ORIGINAL PAPER

Bimodal evolution of the killer cell Ig-like receptor (KIR)

family in New World primates

Luis F. Cadavid & Catalina Palacios & Juan S. Lugo

Received: 26 April 2013 /Accepted: 26 June 2013

#

Springer-Verlag Berlin Heidelberg 2013

Abstract The immunoglobulin-like receptor (KIR) gene

family in New World primates (Platyrrhini) has been

activating and inhibitory KIRs that diversified independently

from their Catarrhini counterparts. They had a unique mech-

anism to generate activating receptors from inhibitory ones,

involving a single nucleotide deletion in exon 7 and a change

in the donor splice site of intron 7. The A. geoffroyi haplotype

contained at least six gene models including a pseudogene,

two coding inhibitory receptors, and three coding activating

receptors. The centromeric region was in a tail-to-tail orienta-

tion with respect to the telomeric region. The owl monkey

KIR haplotype shared this organization, and in phylogenetic

ability between paralogous genes but not between alleles in a

locus. Atelidae, therefore, has expanded the KIR family in a

remained relatively conserved and the telomeric region has

diversified by a rapid process of gene duplication and diver-

gence, likely favored by positive selection for ligand binding.

Keywords KIR

.

Killer cell Ig-like receptor

.

New World

primates

.

Atelidae

(MHC) class I molecules (McQueen and Parham 2002). An

altered expression of MHC class I molecules on the target

cell initiates an activating signal cascade leading to cytotox-

icity, whereas normal expression of MHC class I molecules

triggers an inhibitory signaling resulting in anergy (Cheent

and Khakoo 2009). NK cell receptors belong to two struc-

tural classes, the lectin-like and the immunoglobulin (Ig)-

like receptors (KIRs). The former are type II transmembrane

proteins represented by the CD94/NKG2 family and are

encoded in the natural killer complex on human chromo-

Electronic supplementary material The online version of this article

(doi:10.1007/s00251-013-0719-4) contains supplementary material,

which is available to authorized users.

L. F. Cadavid (*)

Department of Biology and Institute of Genetics, Universidad

Nacional de Colombia, Cr. 30 no. 45-08, Bogotá, Colombia

e-mail: [email protected]

Present Address:

a transmembrane domain, and a short or long cytoplasmic

domain (Barrow and Trowsdale 2008). KIRs with long cy-

toplasmic domains function as inhibitory receptors due to the

presence of two immunoreceptor tyrosin-based inhibitory

motifs (ITIMs), whereas KIRs with short cytoplasmic do-

mains function as activating receptors due to the presence of

a positively charged residue at the transmembrane domain

allowing the interaction with DAP12 molecules, which

posses immunoreceptor tyrosin-based activating motifs (La-

nier 2003).

at the centromeric end (KIR3DL3), central region (KIR3DP1

and KIR2DL4), and telomeric end (KIR3DL2) of the cluster

(Parham and Moffett 2013). The 13 genes and 2 pseudogenes

identified in the human KIR region are grouped into two types

of haplotypes (A and B). Haplotype A mainly contains genes

encoding inhibitory receptors, whereas haplotype B primarily

contains genes encoding activating receptors (Parham and

Moffett 2013). This KIR cluster organization has been rela-

tively well conserved in Catarrhini primates (humans, apes,

and Old World monkeys) with species-specific differences

molecule MHC-G. Lineage II KIRs are represented by the

human KIR3DL1 and KIR3DL2 and recognize MHC-A and

MHC-B molecules, whereas lineage III KIRs (i.e., KIR2DL2

and KIR2DS1) recognize MHC-C and certain MHC-B allo-

types. Finally, lineage V is represented by the human KIR3DL3

with no known MHC specificity.

In contrast with the solid understanding of the KIR system

in Catarrhini, KIR genes in Platyrrhini (New World pri-

mates) have only been reported in one species, the owl

monkey (Aotus sp.) (Cadavid and Lun 2009). Genomic

direct orthology to any of the genes from the other four

lineages. In order to have a better understanding of the compo-

sition and diversification patterns of the KIR system in

Platyrrhini, we have analyzed the KIR homologous genomic

region in the black-handed spider monkey (Ateles geoffr oyi),

woolly monkey (Lagothrix la gotricha). The Atelidae family in-

cludes five genera (Alouatta, Ateles, Brachyteles, Lagothrix, and

1.1±0.2 MYA (Fabre et al. 2009).

Materials and methods

Genomic sequence of the Ateles geoffroyi KIR haplotype

A draft sequence of the bacterial artificial chromosome

(BAC) clone UC1-11A1 containing the black-handed spider

monkey (A. geoffroyi) KIR haplotype was obtained from

publicly available databases (GenBank accession number

AC234015). This BAC clone was sequenc ed by the NIH

Intramural Sequencing Center ( www.nisc. nih.gov) as part of

del Alto Magdalena) under the animal care and use committee

policies of the respective institutions. Total RNA was isolated

with the Trizol reagent (Invitrogen) and used for complemen-

tary DNA (cDNA) synthesis with the Reverse Transcription

System kit (Fermentas). Full-length KIR cDNAs were

amplified by PCR with forward primer AteKIRPS-F

5′-ATGTCGCTCATGGTCRTCA GCATG-3′ and reverse

primer NWPKIRL1-R 5′-CTAGAGGACCCCTCAGGG-

3′, which anneal at KIR gene exons 1 and 9, respec-

tively. The reaction contained 400 μM dNTPs, 2 mM MgCl

KIR3DS1) (Cadavid and Lun 2009), indicating that an an-

cient inversion of the KIR centromeric region occurred in the

common ancestor of Ateli dae and Aotinae at least some

20 MYA. The next three gene models downstream in the A.

geoffroyi KIR cluster, Atge-KIR3DS1-3, were predicted to

encode activating receptors with three Ig domains, the pos-

itively charged residue arginine at the fourth position of the

transmembrane region, and a short cytoplasmic domain. The

truncated cytoplasmic domain was the result of a series of

mutations in exon 7 and intron 7 that included a single

putative secondary donor splice site (AG^GTGT) at posi-

tions 66–71 of intron 7, predicted at a 54 % of confidence

using the software GeneSplicer (Pertea et al. 2001). As a

consequence of these mutations, exon 7 was predicted to be

transcribed together with the first 65 nucleotides of intron 7,

and joined to exon 8 during messenger RNA processing in an

altered phase, generating a premature stop codon at positions

10–12 of this exon (Fig. 2). The owl monkey activating KIR

Fig. 1 Gene organization of the KIR cluster in the spider monkey

(Ateles geoffroyi) and the owl monkey (Aotus sp.) based on BAC clone

draft sequences AC234015 and AC234076/AC193069, respectively.

Transcription orientation is indicated for each gene model, as well as

exon, repetitive element, and retroelement localization along the chro-

mosomal region. The predicted receptor structure is shown above each

gene model

Fig. 2 Ateles geoffroyi KIR gene models predicted to encode activating

positions 66–73 (gray background). These alterations generated recep-

tors with cytoplasmic domains having a 22-amino acid insertion in the

cytoplasmic domain derived from intron 7 and a premature termination

due to a stop codon in exon 8

gene model Om-KIR4DS1 had the same modifications in

exon 7 and intron 7 (Fig. 2), suggesting that this mechanism

of switching from an inhibitory to an activating receptor is

prevalent in New World primates. Finally, the last KIR gene

model at the 3′ end of the cluster (Atge-KIR3DL2) was

predicted to encode a receptor with three Ig domains and a

long cytoplasmic tail with two putative ITIMs, suggesting

that it functions as an inhibitory receptor.

Atelidae express a variable set of activating and inhibitory

KIRs

A total of 29 unique full-length KIR cDNAs were sequenced

in the three Atelidae species, 13 corresponding to the two A.

hybridus individuals, 4 to the A. belzebuth individual, and 12

to the two L. lagotricha individuals (Supplementary Fig. 1).

A. belzebuth (Supplementary Fig. 1). SV1 lacked exon 7

encoding the transmembrane domain, SV2 lacked exon 6

encoding the stem domain, SV3 lacked 36 bp at the 5′ end of

exon 4 (D1 domain), and SV4 lacked the last 200 bp of exon

3 (D0 domain). The structure of predicted KIR proteins was

modeled by homology, showing a highly similar folding to

that of KIR3DL1*001 (PDB 3VH8, average RMSD=0.076 Å,

hybridus individuals were assigned to 5 putative loci based on

the phylogenetic analyses (see below) and distinctive sequence

KIR3DS1-3. The transmembrane domain of these activating

fourth position instead of a lysine at the ninth position, typical of

KIR3DS2 lacked exon 6 encoding the stem domain, a feature

(Palacios et al. 201 1). The nucleotide similarity between the five

A. hybridus putative loci averaged 90.1 % (94.8–86.6 %, average

distance d=0.105), similar to that observed between A. geoffroyi

loci, whereas individuals 110 and 180 lacked sequences from

Athy-KIR3DS2 and Athy-KIR3DL1 loci, respectively (Table 1).

A. belzebuth cDNAs were assigned to three putative loci

(Table 1) predicted to encode an inhibitory receptor (Atbe-

KIR3DL2) and two activating receptors (Atbe-KIR3DS3 and

Atbe-KIR2DS3). Although Atbe-KIR2DS3 was the only

Atelidae KIR gene predicted to encode a receptor with a

D1+D2 configuration, it could represent a splicing variant

of Atbe-KIR3DS3 given their high sequence similarity (Sup-

plementary Fig. 1). In this species, the interlocus nucleotide

activating receptors (Lala-KIR3DS1-3). The latter had

Table 1 Putative KIR loci/alleles assigned to the Atelidae cDNA sequences

A. hybridus

Individual identification number for each species

Putative loci (italic type) were assigned according to clustering patterns in gene trees and sequence distinctive features. For each putative locus,

alleles or splicing variants (sv) were designated by numbers (01, 02, etc.)

Uncertainty about its status as a locus as it might be an allele of Lala-KIR3DL1

arginine at the fourth position of the transmembrane domain,

the intronic insertion at the cytoplasmic domain, and had in

common a unique single amino acid deletion at position 16th,

a feature shared by human inhibitory receptors KIR2DL1 and

models. a Platyrrhini KIRs form a monophyletic group conforming the

evolutionary lineage VI (blue background), with the Catarrhini evolu-

tionary lineages ( gray background) organized as sister groups. Lineage

IV, containing Old World primate KIRs, is considered a lineage II

subgroup based on intronic sequences (Guethlein et al. 2007b; Blokhuis

et al. 2011). b KIR cDNAs and gene models from Atelidae species and

from the owl monkey are clustered into four main clades. A. geoffroyi

(Atge) KIRs are shown in black type, A. hybridus (Athy) in red, A.

belzebuth (Atbe)inblue, L. lagotricha (Lala)ingreen, and owl monkey

(Om)inpurple. Numbers of branches indicate posterior probability for

and a 65-bp intronic insertion at the end of exon 7, generat-

ing a shift in the reading frame and a premature stop codon in

exon 8 (Supplementary Fig. 1), just as it was described for

the Atge-KIR3DS gene models (Fig. 2). A. hybridus and A.

belzebuth KIR3DS loci had a splice variant (SV1) that

skipped exon 7 and mai ntained the reading fram e to generate

predicted proteins with no transmembrane domain but with

long cytoplasmic domain having two ITIMs (Supplementary

Fig. 1). This supports the notion that modifications in exon 7

and intron 7 were responsible to generate truncated cytoplas-

receptor and suggesting that it is probably an allele from

the Lala-KIR3DL1 locus.

Platyrrhini KIRs form an independent evolutionary lineage

and are organized into four clades

Gene trees generated by distance, maximum likelihood, and

Bayesian approaches showed that Platyrrhini KIRs formed a

monophyletic group with no direct orthology to any of their

Catarrhini counterparts (Fig. 3a). This Platyrrhini clade

conformed the primate KIR lineage VI previously reported

(Cadavid and Lun 2009 ), which likely diversified from the

contained sequences predicted to encode inhibitory receptors

from the four Atelidae species, with the single exception of

the L. lagotricha cDNA Lala-KIR3DS2*01

, which, as men-

tioned be fore, might be an allele of the putat ive inhibitory

locus Lala-KIR3DL1. Clade 3 contained all the telomeric

gene models from the owl monkey and clade 4 clustered the

inverted centromeric gene models from the owl monkey

(Om-KIR3DP1, Om-KIR3DS1, and Om-KIR3DL2) and from

A. geoffroyi (Atge-KIR3DL1 and Atge-KIR3DP1), together

Atge-KIR3DP1/Aona-KIR3DP1 (Fig. 3b). No direct orthologous

Rapid diversification of KIR genes in Atelidae

both episodic and pervasive selection at the level of individ-

ual codon positions. Alignment-wise analysis of selection

was done by species, phylogenetic clades, and with the entire

Platyrrhini dataset (Table 2). Evidence of positive selection

was found at significant levels only in comparisons from A.

hybridus, A. geoffroyi, clade 1, and with all Platyrrhini se-

quences. In addition, in all but one category (A. belzebuth

sequences), there were individual sites under positive selec-

tion, including positions predicted to interact with the MHC

class I molecules (Table 2). Forty-three positively selected

positive selection. a Positively selected positions in D0, D1, and D2

domains identified in comparisons between all Platyrrhini sequences

at a significance level of p<0.05 are indicated over the human

KIR3DL1*0101-HLA-B27*01 structure (PDB 3VH8). Positions un-

der positive selection that are predicted to contact the MHC are

highlighted in green, whereas those that are not in contact with the

MHC are shown in yellow. Other positions that contact the MHC but

are not positively selected are highlighted in blue. The D0 domain is

shown in green,D1inblue,D2inlight purple, the MHC’s peptide

binding region in red, and the peptide in white. b Variation in