Studies on the Expression of the Deleted KIR2DS4*003 Gene Product and Distribution of KIR2DS4 Deleted and Nondeleted Versions in Different Populations
Introduction
The killer immunoglobulinlike receptor (KIR) gene family, located within the leukocyte receptor cluster on chromosome 19q13.4, encodes cell surface receptors that are found on natural killer (NK) cells and certain T-cell subsets. NK cells mediate a variety of responses to tumor cells, viruses, and parasites via the release of cytokines and chemokines and the establishment of appropriate adaptive immune responses through cross-talk with dendritic cells. NK cell effector functions are regulated by positive and negative signals transmitted through paired activating and inhibitory receptors. In vivo, NK cells are under the constitutive dominance of inhibitory receptors for self major histocompatibility complex (MHC) Class I ligands [1] ensuring that effector functions occur only when activating signals are able to overcome inhibitory signaling. This is achieved by either a predominance of activating receptor-ligand interactions or a lack of inhibitory receptor-ligand interactions [2, 3, 4, 5, 6].
Intense research interest over the last few years has seen a clearer picture emerge on the genomic organization of the KIRs [7, 8, 9], molecular structure of the KIR-human leukocyte antigen (HLA)-ligand interaction [10, 11] and the extent of KIR diversity within the human population [12, 13, 14, 15, 16]. Seventeen distinct KIR gene loci have now been identified that vary with respect to their presence or absence on different KIR haplotypes, creating considerable diversity in the number of KIR phenotypes observed. Allelic polymorphism is detectable in individual KIR genes, which contributes further to the level of KIR diversity observed [17].
Inhibitory and activating KIR genes are distinguished by the presence of long (L) or short (S) tails, respectively. The inhibitory signal is transduced by the presence of immune receptor–tyrosine–based inhibitory motives (ITIM) located in the long tail, whereas the activating signal transduces their positive signal via an association with the signal transduction protein DAP12 that contains an immune receptor–tyrosine–based activating motive (ITAM) in its cytoplasmic domain. MHC ligands for the inhibitory receptors have been well defined; HLA-C1 group (lysine at position 80) for KIR2DL2 and KIR2DL3; HLA-C2 group (asparagine at position 80) for KIR2DL1, and HLA-Bw4 for KIR3DL1 [18]. However the ligands for the activating receptors are poorly defined. Studies have indicated that KIR2DS4 may have a restricted affinity for HLA-C ligands interacting specifically and with low affinity with HLA-Cw4 rather than HLA-Cw6 [19]. More recently a nonclass I MHC ligand for KIR2DS4 has been shown, on melanoma cell lines [20].
Two haplotype arrangements (A and B) have been identified in relation to the 17 KIR genes [21]. Each haplotype has the four framework genes, KIR2DL4, KIR3DL2, KIR3DL3, and KIR3DP1. The A haplotype is generally less variable in its gene organization, containing, in addition to the framework genes KIR2DL1, KIR2DL3, and KIR3DL1, only one activating gene, KIR2DS4, although KIR2DL4 is believed to have both inhibitory and activating properties. On the other hand the B haplotype is defined by the presence of one or more activating genes KIR2DS1/2/3/5, KIR3DS1, and the inhibitory genes KIR2DL5A/B and KIR2DL2.
Previously we reported a polymerase chain reaction–sequence-specific oligonucleotide probe (PCR-SSOP) approach for determining the presence or absence of the individual KIR gene loci and observed considerable diversity in the KIR phenotype profiles within the Northern Ireland [22, 23] population. Utilization of this KIR typing system identified a novel form of KIR2DS4 (2DS4*003) that has a 22 bp deletion in exon 5 [14]. Subsequently this novel deletion was confirmed by others [24]. Cloning and sequencing of this KIR2DS4 variant showed that it encoded an entirely novel C-terminus [25]. The new reading frame that is generated because of the 22 bp deletion in 2DS4*003 terminates at the first in-frame stop codon (TGA), located just downstream (25 nucleotides) of the start of exon 7, which encodes the transmembrane anchor. The majority of the protein sequence of the D2 domain (∼70%; 63 of 91 residues), and all the linker/stalk protein sequence, becomes radically altered. The new C-terminal protein sequence contains no hydrophobic regions that would be capable of acting like a transmembrane region, indicating that this truncated version of KIR2DS4 (devoid of transmembrane and cytoplasmic domains), if expressed, would fail to anchor in the membrane during translation and be secreted (a soluble KIR molecule).
A high prevalence (80%) of the KIR2DS4*003 deletion variant in the Northern Ireland population has been reported along with the identification of two further deleted variants of KIR2DS4, named KIR2DS4*004 and KIR2DS4*006, present at a frequency of 7% and 33%, respectively [26]. The presence of mRNA for the KIR2DS4*003 allele, as demonstrated by the ease with which cDNA clones were obtained, suggests expression. The aim of the present study is to provide formal proof of KIR2DS4*003 expression as a secreted protein, in contrast with the membrane-bound KIR2DS4*00101, by using an in vitro cell line model. In addition we have examined the frequency of the nondeleted and deleted versions of KIR2DS4 in different populations, paying particular attention to the fact that individuals homozygous for the A haplotype and with the deleted version of KIR2DS4 may not have any KIR functional activating gene.
Section snippets
Materials and methods
cDNAs known to encode the deleted (*003) and full-length forms (*00101) of KIR2DS4 were amplified by PCR. The pCDNA3.1-KIR2DS4 mammalian expression constructs were generated by reverse transcriptase–polymerase chain reaction (RT-PCR) using the following primer pairs: 2DS4FOR 5′gccgccatgtcgctcatggtcatcatcatggcgtgtgttggg3′; 2DS4*003REV 5′gaacatgtaggtgtctggggttaccgg3′ (for the deleted construct) 2DS4*00101REV 5′tgcgtatgacacctcctgatggtc3′ (for the full-length construct). PCR conditions were as
Results
Amplification of cDNAs encoding the coding sequences for KIR2DS4 variants *003 and *00101 yielded PCR products of 696 and 869 bp, respectively, as seen by agarose gel electrophoresis (Figure 1A). Transcription and translation of the corresponding constructs was demonstrated with the IVT rabbit reticulocyte system. The constructs included a V5 tag at the C-terminus of the KIR sequence, generating V5-tagged *003 and *00101 KIR2DS4 proteins (fusion proteins), which allowed their detection by
Discussion
The present results indicate that the fusion protein encoded by the *003-V5, deleted form of KIR2DS4, is expressed and shed from the cell surface into the culture medium, whereas that encoded by the *00101-V5, the full-length KIR2DS4, remains anchored on the cell surface. The kinetics of expression in our cell model system are compatible with this view. Thus, the gradual decline of *00101 expression at 96 and 144 hours posttransfection, following a peak at 72 hours, may be indicative of the
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