Gene number determination and genetic polymorphism of the gamma delta T cell co-receptor WC1 genes

Abstract

Background: WC1 co-receptors belong to the scavenger receptor cysteine-rich (SRCR) superfamily and are encoded by a multi-gene family. Expression of particular WC1 genes defines functional subpopulations of WC1(+) γδ T cells. We have previously identified partial or complete genomic sequences for thirteen different WC1 genes through annotation of the bovine genome Btau_3.1 build. We also identified two WC1 cDNA sequences from other cattle that did not correspond to sequences in the Btau_3.1 build. Their absence in the Btau_3.1 build may have reflected gaps in the genome assembly or polymorphisms among animals. Since the response of γδ T cells to bacterial challenge is determined by WC1 gene expression, it was critical to understand whether individual cattle or breeds differ in the number of WC1 genes or display polymorphisms.

Results: Real-time quantitative PCR using DNA from the animal whose genome was sequenced ("Dominette") and sixteen other animals representing ten breeds of cattle, showed that the number of genes coding for WC1 co-receptors is thirteen. The complete coding sequences of those thirteen WC1 genes is presented, including the correction of an error in the WC1-2 gene due to mis-assembly in the Btau_3.1 build. All other cDNA sequences were found to agree with the previous annotation of complete or partial WC1 genes. PCR amplification and sequencing of the most variable N-terminal SRCR domain (domain 1 which has the SRCR "a" pattern) of each of the thirteen WC1 genes showed that the sequences are highly conserved among individuals and breeds. Of 160 sequences of domain 1 from three breeds of cattle, no additional sequences beyond the thirteen described WC1 genes were found. Analysis of the complete WC1 cDNA sequences indicated that the thirteen WC1 genes code for three distinct WC1 molecular forms.

Conclusion: The bovine WC1 multi-gene family is composed of thirteen genes coding for three structural forms whose sequences are highly conserved among individual cattle and breeds. The sequence diversity necessary for WC1 genes to function as a multi-genic pattern recognition receptor array is encoded in the genome, rather than generated by recombinatorial diversity or hypermutation.

Figure 1

Schematic representation and primer placement of the predicted structure of WC1 Domain 1. Secondary structure motifs are indicated above (beta-strand arrows (β1-β6) and an alpha-helix arrow). The RVEVLxxxxW bacterial binding motif is circled. The two regions (VR1 and VR2) that contain most of the sequence diversity between WC1 SRCR domain 1s are boxed. Cysteines are indicated in black [29]. The common forward and reverse primers were designed based on the conserved region and denoted by a single asterisk (*), while the specific forward primers for each WC1 gene are denoted by double asterisks (**).

Figure 2

Q-PCR for WC1 gene number. (A) The standard curves for primer sets of WC1s (WC1-com) and other control genes. The standard curves were constructed with a series of 10-fold dilution of cDNA derived from ex vivo PBMC. Each standard dilution was amplified by real-time QPCR in duplicate. For each primer set, C_T values determined from real-time QPCR were plotted against the logarithm of their known initial gene numbers. A standard curve was generated by linear regression through these points. (B) The slopes of the standard curves for primers of WC1s (WC1-com) and other control genes. From the slopes, amplification efficiencies were also determined. (C) Primer specificities of WC1s (WC1-com) and other control genes’ primer sets. Confirmation of PCR amplification specificities by melting curve for primer sets of WC1 common (WC1-com), bovine TRDJ1, bovine GAPD, bovine IFNA, bovine IFNB, bovine IFNE, and bovine IFNW. Melting peaks were examined for WC1 common (WC1-com, solid line with empty diamond marker), bovine TRDJ1 (solid line with solid circle marker), bovine GAPD (solid line), bovine IFNA (solid line with solid square marker), bovine IFNB (solid line with solid triangle marker), bovine IFNE (solid line with empty circle marker), and bovine IFNW (solid line with empty square marker). (D) Gene numbers of bovine WC1 genes and other control genes. The breeds of tested cattle are indicated in the figure. The ΔΔC_T method was applied for relative quantification. Some breeds contained more than one animal, and each evaluation was performed at least twice, yielding similar results.

Figure 3

Specific amplification of each WC1 Domain 1. (A) Primer specificities of thirteen WC1 specific primer sets. Confirmation of PCR amplification specificities by gel electrophoresis for thirteen WC1 specific primer sets (WC1-1, WC1-3, WC1-4, WC1-5, WC1-6, WC1-7, WC1-9, WC1-10, WC1-11, WC1-12, WC1-13, WC1-nd1, and WC1-nd2) and the bovine IFNB primer pair. Plasmids containing thirteen WC1 domain 1 gene sequences were used as templates in PCR reactions. For each primer set, the identities of the amplified products were confirmed by DNA sequencing analysis. (B) Genomic DNA and cDNA evidence for Dominette. PCR analysis was conducted by using genomic DNA and cDNA derived from the reference animal Dominette of the Hereford breed (designed with prefixes of “D_gDNA and D_cDNA”, respectively). Primer pairs for amplification of thirteen WC1 SRCR domain 1 sequences with the specific primer sets tested in (A). For each primer set, the identities of the amplified products were confirmed by DNA sequencing analysis.

Figure 4

Amplification of full-length transcripts for WC1s. (A) Primers designed for complete coding sequences of all thirteen WC1 genes. Schematic representations of the molecular forms of archetypal WC1 genes and WC1-11 with primer placement indicated. The WC1 common forward primer (WC1atg-for) for complete coding sequences was designed based on the conserved region in the signal sequences, while the reverse primers (WC1group1,2-rev and WC1group3-rev) were based on the end of the 3^′ coding sequences. Abbreviations are as follows: ID, inter-domain sequence; TM, transmembrane region; ICD, intracytoplasmic domain. (B) cDNA evidence for WC1 genes. Primer pairs WC1atg-for/WC1group1,2-rev (designed for WC1group1,2) and primer set WC1atg-for/WC1group3-rev (designed for WC1group3) were used to amplify all the complete coding sequences of WC1 transcripts as described in the previous study [3]. (C) Confirmation of complete coding sequences for WC1-nd1 and WC1-nd2. Four different templates used in PCR for all thirteen WC1 domain 1 specific primer pairs are indicated in the left part of each gel. (D) Agarose gel electrophoresis evidence for complete coding sequences of WC1-nd1 and WC1-nd2. Complete coding sequences of WC1-nd1 and WC1-nd2 amplified by primer pairs of specific forward primers and common reverse primers (WC1group1,2-rev). The cDNA isolated from sorted WC1.1⁺ γδ T cells was used as a template. Gel electrophoresis of the PCR products was performed on 1% agarose gel. For each primer set, the identity of the amplified products was confirmed by DNA sequencing analysis.

Figure 5

Complete sequences of cDNA clones CC6 (WC1-6), CC7 (WC1-7), CC12 (WC1-12), CCnd1 (WC1-nd1) and CCnd2 (WC1-nd2). Deduced amino acids sequence from the coding sequences of WC1-1, WC1-2, WC1-8, and WC1-13, and the archetypal WC1 (Wc1.1) sequence were aligned using ClustalW2 and the default parameters and refined manually. GenBank accession numbers for amino acid sequences used for comparison are described in Materials and Methods. 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 archetypal WC1 sequence. Continued in Figure 6.

Figure 6

Complete sequences of cDNA clones CC6 (WC1-6), CC7 (WC1-7), CC12 (WC1-12), CCnd1 (WC1-nd1) and CCnd2 (WC1-nd2), continued. Deduced amino acids sequence from the coding sequences of WC1-1, WC1-2, WC1-8, and WC1-13, and the archetypal WC1 sequence (WC1.1) were aligned using ClustalW2 and the default parameters and refined manually. GenBank accession numbers for amino acid sequences used for comparison are described in Materials and Methods. 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 archetypal WC1 sequence.

Figure 7

Structure of the mis-assembled WC1-2. Schematic representation of WC1-2 with the eleven exons annotation as in a previous study (not shown to scale, [3]). The gap adjacent to the coding region of WC1-2 in the Btau 3.1 assembly is indicated by double slashes (//). Abbreviations are as follows: D, SRCR domain; ID, inter-domain sequence; TM, transmembrane region; T, intracytoplasmic tail exon numbers.

Figure 8

Genomic DNA and cDNA evidence for the 13 WC1 genes in two bovine breeds. PCR analysis was conducted by using genomic DNA and cDNA derived from cattle of two different breeds (Belted Galloway, designed with prefixes of “Y_gDNA and Y_cDNA”, respectively; Holstein, designed with prefixes of “B_gDNA and B_cDNA”, respectively). Primer pairs for distinguishing thirteen WC1 domain 1s were thirteen WC1 specific primer sets. For each primer set, the identities of the amplified products were confirmed by DNA sequencing analysis.

Figure 9

Comparison of genomic and cDNA WC1 domain 1 sequences for different bovine breeds. WC1 domain 1 deduced amino acid sequences were aligned with ClustalW2 using the default parameters and visualized with Bioedit. Analysis includes all non-redundant genomic sequences (denoted by gray shades) and cDNA sequences. Annotated sequences were designed with a prefix of “A”. Sequences obtained from individuals of three breeds (Hereford, Belted Galloway, and Holstein) were designed with prefixes of “D”, “Y”, and “B”, respectively. Identities are indicated by dots (.). Gaps resulting from the alignment are indicated by tildes. Amino acids representing heterozygous alleles are separated by a slash.

Figure 10

Phylogenetic tree of WC1 domain 1 sequences. The evolutionary history of 90 taxa was inferred using Bayesian analysis in MrBayes3.2 [33]. Annotated sequences were designed with a prefix of “A”. Sequences obtained from individual animal of three breeds (Hereford, Belted Galloway, and Holstein) were designed with prefixes of “D”, “Y”, and “B”, respectively. Genomic DNA and transcripts sequences were designed with prefixes of “G and cDNA”, respectively. Markov chain Monte Carlo analysis was performed for 830,000 cycles, using 2 runs of 4 chains each, a temperature setting of 0.2, and an amino acid mixed model to approximate the posterior probabilities of trees, shown at branch nodes. The average standard deviation of split frequencies was 0.01, which was diagnostic of convergence at <0.05. Bootstrap values >70 are shown.

Figure 11

Schematic representations of three types of WC1 molecules. The WC1 genes corresponding to the schematics are indicated. WC1⁺ γδ T cells were defined based on mAb reactivity as WC1.1⁺, WC1.2⁺, and WC1.3⁺ wherein the WC1.3⁺ population is a subpopulation of WC1.1⁺ cells [7]. According to their domain 1 sequence similarities with bovine archetypal WC1.1 (WC1-3), archetypal WC1.2 (WC1-4) or swine WC1 domain 1 sequences, WC1 genes were further classified into groups: WC1.1-like, WC1.2-like, unique, and swine-like. Among WC1.1-like WC1 genes, WC1-8 (nd2) represents the gene product which reacts with the anti-WC1.3 mAb (Chuang and Baldwin, unpublished data) and which is different from the WC1.3 gene sequence reported by Wijngaard et al. [7]. No WC1 genes have the SRCR domain 1 sequences reported by Wijngaard et al. [7]; instead, the most similar sequence is that of SRCR domain 6 of WC1-4 and WC1-9, and we thus suggest part of the published WC1.3 sequence is erroneous.