The complete inventory of receptors encoded by the rat natural killer cell gene complex

Abstract

The natural killer cell gene complex (NKC) encodes receptors belonging to the C-type lectin superfamily expressed primarily by NK cells and other leukocytes. In the rat, the chromosomal region that starts with the Nkrp1a locus and ends with the Ly49i8 locus is predicted to contain 67 group V C-type lectin superfamily genes, making it one of the largest congregation of paralogous genes in vertebrates. Based on physical proximity and phylogenetic relationships between these genes, the rat NKC can be divided into four major parts. We have previously reported the cDNA cloning of the majority of the genes belonging to the centromeric Nkrp1/Clr cluster and the two telomeric groups, the Klre1-Klri2 and the Ly49 clusters. Here, we close the gap between the Nkrp1/Clr and the Klre1-Klri2 clusters by presenting the cDNA cloning and transcription patterns of eight genes spanning from Cd69 to Dectin1, including the novel Clec2m gene. The definition, organization, and evolution of the rat NKC are discussed.

Fig. 1

Amino acid sequence alignments of the CLSF receptors. r rat, m mouse, h human. Dashes mean identity with top sequence. Points indicate gaps. Ex1, Ex2, etc. on top of alignment show start site of corresponding exons. Predicted transmembrane regions (TMpred) are underlined. ITAMs and ITIMs are indicated. Potential N-glycosylation sites are boxed. Membrane external cysteines are highlighted in yellow. Numbers connected with black lines on top of the CD69 and Dectin1 sequences indicate SS-bonds as demonstrated for crystal structures of human CD69 and mouse Dectin1 (http://www.ncbi.nlm.nih.gov/ Genbank/). For the other genes, the corresponding cysteines are similarly numbered. X above cysteines in Clec1a indicates probable extra disulphide bridge, which in addition to the bridge between the cysteines labeled 2, links the first α helix with the last β strand of the lectin-like domain. GenBank accession numbers: rat CD69–GU357488, rat Clec2m–GU357487, mouse Clec2m–GU553093, rat Clec12a–GU357484, rat Clec12b–GU357483, rat Clec1b–GU357482, rat Clec9a–GU357486, rat Clec1a–GU357481, rat Dectin-1–GU357485. The rat strains from which the sequence was derived are shown in parentheses (PVG, DA, or both). It should be noted that the reported coding sequences were identical to the corresponding parts of the genomic rat sequence based on the BN strain. The single exception was rat Dectin1, with two single base substitutions in exon 6, one silent and one missense giving rise to an A225G substitution (identical for four independent cDNAs sequenced)

Fig. 1

Amino acid sequence alignments of the CLSF receptors. r rat, m mouse, h human. Dashes mean identity with top sequence. Points indicate gaps. Ex1, Ex2, etc. on top of alignment show start site of corresponding exons. Predicted transmembrane regions (TMpred) are underlined. ITAMs and ITIMs are indicated. Potential N-glycosylation sites are boxed. Membrane external cysteines are highlighted in yellow. Numbers connected with black lines on top of the CD69 and Dectin1 sequences indicate SS-bonds as demonstrated for crystal structures of human CD69 and mouse Dectin1 (http://www.ncbi.nlm.nih.gov/ Genbank/). For the other genes, the corresponding cysteines are similarly numbered. X above cysteines in Clec1a indicates probable extra disulphide bridge, which in addition to the bridge between the cysteines labeled 2, links the first α helix with the last β strand of the lectin-like domain. GenBank accession numbers: rat CD69–GU357488, rat Clec2m–GU357487, mouse Clec2m–GU553093, rat Clec12a–GU357484, rat Clec12b–GU357483, rat Clec1b–GU357482, rat Clec9a–GU357486, rat Clec1a–GU357481, rat Dectin-1–GU357485. The rat strains from which the sequence was derived are shown in parentheses (PVG, DA, or both). It should be noted that the reported coding sequences were identical to the corresponding parts of the genomic rat sequence based on the BN strain. The single exception was rat Dectin1, with two single base substitutions in exon 6, one silent and one missense giving rise to an A225G substitution (identical for four independent cDNAs sequenced)

Fig. 2

Dendrogram of the novel receptors (plus Lox1) and their human and mouse orthologs (based on amino acid sequences). Numbers indicate bootstrap values (1,000 reiterations) and Bayesian clade credibility values. Values shown only for branching points with bootstrap values >900 or credibility values >90

Fig. 3

Gene transcription as measured by quantitative PCR. The transcriptions of genes were calculated as the ratio of copy number of target gene to the copy number of endogenous reference gene, PMCA4. Thresholds for intermediate and high transcription were set to 1/100 and 1 relative to PMCA4. Not detected no PCR product detected after 40 cycles, DC dendritic cells, PM peritoneal macrophages, RNK16 NK cell line, LAK IL2-stimulated NK cells. Similar results were obtained using the HPRT1 gene (hypoxanthine guanine phosphoribosyl transferase 1) as the endogenous reference gene

Fig. 4

Dendrogram based on deduced protein sequences of all the CLSF genes encoded by the rat NKC (only eight of the predicted 34 Ly49 receptors shown). Also included are Mafa1/Klrg1 and Mdl1/Clec5a, plus the seven predicted functional APLEC genes. Vertical black lines indicate major branches of the NKC receptors. The genes containing five coding exons are indicated with 5 ex. Only bootstrap values >900 from 1,000 reiterations are shown

Fig. 5

Map of the rat NKC. Arrows indicate gene orientation. Numbers below the line indicate distance from the starting point (the Nkrp1a gene at 165.637 Mb according to the current release of the rat genome). The four major clusters are color-coded: green Nkrp1/Clr gene cluster (including Cd69 and Clec2m), magenta Clec12a–Lox1 cluster, red Klre1–Klri2 cluster, blue Ly49 cluster. The topography of the chromosomal map with respect to major clusters is seen to be congruent with the major branches of the phylogenetic tree in Fig. 4. It should be emphasized that most of the genes shown here have been cDNA cloned primarily in the DA or PVG strain, so that the map shown here represents the positions of the predicted alleles from the BN strain, from which the rat genome sequence is derived. The extent of intestrain variation, including copy number variation, is so far largely uncharted