI 8866 cells (1 x 105) in IMDM with 10% BCS, 2% human AB serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and 50 U/ml of rIL-2 for 10 days. RNA was prepared using RNAzol (Tel-Test, Friendswood, TX) according to manufacturer’s instructions. Total RNA was used for cDNA preparation. Either avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI) or Moloney murine leukemia virus reverse transcriptase (Life Technologies, Rockville, MD) was used for generation of cDNA according to the manufacturer’s protocols.
PCR and cloning of NK receptor genes
Lectin-like receptors. A panel of five individuals was sampled in cloning the lectin-like receptors. Oligonucleotide primers based on conserved sequences of CD94, NKG2 family members or Ly49L were used in PCR amplification of cDNA to obtain products corresponding to the lectin-like receptor genes. The primers for CD94 and NKG2D were the same as described previously (10). For other members of the NKG2 family and Ly49L, new primers were designed. The primer pairs are as follows: for NKG2A, 5'-CTG CGG ACA GAA GAG TAC AGT-3' and 5'-GCC CGA CAC AAA TGC TAG GAT-3'; for NGK2CE, 5'-AGA AGT GAG TCT GGC CCA GGA-3' and 5'-TCA CCC ATG GAT GAT GAC TGC-3'; for NKG2F, 5'-ACC CAA AGA GGC AGC AAA GGA AAC T-3' and 5'-ATG CCA ACC CAT GAG GGA ACT G-3'; and for Ly49L, 5'-GGA TTT GAA TGC AAA ACT TA-3' and 5'-TTC CAT TTT CAC CTT TTC TT-3'. Amplification conditions for Ly49L were 94°C for 1 min, 30 cycles of 94°C for 30 s, 47°C for 30 s, 72°C for 1 min, followed by a final extension at 72°C for 10 min. For the other primer pairs, the amplification conditions were 94°C for 1 min, 30 cycles of 94°C for 15 s, 60°C for 15 s, 72°C for 1 min, followed by a final extension at 72°C for 10 min. Amplification products were cloned into the pCR4-TOPO vector using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA). Clones were analyzed by restriction digest and/or partial nucleotide sequencing using T3 and T7 primers, followed by complete sequencing of selected clones using dye terminator automated sequencing on an ABI377 sequencer (PE Applied Biosystems, Foster City, CA).
Killer cell Ig-like receptor
KIR sequences were amplified initially using primers that had successfully amplified KIR in chimpanzee and human (9, 16). A panel of nine individuals was sampled. A single 3' primer (NKR) was used in combination with one of two 5' primers: the 2IgF primer amplifies both KIR2D and KIR3D, whereas the 3Ig5' primer was originally developed to be KIR3D specific. The priming site for 3Ig5' is situated 3' of the site for the 2IgF primer. When sequence analysis of cDNA clones obtained with the 2IgF/NKR primers showed that orangutan KIR differ at several positions within the 3Ig5' priming site, a new Popy-3Ig5' primer was made which incorporated the differences (5'-CAC ACA TGG GTG GTC AGG GAC-3'). The Popy-3Ig5'/NKR primer combination also amplified Popy-KIR2DL4. The following conditions were used for the amplification, 94°C for 5 min, 30 cycles of 94°C for 30 s, 60°C for 30 s, 72°C for 2 min, followed by a final extension at 72°C for 7 min. Amplification products were subjected to agarose gel electrophoresis and bands of the appropriate size were purified and cloned and sequenced as described above. A minimum of 96 clones was examined from each individual orangutan.
Phylogenetic analysis
Sequences were manually aligned using the GCG Wisconsin Package (Wisconsin Package Version 10.2; Genetics Computer Group, Madison, WI). Codon-aligned sequences were submitted to the SNAP server for dN/dS analysis (hiv-web.lanl.gov/SNAP/WEBSNAP/SNAP.html) (17, 18). Neighbor joining trees were constructed using the PAUP (v4.0b8) program suite (19). Confidence in the branching of the tree was assessed by bootstrap analysis using 1000 replicates.
In this study, we use the term orthologous to describe genes derived from a common ancestor for which there is a single example in each of the daughter species. Paralogous refers to genes resulting from duplication within the common ancestor.
Popy-C motif analysis
Genomic DNA from a panel of 18 orangutans was analyzed for the presence of Popy-C as well as the identity of the KIR-binding motif present. First, a general amplification of a product corresponding to exons 2 and 3 of Popy-MHC class I loci was performed. The primers used were Exon2F (5'-AGG CTC CCA CTC CAT GAG GTA-3') and Exon3R (5'-CTT CCC GTT CTC CAG GTA TC-3') with the following conditions, 95°C for 5 min, 10 cycles of 95°C for 20 s, 62°C for 45 s, 72°C for 2 min, followed by a final extension at 72°C for 10 min. A second set of amplifications was performed on the product of the first PCR and typed for the presence of Popy-C as well as the identity of the motifs. Amplification conditions modified from the protocol described by Frohn et al. (20) were used. A common forward primer HLA-CF (5'-CGC CGC GAG TCC GAG AGG-3') was used in combination with three different reverse primers. These primers were as follows: specific for Popy-C, PopyCR (5'-GCG TAC TGG TTA TAC CCG CT-3'), specific for Asn80, NK1 (5'-GTT GTA GTA GCC GCG CAG T-3'), and specific for Lys80, NK2 (5'-GTT GTA GTA GCC GCG CAG G-3'). A 1/10 dilution of the product resulting from the first amplification was used with the following amplification conditions: 95°C for 1 min, 10 cycles of 95°C for 10 s, 67°C for 30 s, 72°C for 30 s, 20 cycles of 95°C for 10 s, 62°C for 30 s, 72°C for 30 s followed by a final extension at 72°C for 10 min. Products were visualized by agarose gel electrophoresis. The Popy-C amplification product from individuals not previously analyzed was cloned and sequenced as described above.
Accession numbers
GenBank accession numbers for the sequences described in this study are as follows: Popy-CD94 (AF470380-5), Popy-NKG2A (AF470391-7), Popy-NKG2CE (AF470398-402), Popy-NKG2D (AF470403-4), Popy-NKG2F (AF470405-6), Popy-Ly49L02 (AF470390), Popy-2DL (AF470358-9), Popy-2DS (AF470360-4), Popy-3DL (AF470365-74), Popy-3DSA (AF470375), Popy-2DL4 (AF470388-9), Popy-C (AF470376-9), and Popy-C intron 2 (AF470386-7).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A). All orangutan
sequences have been given the prefix Popy (P. pygmaeus)
followed by the name consistent with the family to which the sequence
was assigned. Alleles of the CD94, NKG2 family members could
be confidently assigned to single loci and were given numerical
designations. In the KIR family, where locus assignment was
ambiguous, sequences of each lineage were given letter designations. In
comparison to humans and chimpanzee, overall sequence similarity was
highest for the C-type lectin family and KIR lineage I. In contrast,
KIR lineages II and III exhibited considerable variation (Fig. 1B).
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Complementary DNA clones corresponding to CD94,
NKG2D, NKG2F, and NKG2A were
identified. Nucleotide differences from their human counterparts are
2.2, 3.4, 4.5, and 4.6%, respectively, consistent with an orthologous
relationship (Fig. 1
). Clones corresponding to a fourth member of the
orangutan NKG2 family are closely related to human
NKG2C and NKG2E, and on phylogenetic analysis was
found to be equidistant from them (Fig. 2). These relationships indicate that the
NKG2C and NKG2E genesare the products
from duplication of an ancestor resembling the orangutan gene, which we
have called Popy-NKG2CE.
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). The differences are particularly
evident in the region implicated in ligand binding (residues 221–41 in
Fig. 3) (21, 22, 23). In the rhesus monkey, this region of
MmNKG2A (24) is also more similar to human and chimpanzee
NKG2E, indicating that this sequence may more closely represent the
ancestral NKG2A sequence. Popy-NKG2A terminates at the same position as
other NKG2A and NKG2C sequences (10, 25) and does not use
the splice site (26) that results in the divergent end
sequence of NKG2E in the human and chimpanzee sequences, indicating
that this splice site usage has arisen in the lineage leading to the
chimpanzee and human sequences.
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Polymorphism in orangutan CD94 and NKG2
genes was assessed by analysis of cDNA clones from five individuals.
Multiple alleles for CD94, NKG2A, and
NKG2CE were defined. For each locus, the alleles differ by
one to five nucleotides. Unexpected was finding six alleles for
CD94, a gene that appears monomorphic in humans and
chimpanzees (10). By contrast, only two orangutan
NKG2D alleles and two NKG2F alleles were defined
(Fig. 1). No more than two variants for each lectin-like receptor were
expressed by individual orangutans, consistent with each of the
lectin-like receptors being encoded by a single locus.
Popy-Ly49L, a potentially functional gene
Although the Ly49L genes of human, chimpanzee, and
gorilla appear nonfunctional, that is not the case for all primate
species. Recently, expression of an intact Ly49L transcript
in the baboon was demonstrated and partial genomic analysis showed that
the disabling mutations present in human, chimpanzee, and gorilla
Ly49L were absent from an orangutan (28). To
determine the complete coding sequence of Popy-Ly49L, we
used RT-PCR to amplify full-length products from mRNA. Amplification
from mRNA prepared from a culture of activated NK cells gave a weak but
specific amplification, whereas mRNA prepared from PBMC gave no signal.
The amplification product from the activated NK cells was cloned and
sequenced. Analysis of the complete Popy-Ly49L sequence
showed that it, like that of the baboon, is predicted to encode a
functional cell surface glycoprotein. Comparison of primate sequences
reveals Ly49L to be highly conserved, to an extent well
within the range seen for CD94 and NKG2 family
members: the nucleotide sequence of Popy-Ly49L differs from
the human and baboon sequences by just 3.5 and 5.1%, respectively
(Fig. 1). Furthermore, the dN/dS ratios obtained from these comparisons
are much <1 (0.4–0.6), indicating that purifying selection has
operated upon the Ly49L gene.
Orangutan KIR
KIR cDNA clones were obtained from nine orangutans. For
eight individuals, mRNA was derived from PBMCs and for one individual,
Allen, cultured NK cells. The oligonucleotide primers used in RT-PCR
were based on sequences conserved in human and chimpanzee
KIR. Orangutan KIR corresponding to all three of
the expressed KIR lineages defined in humans and chimpanzees
were obtained (Fig. 4).
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Lineage I KIR have the D0 + D2 configuration of Ig-like domains and are represented by KIR2DL4 and KIR2DL5 in humans (29, 30). Seven of the nine orangutans gave clones corresponding to orangutan KIR2DL4, which has 96.4% nucleotide sequence similarity to human KIR2DL4. No orangutan cDNA clone resembling human KIR2DL5 was obtained.
Two Popy-KIR2DL4 sequences that differ by nine nucleotide (2
aa) substitutions were defined, one individual giving both types of
sequence. These sequences likely represent alleles of
Popy-KIR2DL4. Of note is that neither Popy-KIR2DL4 allele
encodes a protein corresponding to the full-length human KIR2DL4 (Fig. 5A). Popy-KIR2DL4A
contains a point substitution that leads to premature termination in
the D2 domain, at a position just before the end of the final
-strand. Therefore, this allele may encode a soluble protein having
extracellular domains with normal conformation.
Popy-KIR2DL4B contains a frameshift at the beginning of the
region encoding the cytoplasmic tail that causes premature termination
three codons downstream. As a consequence, the protein tail is
predicted to be membrane bound but to have a highly foreshortened
cytoplasmic tail lacking any immunoreceptor tyrosine-based inhibition
motifs (ITIM). The frameshift in the Popy-KIR2DL4B allele is
due to deletion of a single A in a series of 11 As and is identical
with that described in human KIR2DL4 alleles
(30) (directly deposited by Selvakumar et al. in GenBank,
accession no. AF276292).
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B). Popy-KIR2DL4
combines the intact ITIMs from both the human and chimpanzee, as does
the rhesus monkey ortholog, Mm-KIR2DL4 (32). However, the
gene encoding the latter KIR has neither of the disabling mutations
found in the orangutan sequence and is predicted to encode a
full-length protein with two ITIMs in the cytoplasmic tail (Fig. 5B). These comparisons suggest that the
Popy-KIR2DL4 gene has been subject to a process of
modification or inactivation during evolution of orangutans.
Analysis of the frequencies of synonymous and nonsynonymous
substitutions indicates that KIR2DL4 has been subject to
purifying selection, as seen from dN/dS ratios that are much <1. This
is true for both interspecies and intraspecies comparisons (Fig. 5C) with the exception of the intraspecific comparison for
the rhesus monkey sequences where the value is 1.03, suggesting neutral
evolution within this species. Inspection of the aligned sequences
shows that substitutions are distributed relatively evenly throughout
the coding region, with the exception of exon 3 encoding the D0 domain
where substitutions are rare. Codon-by-codon analysis supports this
conclusion providing no evidence for any mutational hotspot, although
the region encoding the stem and the first part of the transmembrane
region lacks nonsynonymous substitutions (data not shown).
Lineage II KIR
In humans and chimpanzees, the lineage II KIR have three Ig-like
domains and include inhibitory receptors specific for MHC-A and -B
(9, 33, 34, 35). KIR of this lineage were most
abundant within the population of orangutan KIR cDNA clones
we sequenced. Clones encoding KIR3DL were obtained from all 9
individuals, and 10 different KIR3DL variants were defined
(Fig. 6A). Of these, the
D1/D2 and E1/E2 pairs of variants are only
distinguished by single synonymous substitutions. Although the
KIR3DL variants are closely related (95–99.9% nucleotide
sequence identity), they cannot all be alleles of a single locus as two
individuals (JingJing and LokLok) expressed three KIR3DL
variants. Indeed, comparison of the genotype of Lokan with his
offspring Loklok confirms the inheritance of one haplotype containing
the single KIR3DL, KIR3DLC from Lokan; and therefore, the
inheritance of a haplotype containing the other two KIR3DL
from the other parent. Of the remaining seven orangutans, six expressed
two forms of KIR3DL while one individual (Allen) expressed a
short-tailed KIR3DS form as well as two KIR3DL.
Thus, there is evidence for two genes encoding KIR3DL, at
least on some haplotypes.
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7% of their nucleotide sequence.
Phylogenetic analysis shows that they group separately from the human
and chimpanzee sequences (Fig. 7). Thus,
the orangutan sequences cannot be assigned as orthologous to any of the
human or chimpanzee sequences on the basis of sequence comparison. This
is consistent with the lack of orthology seen between human and common
chimpanzee KIR of lineage II (9). The orangutan sequences
of this lineage exhibit a patchwork pattern of substitution, as found
in other KIR genes, which provides evidence for
microrecombination having contributed to the diversity within
Popy-KIR3D (Fig. 6A). Evidence for interlocus
recombination is that Popy-KIR3DLE has a D2 domain more closely related
to that of Popy-KIR2D of lineage III than to other KIR3D. This
implicates a conversion between KIR3D and KIR2D
genes, involving exon 5, in the formation of Popy-KIR3DLE.
The Popy-KIR3DS sequence also appears to be a product of
recombination. Although it is highly homologous to Popy-KIR3DL
sequences in the extracellular domains, the transmembrane portion of
the sequence is closely related to the KIR2DS sequences, having a
termination codon that is, relative to KIR3D, introduced by
point substitution. Popy-KIR3DS also has the characteristic lysine
residue in the transmembrane segment that is associated with DAP12
interaction (36).
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B), indicative of purifying selection,
although the values are greater than seen for the lineage I
KIR (Fig. 4C). This is true both within and between
species. Lineage III KIR
In humans, lineage III consists of eight KIR having two
extracellular Ig-like domains in the D1 + D2 configuration (33, 38). By contrast, the six chimpanzee KIR of this lineage
comprise two KIR2D and four KIR3D (9). In both species,
lineage III includes the MHC-C-specific KIR. Only one lineage III KIR,
KIR2DS4, has human and chimpanzee orthologs. All other loci appear to
have arisen since the divergence of the human and chimpanzee lineages
5 MYA (39). There is one exception to this, a
pseudogene, KIR3DP1 (also called KIRX), that appears to be
present on all human KIR haplotypes examined at the genomic
level (40). In our phylogenetic analysis,
KIR3DP1 groups with the chimpanzee KIR of lineage
III (bootstrap value of 85%) indicative of its common origin with
these KIR (Fig. 7).
In the orangutan, clones corresponding to lineage III KIR
were relatively infrequent. Seven different KIR were
characterized, all being KIR2D with a D1 + D2 configuration. These
comprised five KIR2DS and two KIR2DL. Popy-KIR2D
must be encoded by at least two loci, as individual orangutans
expressed three of these KIR (Fig. 1A). As a
group, the orangutan KIR2D form a cluster more closely
related to human and chimpanzee KIR2DS4 than to any other
KIR. In phylogenetic analysis, a 91% bootstrap value
supports grouping of the orthologous human and chimpanzee
KIR2DS4. When Popy-KIR2D are included, their
grouping with human and chimpanzee KIR2DS4 is supported with
71% bootstrap value (Fig. 7). These data favor a model in which all
Popy-KIR2D have evolved from a common
KIR2DS4-like ancestor.
Within the Popy-KIR2D of lineage III group are good examples
of recombinant KIR in which different extracellular domains are
associated with the same stem, transmembrane, and cytoplasmic domains,
and vice versa (Fig. 8). For example, in
the extracellular domains, 2DLA is more like 2DSD than 2DLB. Similarly,
2DLB is more like 2DSC in this same region. For all Popy-KIR2D, the
transmembrane and cytoplasmic domains contain the characteristic
features associated with other long- and short-tailed KIR. The
Popy-KIR2DS have a charged residue (Lys233) in
the transmembrane region that could facilitate interaction with the
DAP12 adapter molecule and the sequences terminate in what would be the
first ITIM sequence of a KIR2DL. The lineage III Popy-KIR2DL are all
predicted to have two ITIMs.
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), but no Popy-KIR2D has methionine 44.
Among the orangutan KIR2D, both KIR2DL and KIR2DS have glutamate 44,
raising the possibility that this residue is associated with a distinct
type of ligand specificity. Unlike the lineage I and II KIR, an Ig domain with invariant sequence is not a feature of the lineage III KIR, regardless of whether the short- and long-tailed KIR are considered separately or together. The dN/dS ratios of the lineage III KIR are comparable to those for the lineage II KIR when compared within groups with a similar cytoplasmic tail (0.73 for the 2DL and 0.72 for the 2DS).
Popy-C allotypes all have the C1 KIR-binding motif
Whereas MHC-C is fixed in the human and chimpanzee
MHC, it is present only on 50% of orangutan
MHC haplotypes. Moreover, a preliminary analysis revealed
only Popy-C allotypes having asparagine at position 80 and none having
lysine at position 80 (11, 14). To explore further the
apparent reduction in orangutan KIR epitopes compared with human and
chimpanzee, we typed 18 orangutans, including 4 of those from
whom KIR cDNA clones were isolated, for Popy-C
and for presence of the C1 (Asn80) and C2
(Lys80) motifs. Eleven individuals had
Popy-C and all of them typed positively for C1 and
negatively for C2. These results indicate that Popy-C
alleles encoding the C2 motif are either not present or are at low
frequency in the orangutan. That this species also has KIR2D with
potential specificity for C1, but lacks KIR2D with specificity for C2,
suggests that MHC-C mediated regulation of NK cells in the orangutan
uses only one of the two ligand-receptor combinations available to
humans.
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Discussion |
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50% of Popy-MHC
haplotypes (14); but in contrast, Popy-B
appears duplicated such that individual orangutans express three or
four Popy-B alleles (14, 44). We demonstrate in
this study that the orangutan differs from human and chimpanzee to even
greater extent in its NK cell receptors. The differences are greater
for KIR than for the lectin-like receptors, consistent with previous
phylogenetic comparison (9, 10, 45), but neither set of
differences appears trivial.
The main points of difference in the lectin-like receptors are as
follows. Orangutan NK cells express Ly49L, a gene that
appears nonfunctional in humans and chimpanzee. The NKG2C
and NKG2E genes are represented by a single
Popy-NKG2CE gene that has similarity to both of the human
genes. A reverse trend occurs in the chimpanzee NKC where
there are two genes encoding Pt-NKG2C forms as well as the
Pt-NKG2E gene (10). Popy-NKG2A diverges from
human and chimpanzee NKG2A in the ligand-contact region of the CRD
where it is similar to NKG2E. Superficially, this might appear the
product of a gene conversion. However, Popy-NKG2A shares this feature
with rhesus monkey Mm-NKG2A (24), favoring the
interpretation that the orangutan has retained the ancestral form of
the CRD and the human and chimpanzee NKG2A CRD is the more recently
derived. Correlating with this species difference in the NKG2A receptor
are three positions in the MHC-E peptide-binding domain which
distinguish orangutan and rhesus from chimpanzee and human (Fig. 9). Of these, Pro57
and Arg169 are likely to be surface accessible to
the CD94:NKG2A heterodimer (46), and perhaps have some
modulating influence on its affinity or specificity for MHC-E. The
threonine at position 73 is located in the P6 binding pocket, and by
altering the hydrophobicity of that pocket, may have some effect on
peptide-binding; and therefore, indirectly influence the binding of the
CD94:NKG2A heterodimer.
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). Thus, there has been a
general trend toward inactivation of the ITIMs in this gene in
hominoids. Indeed, the observation that anti-KIR2DL4 Abs can
stimulate IFN-
production by NK cells (47) and the
presence in the KIR2DL4 transmembrane domain of an arginine residue
absent from other KIR (48) point to KIR2DL4 having
activating function. KIR2DL4 is proposed as a receptor for the MHC-G
nonclassical class I molecule (49). Striking in this
regard is that MHC-G is a pseudogene in rhesus macaque while being an
active expressed gene in the hominoids (13). This
difference may reflect functional differences in the KIR2DL4 receptors
of hominoids and macaque. The impression gained from our study is that the KIR system of the orangutan is markedly simpler than that encountered in either human or common chimpanzee. Major lineages are all represented, but within each lineage the number of loci is more restricted as is their divergence. At present our data can be accommodated minimally by five KIR genes: KIR2DL4, two KIR3D, KIR2DL, and KIR2DS. Of these the two Popy-KIR2D genes appear paralogs of KIR2DS4 and the two Popy-KIR3D are paralogous with KIR3DL1 and 2. Were these five genes to be organized into a single haplotype, it could resemble a hybrid consisting of the left hand (5') half of a human B haplotype (containing KIR2DL2) in combination with the right hand (3') half of a human A haplotype (having KIR2DL4, KIR3DL1/3DS1, KIR2DS4, and KIR3DL2) (50, 51).
A major implication of our data is that MHC-C-mediated regulation of NK cells is also simpler in orangutan than either human or chimpanzee. Several points suggest that HLA-C provides the more important inhibitory HLA class I ligands for KIR: all HLA-C allotypes are KIR ligands, all individuals can use at least one KIR2D as a self-receptor for HLA-C, the low expression of HLA-C is compatible with this locus having become more specialized toward the NK cell response. HLA-C is also the most recently evolved of the expressed HLA class I genes, and it is only present in humans, African apes, and orangutan (14). Humans and chimpanzees have the C1 and C2 groups of HLA-C allotype (52) along with two types of MHC-C-specific KIR that complement them (9), whereas orangutan has only the C1 group of MHC allotype. Reflecting this difference, there are orangutan KIR with the potential for C1 specificity, but none with the potential for C2 specificity. Thus, the orangutan has a system of MHC-C and specific KIR that resembles a halfway intermediate in the evolution of the human system.
Contemporaneous with the emergence of MHC-C as the dominant ligand for KIR, we see radiation of lineage III KIR, which include the MHC-C receptors. We find two genes of this lineage in the orangutan, one KIR2DL and one KIR2DS. In the chimpanzee, there are six members of this lineage and in human eight (9). In addition to differences in number, the majority of the chimpanzee KIR are long-tailed, whereas in human five of the eight genes encode short-tailed KIR. The rapid emergence of new loci for ligands and receptors and a stepwise increase in the complexity of the system is evidence for coevolution of HLA-C and certain types of lineage III KIR.
In human and chimpanzee the lineage II KIR have specificity for MHC-A and -B. Therefore, the parsimonious hypothesis is that Popy-KIR3D will include any receptors for polymorphic Popy-A and -B molecules. Striking is that neither lineage II nor lineage III KIR are represented in rhesus monkey, which instead has a distinct and more diverse lineage of KIR3D related to both lineage I KIR and to the nonfunctional human KIR3DL3 (also called KIRCI or KIR3DL7) gene (53). Correlating with this difference is the lack of orthology between HLA-A and -B and the Mamu-A and B-like genes of the rhesus monkey (11). Thus, both lineage II and III KIR may be specific to the hominoids.
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2 Address correspondence and reprint requests to Dr. Peter Parham, Department of Structural Biology, Stanford University School of Medicine, Sherman Fairchild Building D-157, 299 Campus Drive West, Stanford, CA 94305-5126. E-mail address: peropa{at}leland.stanford.edu
3 Abbreviations used in this paper: NKC, NK cell complex; LRC, leukocyte receptor complex; KIR, killer cell Ig-like receptor; CRD, carbohydrate recognition domain; ITIM, immunoreceptor tyrosine-based inhibition motif.
Received for publication February 1, 2002. Accepted for publication April 19, 2002.
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