The turkey Ig-like receptor family

Es wurden sechs turkey-Ig-like receptor (TILR) Sequenzen identifiziert, bestehend aus einer TILR-A, einer TILR-B und vier TILR-AB Sequenzen. Bei allen vier TILR-AB Sequenzen sind die fünf für die IgY-CHIR-AB-Bindung essentiellen Aminosäuren konserviert und für TILR-AB1 konnte die Bindung an IgY nachgewiesen werden. Interessanterweise bindet TILR-AB1 nicht nur IgY der Pute, sondern auch an IgY von Huhn, Wachtel und Fasan, die alle der Vogelfamilie der Phasanidae angehören. Bei drei ausgewählten Vertretern außerhalb dieser Familie (Graupapagei, Falke und Ente) konnte die IgY-Bindung an TILR-AB1 nicht nachgewiesen werden. Im Gegensatz zum Huhn, dessen CHIR-Familie auf einem Mikrochromosom lokalisiert ist, werden die in den Datenbanken vorhandenen TILR-Sequenzen und Fragmente dem Makrochromosom 3 zugeordnet. Auch im Expressionsmuster unterscheiden sich CHIR und TILR deutlich. Während die TILR-B-Expression nicht näher untersucht werden konnte, zeigte sich für TILR-A und TILR-AB, dass diese weder auf T- noch auf B-Zellen exprimiert werden. Auf Thrombozyten und Monozyten ist eine sehr hohe Expressionsrate sichtbar. Auffällig ist, dass der Großteil der TILR-A positiven Zellen auch TILR-AB coexprimiert. In den Datenbanken finden sich derzeit keine Hinweise auf das Vorhandensein von CHIR-Homologen bei Zebrafink, Fliegenschnäpper, Wellensittich und Ente. Auch die verfügbaren gegen CHIR gerichteten monoklonalen Antikörper zeigten auf Entenblut keine Kreuzreaktivität. Erst eine genaue Annotation der genomischen Daten wird Aufschluss darüber geben, inwiefern CHIR-homologe Rezeptorfamilien bei anderen Vogelspezies vorhanden und expandiert sind.Six turkey Ig-like receptor (TILR) sequences were identified, composed of one TILR-A, one TILR-B and four TILR-AB sequences. The five amino acid residues, that are essential for the binding to IgY, were conserved in all TILR-AB sequences and the IgY-TILR-AB1 interaction was detectable. Interestingly, TILR-AB1 does not only bind to turkey-IgY, but also to IgY from chicken, quail and pheasant. All of them belong to the bird family phasanidae. The binding of TILR-AB1 to IgY of three particular members outside of this family (grey parrot, hawk and duck) was not measurable. The chicken CHIR family is located on microchromosome 31, in contrast, TILR sequences received from data bases were annotated to macro-chromosome three. Furthermore the expression pattern of CHIR and TILR are different. TILR-A and TILR-AB were shown to be mainly expressed on thrombocytes and macrophages, but not on T- or B-cells. The expression of TILR-B could not be analyzed further due to lack of cross-reactive mab. Most of the TILR-A positive cells also coexpress TILR-AB. There was no evidence in data bases for the existence of CHIR homologues in zebra finch, flycatcher, budgerigar and duck. Also by means of the available anti-CHIR monoclonal antibodies no cross reaction with blood cells of the duck was detectable. Further annotation of different bird genomes will shed light on the situation in other bird species, if they have CHIR-homologue receptor molecules and if so to what extent

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

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

Double fluorescence analyses of turkey PBMC.

<p>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.</p

Alignment of the TILR-A sequences.

<p>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.</p

Immunofluorescence staining of duck and turkey PBMC.

<p>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.</p

Alignment of the TILR-B sequences.

<p>The annotation of sequence features is similar to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059577#pone-0059577-g001" target="_blank">Fig. 1</a>. 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.</p

Inaugural-Dissertation zur Erlangung der tiermedizinischen Doktorwürde

II. LITERATURÜBERSICHT..........................................................................