Unusual evolutionary conservation and further species-specific adaptations of a large family of nonclassical MHC class Ib genes across different degrees of genome ploidy in the amphibian subfamily Xenopodinae

Abstract

Nonclassical MHC class Ib (class Ib) genes are a family of highly diverse and rapidly evolving genes wherein gene numbers, organization, and expression markedly differ even among closely related species rendering class Ib phylogeny difficult to establish. Whereas among mammals there are few unambiguous class Ib gene orthologs, different amphibian species belonging to the anuran subfamily Xenopodinae exhibit an unusually high degree of conservation among multiple class Ib gene lineages. Comparative genomic analysis of class Ib gene loci of two divergent (~65 million years) Xenopodinae subfamily members Xenopus laevis (allotetraploid) and Xenopus tropicalis (diploid) shows that both species possess a large cluster of class Ib genes denoted as Xenopus/Silurana nonclassical (XNC/SNC). Our study reveals two distinct phylogenetic patterns among these genes: some gene lineages display a high degree of flexibility, as demonstrated by species-specific expansion and contractions, whereas other class Ib gene lineages have been maintained as monogenic subfamilies with very few changes in their nucleotide sequence across divergent species. In this second category, we further investigated the XNC/SNC10 gene lineage that in X. laevis is required for the development of a distinct semi-invariant T cell population. We report compelling evidence of the remarkable high degree of conservation of this gene lineage that is present in all 12 species of the Xenopodinae examined, including species with different degrees of ploidy ranging from 2, 4, 8 to 12 N. This suggests that the critical role of XNC10 during early T cell development is conserved in amphibians.

Fig 1. Genomic organization of X. tropicalis and X. laevis nonclassical MHC genes (SNC and XNC)

Organization of the 29 SNC and 21 XNC genes including pseudogenes, indicated by Ψ. SNC genes are located on chromosome 8 and XNC genes are located on two different scaffolds encompassing a total of 0.8 Mbp (drawn to scale). Scaffold numbers for X. laevis are based on the Xenopus laevis Genome project http://xenopus.lab.nig.ac.jp, XenVis 2.0 assembly and chromosome number and gene location for X. tropicalis are based on the USSC genome bioinformatics Xenopus genomes hosted at NIMR (http://genomes.nimr.mrc.ac.uk, July, 2013 assembly. Expressed genes, as supported by EST identifications and/or RT-PCR, are indicated by * and arrows indicate transcriptional orientation. Flanking non-class Ib genes are shown as white boxes. Orthologous relationships defined by multiple sequence alignments and phylogenetic analysis between XNC and SNC genes are indicated by dashed lines.

Fig 2. The phylogenetic relationships of XNC and SNC α1/α2 domains compared to selected vertebrate classical and nonclassical MHC genes

The neighbor-joining tree was constructed from amino acid alignments of the alpha 1 and alpha 2 domains using pairwise gap deletions and the p-distance method to estimate evolutionary distance. The tree was drawn using MEGA 5.2. and confidence values were measured using 10,000 bootstrap replications with the values indicated at key nodes and * indicating values < 50. Arrows indicate non-classical subfamilies within the Xenopodinae without clear orthologous relationships between X. laevis and X.tropicalis. Species abbreviations are: Xl, X. laevis, Xt, X. tropicalis; Hs, human; Mm, mouse; Ss, pig; Gg, chicken; Me, Tammar wallaby; Wd, short-tailed opossum, Oa, sheep; Ec, horse; Cs, rhinoceros; Dn, nine-banded armadillo; Rp, northern leopard frog and Amcr, Galapagos marine iguana.

Fig 3. Multiple deduced amino acid sequence alignment and phylogenetic relationships of the α3 domains of X. tropicalis and X. laevis nonclassical MHC genes (SNC and XNC)

(A) Deduced amino acid alignment of XNC and SNC α3 domains with X. laevis and X. tropicalis MHC class Ia. A consensus sequence is shown at the top and dots indicate amino acids identical to this sequence; (-) represent gaps in the alignment and conserved cysteines are in bold and underlined. The MHC class I CD8 binding site is boxed and indicated in bold. (B) The neighbor-joining tree was constructed from amino acid alignments of the alpha 3 domains using pairwise gap deletions and the p-distance method to estimate evolutionary distance. The tree was drawn using MEGA 5.2. and confidence values were measured using 10,000 bootstrap replications with the values indicated at key nodes with * indicating values <50. Species abbreviations are: Xl, X. laevis, St, S. tropicalis; Hs, human; Mm, mouse; Ss, pig; Gg, chicken; Me, tammar wallaby; Wd, short-tailed opossum, Oa, sheep; Ec, horse; Cs, rhinoceros; Dn, nine-banded armadillo; Rp,northern leopard frog and Amcr, Galapagos marine iguana.

Fig 3. Multiple deduced amino acid sequence alignment and phylogenetic relationships of the α3 domains of X. tropicalis and X. laevis nonclassical MHC genes (SNC and XNC)

(A) Deduced amino acid alignment of XNC and SNC α3 domains with X. laevis and X. tropicalis MHC class Ia. A consensus sequence is shown at the top and dots indicate amino acids identical to this sequence; (-) represent gaps in the alignment and conserved cysteines are in bold and underlined. The MHC class I CD8 binding site is boxed and indicated in bold. (B) The neighbor-joining tree was constructed from amino acid alignments of the alpha 3 domains using pairwise gap deletions and the p-distance method to estimate evolutionary distance. The tree was drawn using MEGA 5.2. and confidence values were measured using 10,000 bootstrap replications with the values indicated at key nodes with * indicating values <50. Species abbreviations are: Xl, X. laevis, St, S. tropicalis; Hs, human; Mm, mouse; Ss, pig; Gg, chicken; Me, tammar wallaby; Wd, short-tailed opossum, Oa, sheep; Ec, horse; Cs, rhinoceros; Dn, nine-banded armadillo; Rp,northern leopard frog and Amcr, Galapagos marine iguana.

Fig 4. XNC/SNC10 sequence analysis

Deduced amino acid alignment of (A) alpha1/exon 2 and (B) alpha 2/exon 3 of XNC/SNC10 genes from different Xenopodinae species. Dots indicates amino acid identical to X. laevis XNC10.1 and grey shading indicate amino acids identical to X. tropicalis SNC10 while (-) represents gaps in the alignment. Sequences from the different species grouping as either XNC10.1, XNC10.2 or SNC10 are indicated on the right and * indicate that the gene is expressed. Putative peptide anchoring residues, based on alignment with human HLA-A, are boxed and indicated at the bottom.

Fig 4. XNC/SNC10 sequence analysis

Deduced amino acid alignment of (A) alpha1/exon 2 and (B) alpha 2/exon 3 of XNC/SNC10 genes from different Xenopodinae species. Dots indicates amino acid identical to X. laevis XNC10.1 and grey shading indicate amino acids identical to X. tropicalis SNC10 while (-) represents gaps in the alignment. Sequences from the different species grouping as either XNC10.1, XNC10.2 or SNC10 are indicated on the right and * indicate that the gene is expressed. Putative peptide anchoring residues, based on alignment with human HLA-A, are boxed and indicated at the bottom.

Fig 5. Phylogenetic relationships between XNC/SNC10 gene lineage within multiple species of the Xenopodinae subfamily

Neighbor-joining trees were constructed based on amino acid alignments of (A) alpha1/exon 2 and (B) alpha 2/exon 3 of XNC/SNC10 sequences from different Xenopodinae species. Trees were rooted with X. laevis and X. tropicalis MHC class Ia genes, accession numbers: ABA43373.1 and AAP36728.1 respectively. The trees were drawn using MEGA 5.2. using pairwise gap deletions and the p-distance method to estimate evolutionary distance and confidence values were measured using 10,000 bootstrap replications with the values indicated at key nodes. Trees generated based on nucleotide alignments resulted in similar topology (data not shown).

Fig 6. Differential expression of XNC10.1 and XNC10.2

Expression of XNC10.1 and XNC10.2 in various tissues of X. laevis and developmental stage 53/54 tadpoles. Results are normalized to an endogenous control (GAPDH) and expressed as fold change compared to expression of XNC10.2 in spleen. All results are presented as mean ± s.e.m, n = 5 for each group.