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. 2012 Aug 15:13:72.
doi: 10.1186/1471-2156-13-72.

Pseudogenization of the MCP-2/CCL8 chemokine gene in European rabbit (genus Oryctolagus), but not in species of Cottontail rabbit (Sylvilagus) and Hare (Lepus)

Wessel van der Loo  1 , Sandra Afonso, Ana Lemos de Matos, Joana Abrantes, Pedro J Esteves
Affiliations

Pseudogenization of the MCP-2/CCL8 chemokine gene in European rabbit (genus Oryctolagus), but not in species of Cottontail rabbit (Sylvilagus) and Hare (Lepus)

Wessel van der Loo et al. BMC Genet. .

Abstract

Background: Recent studies in human have highlighted the importance of the monocyte chemotactic proteins (MCP) in leukocyte trafficking and their effects in inflammatory processes, tumor progression, and HIV-1 infection. In European rabbit (Oryctolagus cuniculus) one of the prime MCP targets, the chemokine receptor CCR5 underwent a unique structural alteration. Until now, no homologue of MCP-2/CCL8a, MCP-3/CCL7 or MCP-4/CCL13 genes have been reported for this species. This is interesting, because at least the first two genes are expressed in most, if not all, mammals studied, and appear to be implicated in a variety of important chemokine ligand-receptor interactions. By assessing the Rabbit Whole Genome Sequence (WGS) data we have searched for orthologs of the mammalian genes of the MCP-Eotaxin cluster.

Results: We have localized the orthologs of these chemokine genes in the genome of European rabbit and compared them to those of leporid genera which do (i.e. Oryctolagus and Bunolagus) or do not share the CCR5 alteration with European rabbit (i.e. Lepus and Sylvilagus). Of the Rabbit orthologs of the CCL8, CCL7, and CCL13 genes only the last two were potentially functional, although showing some structural anomalies at the protein level. The ortholog of MCP-2/CCL8 appeared to be pseudogenized by deleterious nucleotide substitutions affecting exon1 and exon2. By analyzing both genomic and cDNA products, these studies were extended to wild specimens of four genera of the Leporidae family: Oryctolagus, Bunolagus, Lepus, and Sylvilagus. It appeared that the anomalies of the MCP-3/CCL7 and MCP-4/CCL13 proteins are shared among the different species of leporids. In contrast, whereas MCP-2/CCL8 was pseudogenized in every studied specimen of the Oryctolagus - Bunolagus lineage, this gene was intact in species of the Lepus - Sylvilagus lineage, and was, at least in Lepus, correctly transcribed.

Conclusion: The biological function of a gene was often revealed in situations of dysfunction or gene loss. Infections with Myxoma virus (MYXV) tend to be fatal in European rabbit (genus Oryctolagus), while being harmless in Hares (genus Lepus) and benign in Cottontail rabbit (genus Sylvilagus), the natural hosts of the virus. This communication should stimulate research on a possible role of MCP-2/CCL8 in poxvirus related pathogenicity.

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Figures

Figure 1
Figure 1
The rabbit ortholog of human CCL7 gene exists and is transcribed. The “A11 isoform 1” predicted by GenBank suggests that exon1 of the Rabbit ortholog of CCL7 uses preferentially exon2 and exon3 of CCL11 during transcription (Additional file 2). We show that cDNA of the leporid species (Oryctolagus cuniculus, Lepus granatensis and Lepus europaeus) contain a transcript uniting three exons orthologous to those of human CCL7 gene. The position of missing MCP-3 characteristic N-glycosylation site is underlined (N X S in other mammals). Variable amino acid residues are highlighted in gray.
Figure 2
Figure 2
Lacking of N-terminal X-Pro motifs in Leporid MCP -4/CCL13. All known MCP genes encode a 24GlnPro25 (QP) motif which is required for post-transcriptional maturation and modification of the protein. The Rabbit CCL13 ortholog is unique by encoding a 24GlnThr25 motif (QT). This particularity appears to be shared among leporids, inclusive Sylvilagus. Leporid genomic DNA was amplified using CCL13 primers designed on R-MCPgb. orcuB: Rabbit, subsp. cuniculus, orcuA: Rabbit, subsp. algirus, syfl: Western Cottontail Rabbit, Legr: Granada Hare, Orcu ENS13408: [Ensembl: ENSOCUT00000013408]. The MCP-4 protein sequences of “other” mammals are derived from the CCL13 mRNA sequences listed in Additional file 9. eqca: horse, calu: dog, hosa: human, poab: orangutan, mamu: monkey, aime: giant panda.
Figure 3
Figure 3
Gene orthology of CCL2 ,-7 , -11 , -8 , and -13 of placental mammals supported by phylogeny. The evolutionary history was inferred by using the Maximum Likelihood method conducted in MEGA5 [36]. The tree with the highest log likelihood (-4585.9908) is shown. Bootstrap values are placed next to the branches. Codon positions included were 1st + 2nd + 3rd and were limited to those encoding the mature chemokine. There were 231 positions in the final data set. Positions of leporid branches are highlighted by triangles ▴ for functional, or by ∇ for pseudogenes. Taxon names of sequences obtained in this study are extended by ‘gDNA’ when derived from genomic DNA or by ‘cDNA’ when (also) derived from cDNA. All other sequences represent a sample of mRNA sequences listed in Additional file 9. Mouse CCL12 and CCL8 are located between the CCL11 and CCL1 genes and are likely duplicates of the ortholog of murid CCL8[22].
Figure 4
Figure 4
Fishing the Rabbit CCL8 ortholog using NCBI Blast-Align. Human CCL8 mRNA is compared to the Rabbit genomic fragment containing both the CCL2 and the CCL1 region (R-MCPgb). The graphical representation visualizes the outstanding similarity of one genomic region covering the human RNA transcript almost entirely. At the same time it localizes other structurally related regions and reveals that compared to the coding regions, untranslated region (UTR’s) might be more gene-specific. The graph was produced using the online facilities offered by NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi; option: Align).
Figure 5
Figure 5
The Rabbit ortholog of mammalian CCL8 exist as a pseudogene. The “Orcu WGS” sequence shows parts of the exon embedding fragments of R-MCPgb region with outstanding similarity with mammalian MCP-2/CCL8 gene (Figure 4). The alignment with functional mammalian CCL8 clarifies the pseudogenization of Rabbit CCL8 gene. Exon regions are highlighted in gray, disabling mutations are highlighted in black. orcu: rabbit, hosa: human, susc: swine. The protein sequence shown is inferred from the susc_CCL8 sequence. Insertions were added at exon2 in order to maintain the reading frame. Open spaces are introduced at exon boundaries. ‘.’: identity with leader sequence, ‘-‘: deletions/insertions.
Figure 6
Figure 6
Gene variation at the CCL8 locus within and among leporids. PCR products of leporid specimens were obtained using either gDNA or cDNA and are aligned with the CCL8ps sequence of rabbit WGS. Potential Stop and Translation Initiating codons are underlined. The positions of the cDNA primers are bold underlined. ‘Orcu CCL8ps’ represents the consensus of 11 haplotypes of Oryctolagus cuniculus specimens of both subspecies (O. c. cuniculus and O. c. algirus), after excluding one haplotype that was identical to CCL8ps of WGS (OccTar109allele1) and ignoring singletons (i.e. nucleotide differences observed only once). Occ: Oryctolagus cuniculus; Bumo: Bunolagus monticularis; Syfl: Sylvilagus floridanus; Leti: Lepus timidus; Legr: Lepus granatensis; Leeu: Lepus europaeus. ‘.’: identical to leader sequence; ‘-‘: indel. The data shown are limited to the exon containing fractions. An alignment of the entire 1.6Kb gene region is presented in Additional file 7.
Figure 7
Figure 7
Comparing mature MCP2 proteins of Leporid and Other-Mammals. The consensual numbering of amino acid positions used in functional studies of MCP ligands is shown (e.g. [32]). Pseudogenes are translated according to a nucleotide alignment respecting the reading frame of functional homologs. Positions where leporid MCP-2 differs markedly from the mammalian consensus are highlighted in gray if inferred from ‘functional’ genes, or in black in case differences would be limited to pseudogenes. leeu: European brown hare, legr: Granada hare; syfl: Western cottontail rabbit; orcu: European rabbit, sps. cuniculus; oral: European rabbit, sps. algirus, bumo: South African Riverine rabbit. GenBank Accession of the underlying “Other-Mammal” sequences are listed in Additional file 9.
Figure 8
Figure 8
Schematic presentation of primer positions on the rabbit CCL8 ortholog. Primers were designed to cover all exon and intron regions of the proposed Rabbit ortholog of the Human CCL8 gene (1634 bp) [Genbank NC_013687.1:REGION:23767421.23769054]. ‘>’: position of forward primer; ‘<’: of reverse primer; ‘ps’: PCR fragment length.
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