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Comparative Study
. 2013;8(3):e59577.
doi: 10.1371/journal.pone.0059577. Epub 2013 Mar 18.

The Turkey Ig-like receptor family: identification, expression and function

Katharina Windau  1 , Birgit C Viertlboeck, Thomas W Göbel
Affiliations
Comparative Study

The Turkey Ig-like receptor family: identification, expression and function

Katharina Windau et al. PLoS One. 2013.

Abstract

The chicken leukocyte receptor complex located on microchromosome 31 encodes the chicken Ig-like receptors (CHIR), a vastly expanded gene family which can be further divided into three subgroups: activating CHIR-A, bifunctional CHIR-AB and inhibitory CHIR-B. Here, we investigated the presence of CHIR homologues in other bird species. The available genome databases of turkey, duck and zebra finch were screened with different strategies including BLAST searches employing various CHIR sequences, and keyword searches. We could not identify CHIR homologues in the distantly related zebra finch and duck, however, several partial and complete sequences of CHIR homologues were identified on chromosome 3 of the turkey genome. They were designated as turkey Ig-like receptors (TILR). Using cDNA derived from turkey blood and spleen RNA, six full length TILR could be amplified and further divided according to the typical sequence features into one activating TILR-A, one inhibitory TILR-B and four bifunctional TILR-AB. Since the TILR-AB sequences all displayed the critical residues shown to be involved in binding to IgY, we next confirmed the IgY binding using a soluble TILR-AB1-huIg fusion protein. This fusion protein reacted with IgY derived from various gallinaceous birds, but not with IgY from other bird species. Finally, we tested various mab directed against CHIR for their crossreactivity with either turkey or duck leukocytes. Whereas no staining was detectable with duck cells, the CHIR-AB1 specific mab 8D12 and the CHIR-A2 specific mab 13E2 both reacted with a leukocyte subpopulation that was further identified as thrombocytes by double immunofluorescence employing B-cell, T-cell and thrombocyte specific reagents. In summary, although the turkey harbors similar LRC genes as the chicken, their distribution seems to be distinct with predominance on thrombocytes rather than lymphocytes.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Alignment of the TILR-A sequences.
The signal peptide (SP), Ig domains (IG1, IG2), transmembrane (TM) and cytoplasmic (CY) domains are indicated. Conserved cysteines forming the intrachain disulfide bridges are marked with an asterisk and the cysteine conserved in the transmembrane domain is marked by an arrowhead. The predicted transmembrane region is indicated by a line above the sequence. Note that residues that were not conserved are shaded in black. The predicted exon : intron boundaries are marked by vertical lines in the sequences. For comparison the CHIR with highest homology (CHIR-A2, accession number AJ745093) g002was used. Accession number of TILR-A1: KC201188.
Figure 2
Figure 2. Alignment of the TILR-B sequences.
The annotation of sequence features is similar to Fig. 1. ITIM sequences in the cytoplasmic domain are boxed. For comparison the CHIR with highest homology (CHIR-B4, accession number AJ639839) was used. Accession number of TILR-B1: KC201189.
Figure 3
Figure 3. Alignment of the TILR-AB sequences.
Sequence features are explained in Fig. 1. Note that TILR-AB possess a single Ig domain, only. The residues known to be important for the interaction with IgY are indicated by dots above the sequence. For comparison the CHIR with highest homology (CHIR-AB1, accession number AJ745094) was used. Accession numbers of TILR-AB1: KC201190, TILR-AB2: KC201191, TILR-AB3: KC201192, TILR-AB4: KC201193.
Figure 4
Figure 4. TILR-AB1 reacts with IgY from gallinaceous birds.
Log2 dilution of TILR-AB1-huIg fusion protein on ELISA plates coated with IgY derived from birds as indicated. Mean ± SD of triplicates is shown.
Figure 5
Figure 5. Immunofluorescence staining of duck and turkey PBMC.
Blood was separated via density centrifugation and stained with mab against αVβ3 and various CHIR followed by secondary antibody conjugates. Histograms of one typical out of four separate experiments is shown and frequencies of positive cells are indicated.
Figure 6
Figure 6. Double fluorescence analyses of turkey PBMC.
Cells were labeled with either CHIR-AB1 specific mab 8D12 (A) or CHIR-A2 specific mab 13E2 (B) in combination with the T-cell specific mab CD4 and CD8, the class II specific mab 2G11 and the αVβ3 specific mab 23C6. (C) Double staining of blood leukocytes with 8D12 and 13E2. Gates were set according to forward and sideward scatter features on the lymphocyte gate in A, B and C. (D) Combined immunfluorescence of 8D12 or 13E2 in combination with the MHC class II specific mab 2G11 as in (A) or (B), but gated on the larger monocytes based on forward/side scatter characteristics. Frequencies of positive cells are indicated in the quadrants. One representative of three experiments is shown.
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Funded by Deutsche Forschungsgemeinschaft grant no. GO489/3-7, website: dfg.de. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.