Immunoglobulin-like receptors in chickens: identification, functional characterization, and renaming to cluster homolog of immunoglobulin-like receptors

Abstract

The cluster homolog of immunoglobulin-like receptors (CHIRs), previously known as the "chicken homolog of immunogloublin-like receptors," represents is a large group of transmembrane glycoproteins that direct the immune response. However, the full repertoire of putatively activating, inhibitory, or dual function CHIRA, CHIRB, and CHIRAB on chickens' immune responses is poorly understood. Herein, the study objective was to determine the genes encoding CHIR proteins and predict their function by searching canonical protein structure. A bioinformatics pipeline based on previous work was employed to search for the CHIRs from the newly updated broiler and layer genomes. The categorization into CHIRA, CHIRB, and CHIRAB types was assigned through motif searches, multiple sequence alignment, and phylogeny. In total, 150 protein-encoding genes on Chromosome 31 were identified as CHIRs. Gene members of each functional group (CHIRA, CHIRB, CHIRAB) were classified in accordance with previously recognized proteins. The genes were renamed to "cluster homolog of immunoglobulin-like receptors" (CHIRs) to allow for the naming of orthologous genes in other avian species. Additionally, expression analysis of the classified CHIRs across various reinforces their importance as immune regulators and activation in inflammatory tissues. Furthermore, over 1,000 diverse and rare CHIRs variants associated with differential Marek's disease response (P < 0.05) emphasize the impact of CHIRs on shaping avian immune responses in diverse contexts. The practical applications of these findings encompass advancing immunology, improving poultry health management, optimizing breeding programs for disease resistance, and enhancing overall animal health through a deeper understanding of the roles and functions of CHIRA, CHIRB, and CHIRAB types in avian immune responses.

Keywords: bioinformatics; chicken; cluster homolog of immunoglobulin-like receptors; genome and transcriptome; immunogenetics.

Figure 1

Overview of pipeline and assembly products identified. (A) Process used to identify putative cluster homolog of immunoglobulin-like receptor (CHIR) proteins from GRCg7b and GRCg7w assemblies including “Immunoglobulin domain,” “Region of a membrane-bound protein predicted to be embedded in the membrane,” and “MHC CLASS I NK CELL RECEPTOR” descriptors which were funneled through chromosomal localization, and motif and amino acid search. (B) Counts of predicted protein isoforms and their respective encoding genes. (C) Proportion and counts of genes uncorrected, corrected, newly annotated, or of unknown functional class. (D) Localization of genes on chromosome 31. Blue links denote shared genes.

Figure 2

Phylogenetic analysis and subgrouping of identified cluster homolog of immunoglobulin-like receptors (CHIRs). (A) The phylogenetic tree visually represents the relationship among the identified CHIRs in broiler and layer chickens, accompanied by the refinement of annotations. The functional groups denoted as green, pink, and yellow, correspond to the activatory (CHIRA), inhibitory (CHIRB), or bifunctional (CHIRAB) types, respectively. To simplify the presentation, CHIR-Like proteins (CHIRLs), among others, were collapsed, resulting in 142/367 proteins shown. Predicted CHIRB (XP_04678998.1) and CHIRAB protein (XP_046761963.1) were excluded due to their disparate alignment. (B) The membrane topology representation depicts the arrangement of positive control proteins, specifically CHIRA (NP_001139610.2) and CHIRB (NP_001139612.3), which were utilized for the purpose of subgroup naming. (C) The count of CHIR genes is categorized by functional group, and a word cloud visualization is illustrated to represent the relative sizes of the subgroups.

Figure 3

Conservation of cluster homolog of immunoglobulin-like receptor CHIRs in other poultry species. (A) Phylogenetic tree representing the relationship between putative CHIR in turkey, helmeted guineafowl, zebra finch, and chicken. Highlighted in green (CHIRA, activatory), pink (CHIRB, inhibitory), and yellow (CHIRAB, bifunctional) are representative chicken CHIRs as detailed in Supplementary Table 2. Predicted protein sequences are annotated with immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and positive amino acids. Green circles denote protein sequences that contain a positive amino acid in the transmembrane domain but no ITIM motifs (CHIRA). Pink squares denote the protein sequences with only ITIM motifs and no positive amino acid in the transmembrane domain (CHIRB). The yellow star denotes proteins with ITIM motifs and a positive amino acid of either arginine (R), lysine (K), or histidine (H) in the transmembrane domain (CHIRAB). (B) PCA clustering of CHIR proteins for each species. (C) 3D modeling illustrating the conservation of amino acids (in green) across species on representative chicken CHIRA (XP_046760817.1), CHIRB (XP_040510212.1), and CHIRAB (XP_040512631.1). Positive amino acids within the transmembrane are shown in blue, while ITIMs are depicted in red.

Figure 4

Chromosomal gene synteny and structural clustering of orthologs. (A) Homologous Ig-like receptor gene clusters on chromosome 19 of human (leukocyte immunoglobulin-like receptor, LILR, killer-cell immunoglobulin-like receptor, KIR), chromosome 1 of rat (Lilrs, paired immunoglobulin-like receptors, Pirs, and Kir3dl1), and chromosome 31 of chicken (cluster homolog of immunoglobulin-like receptor, CHIRs), and their gene neighbors. The reference allele and no alternative haplotypes on the current genome assemblies are illustrated (human, GCF_000001405.40, rat, GCF_015227675.2, broiler GCF_016699485.2, and layer GCF_016700215.2), and not all LILRs or KIRs could be shown. (B) Structural clustering of all CHIR proteins from this study with all Ig-like receptor proteins from the human and the rat. A total of 762 proteins from the above genome assemblies were clustered (n = 371 of chicken, n = 342 of human, and n = 49 of rat). Left, Venn diagram illustrating numbers of shared or unique protein clusters. Right, top protein clusters and their representative Swiss-Prot protein hit, along with GO annotation in support of the predicted shared or unique biological function or cellular process. Bottom, phylogenetic analysis describing the relative distance of CHIR protein clusters with human and rat Ig-like receptors clusters.

Figure 5

Expressions of cluster homolog of immunoglobulin-like receptor (CHIRs) in various tissues of the red jungle fowl after alignment to the broiler assembly. (A) Average CHIR gene expression profiles in various male and female tissues. Functional groups are depicted proportionally, with green indicating CHIRA, pink representing CHIRB, and yellow denoting CHIRAB for activatory, inhibitory, and bifunctional types, respectively. The observed high expression threshold serves as a reference scale corresponding to CHIR abundance's inflection point, as seen in Supplementary Figure 4. (B) Relative gene expression levels of CHIRs compared to other regions on chromosome 31. Note that the data relating to CHIR expression in the adrenal gland of the female were not available.

Figure 6

Presence of cluster homolog of immunoglobulin-like receptor (CHIRs) gene variations in inbred chicken line sequences after alignment to the broiler assembly. (A) Schematic of variants across CHIRs. Only frameshift variants of the noncoding region changes were indicated for visualization purposes. (B) CHIR mutations by functional group annotated, normalized for gene number. Homozygous reference alleles (0|0), an allelic variant (0|1), and homozygous for alternative allelic variants (1|1) are indicated. (C) Relative heterozygosity of variant calls along the chromosome. (D) Tajima's D, a measure of observed to expected nucleotide diversity. (E) Inbred chicken CHIR variant clustering through principal component analysis.