Genomic organization and classification of the bovine WC1 genes and expression by peripheral blood gamma delta T cells

Abstract

Background: WC1 co-receptors are group B scavenger receptor cysteine-rich molecules that are found exclusively on gammadeltaT cells and are thought to be encoded by a multi-gene family. Previous studies have shown gammadeltaT cells that respond to a particular stimulus have unique WC1 molecules expressed. Prior to the onset of the studies described here only one full-length WC1 nucleotide sequence was publicly available, though three WC1 molecules had been distinguished based on monoclonal antibody reactivity. Furthermore, the number of WC1 genes found in the bovine genome and their sequences had not yet been resolved.

Results: By annotating the bovine genome Btau_3.1 assembly, here we show the existence of 13 members in the WC1 gene family and their organization within two loci on chromosome 5 including three distinct exon-intron gene structures one of which coded for a potentially more primitive and smaller WC1 molecule that is similar to the swine WC1 gene. We also provide cDNA evidence as verification for many of the annotated sequences and show transcripts for isoforms derived by alternative splicing.

Conclusion: It is possible that WC1 diversity contributes to functional differences that have been observed between gammadeltaT cell populations. The studies described here demonstrate that WC1 molecules are encoded by a large, multi-gene family whose transcripts undergo extensive alternative splicing. Similar to other non-rearranging immunoreceptors, it is likely that the WC1 gene repertoire underwent expansion in order to keep pace with rapidly changing ligands.

Figure 1

Schematic representation of WC1 exon-intron structure. Three structures were identified and were found to contain differing numbers of exons. Representative structures of WC1 genes containing (A) 20 exons (Type I; based on genomic sequence for WC1-5, GLEAN_13176) (B) 21 exons (Type II; based on genomic sequence for WC1-9, GLEAN_12191) (C) 15 exons (Type III, based on genomic sequence for WC1-11, GLEAN_09904 and GLEAN_12182) are shown. Exon numbers and SRCR domain numbers are indicated. Scale is shown in base pair increments beneath the schematic. Abbreviations are as follows: ID, inter-domain sequence; TM, transmembrane region; ICD, intracytoplasmic domain.

Figure 2

Schematic representation of WC1 loci organization. 13 WC1 genes were found within two loci on chromosome 5 with CD163, another group B SRCR molecule, found within one of the WC1 loci. WC1 gene designations, orientations and Bovine Genome Scaffold identifications are as indicated. Diagram is not shown to scale. WC1-11 was identified in two gene prediction models; however, evidence suggests that there is only a single WC1-11 gene.

Figure 3

Alignment of WC1 deduced amino acid sequences identified in the genome (part 1). Full-length deduced amino acid sequences of the annotated WC1 genes were aligned using ClustalW2 and the default parameters and were refined by hand. The archetypal WC1 sequence (WC1.1, GenBank accession number X63723) was included in the analysis for comparison. WC1-2, WC1-3, WC1-6, WC1-8 and WC1-12 sequences are partial due to gaps in the genome sequence. Gene types (I, II or III), as determined based on exon-intron structure are indicated to the left of the sequences. Identities are indicated by dots (.), gaps resulting from the alignment are indicated by tildes (~), gaps resulting from lack of genomic sequence (when the gaps were found adjacent and not within a coding region) are indicated by dashes (-). SRCR domains are indicated in roman numerals and the transmembrane region is shown underlined for the archetypal WC1.1 sequence.

Figure 4

Alignment of WC1 deduced amino acid sequences identified in the genome (part 2). Full-length deduced amino acid sequences of the annotated WC1 genes were aligned using ClustalW2 and the default parameters and were refined by hand. The archetypal WC1 sequence (WC1.1, GenBank accession number X63723) was included in the analysis for comparison. WC1-2, WC1-3, WC1-6, WC1-8 and WC1-12 sequences are partial due to gaps in the genome sequence. Gene types (I, II or III), as determined based on exon-intron structure are indicated to the left of the sequences. Identities are indicated by dots (.), gaps resulting from the alignment are indicated by tildes (~), gaps resulting from lack of genomic sequence (when the gaps were found adjacent and not within a coding region) are indicated by dashes (-). SRCR domains are indicated in roman numerals and the transmembrane region is shown underlined for the archetypal WC1.1 sequence.

Figure 5

Alignment of WC1 deduced amino acid sequences identified in the genome (part 3). Full-length deduced amino acid sequences of the annotated WC1 genes were aligned using ClustalW2 and the default parameters and were refined by hand. The archetypal WC1 sequence (WC1.1, GenBank accession number X63723) was included in the analysis for comparison. WC1-2, WC1-3, WC1-6, WC1-8 and WC1-12 sequences are partial due to gaps in the genome sequence. Gene types (I, II or III), as determined based on exon-intron structure are indicated to the left of the sequences. Identities are indicated by dots (.), gaps resulting from the alignment are indicated by tildes (~), gaps resulting from lack of genomic sequence (when the gaps were found adjacent and not within a coding region) are indicated by dashes (-). SRCR domains are indicated in roman numerals and the transmembrane region is shown underlined for the archetypal WC1.1 sequence.

Figure 6

Alignment of WC1 deduced amino acid sequences identified in the genome (part 4). Full-length deduced amino acid sequences of the annotated WC1 genes were aligned using ClustalW2 and the default parameters and were refined by hand. The archetypal WC1 sequence (WC1.1, GenBank accession number X63723) was included in the analysis for comparison. WC1-2, WC1-3, WC1-6, WC1-8 and WC1-12 sequences are partial due to gaps in the genome sequence. Gene types (I, II or III), as determined based on exon-intron structure are indicated to the left of the sequences. Identities are indicated by dots (.), gaps resulting from the alignment are indicated by tildes (~), gaps resulting from lack of genomic sequence (when the gaps were found adjacent and not within a coding region) are indicated by dashes (-). SRCR domains are indicated in roman numerals and the transmembrane region is shown underlined for the archetypal WC1.1 sequence.

Figure 7

WC1 Domain 1 sequence. (A) WC1 Domain 1 deduced amino acid sequences were aligned with ClustalW2 using the default parameters and visualized with JalView. Analysis includes all non-redundant genomic sequences and all non-redundant cDNA sequences. The archetypal WC1 sequence (WC1.1, GenBank accession number X63723) and swine WC1 sequence (swppWC1, GenBank accession number CAA67710) were included in the analysis for comparison. WC1-6 sequence is partial due to poor genomic sequence integrity. (B) Phylogenetic tree generated using WC1 Domain 1 deduced amino acid sequences and the Neighbor-Joining method [67]. Archetypal WC1 (WC1.1) and swine WC1 (swppWC1) Domain 1 sequences (accession numbers above) were included for comparison and archetypal WC1 Domain 2 (WC1.1D2) sequence was included to root the tree. The optimal tree with the sum of branch length = 2.02452921 is shown with bootstrap values (based on 1000 replicates) reported next to the branches. Positions containing alignment gaps were eliminated only in pairwise sequence comparisons for a total of 112 positions in the final dataset.

Figure 8

WC1 Domain 9 sequence. (A) WC1 Domain 9 deduced amino acid sequences were aligned with ClustalW2 using the default parameters and visualized with JalView. Analysis includes all non-redundant genomic sequences and all non-redundant cDNA sequences. The archetypal WC1 sequence (WC1.1, GenBank accession number X63723) was included in the analysis for comparison. (B) Phylogenetic tree generated using WC1 Domain 9 deduced amino acid sequences and the Neighbor-Joining method [67]. Archetypal WC1 Domain 9 (WC1.1) sequence (accession number above) was included for comparison and archetypal WC1 Domain 2 (WC1.1D2) sequence was included to root the tree. The optimal tree with the sum of branch length = 1.02259498 is shown with bootstrap values (based on 1000 replicates) reported next to the branches. Positions containing alignment gaps were eliminated only in pairwise sequence comparisons for a total of 106 positions in the final dataset.

Figure 9

WC1 intracytoplasmic region sequence. (A) WC1 intracytoplasmic region deduced amino acid sequences were aligned with ClustalW2 using the default parameters and visualized with JalView. Analysis includes all non-redundant genomic sequences and all non-redundant cDNA sequences and sequences were truncated to begin at the second intracytoplasmic tail encoding exon. The archetypal WC1 (WC1.1, GenBank accession number X63723) and swine WC1 (swppWC1, GenBank accession number CAA67710; swWC1-29e1, GenBank accession number CAA67709) sequences were included in the analysis for comparison. (B) Phylogenetic tree generated using WC1 intracytoplasmic tail deduced amino acid sequences and the Neighbor-Joining method [67]. Archetypal WC1 (WC1.1) and swine WC1 (swppWC1 and swWC1-29e1) intracytoplasmic sequences (accession numbers above) were included for comparison. The optimal tree with the sum of branch length = 1.48497595 is shown with bootstrap values (based on 1000 replicates) reported next to the branches. Positions containing alignment gaps were eliminated only in pairwise sequence comparisons for a total of 213 positions in the final dataset.

Figure 10

WC1 isoform generation. Schematic representations of 35 WC1 cDNA sequences derived from pooled mRNA from ex vivo and activated total PBMC taken from a single animal. The cDNA clone names upon which the schematics are based are indicated in parentheses to the right of the schematics. Where available, full-length cDNA sequences are shown with putative alternative splice variants shown below the full-length sequence of the same pattern. cDNA sequences were considered to be alternative splice variants of a particular full-length sequence when percent identity of the existing deduced amino acid sequence was ≥ 98%.