Coevolution of activating and inhibitory receptors within mammalian carcinoembryonic antigen families

Abstract

Background: Most rapidly evolving gene families are involved in immune responses and reproduction, two biological functions which have been assigned to the carcinoembryonic antigen (CEA) gene family. To gain insights into evolutionary forces shaping the CEA gene family we have analysed this gene family in 27 mammalian species including monotreme and marsupial lineages.

Results: Phylogenetic analysis provided convincing evidence that the primordial CEA gene family in mammals consisted of five genes, including the immune inhibitory receptor-encoding CEACAM1 (CEA-related cell adhesion molecule) ancestor. Our analysis of the substitution rates within the nucleotide sequence which codes for the ligand binding domain of CEACAM1 indicates that the selection for diversification is, perhaps, a consequence of the exploitation of CEACAM1 by a variety of viral and bacterial pathogens as their cellular receptor. Depending on the extent of the amplification of an ancestral CEACAM1, the number of CEACAM1-related genes varies considerably between mammalian species from less than five in lagomorphs to more than 100 in bats. In most analysed species, ITAM (immunoreceptor tyrosine-based activation motifs) or ITAM-like motif-containing proteins exist which contain Ig-V-like, ligand binding domains closely related to that of CEACAM1. Human CEACAM3 is one such protein which can function as a CEACAM1 decoy receptor in granulocytes by mediating the uptake and destruction of specific bacterial pathogens via its ITAM-like motif. The close relationship between CEACAM1 and its ITAM-encoding relatives appears to be maintained by gene conversion and reciprocal recombination. Surprisingly, secreted CEACAMs resembling immunomodulatory CEACAM1-related trophoblast-specific pregnancy-specific glycoproteins (PSGs) found in humans and rodents evolved only in a limited set of mammals. The appearance of PSG-like genes correlates with invasive trophoblast growth in these species.

Conclusions: These phylogenetic studies provide evidence that pathogen/host coevolution and a possible participation in fetal-maternal conflict processes led to a highly species-specific diversity of mammalian CEA gene families.

Figure 1

Phylogeny of mammalian carcinoembryonic antigen related cell adhesion molecule (CEACAM) genes. Phylogenetic trees were constructed from N domain exon (A) and B domain exon nucleotide sequences (B) from CEACAM (CC) of cattle, horses, dogs, humans and mice using the neighbour-joining method (MEGA 4.0 software). The reliability of a phylogenetic tree was assessed using the Bootstrap test applying 1000 replicates. The statistical support for selected nodes is shown. Values >70 and >30 are shown in (A) and (B), respectively. Due to space limitations, only selected murine and horse Ceacams/CEACAMs and Psg were included and the topology of the B domain phylogenetic tree is shown (B). N1 domain exons were used from murine Psg genes which contain multiple N domain exons. Five groups of N domain exons can be discriminated. The N domain exon sequences from the CEACAM16, CEACAM18, CEACAM19 and CEACAM20 genes each cluster together, while N exon sequences from CEACAM1-like genes including PSG genes form species-specific groups. Similar groups were discriminated using B domain exons (B). CEACAM1 genes were identified based on the presence of immunoreceptor tyrosine-based inhibition motifs (ITIM)/immunoreceptor tyrosine-based switch motif (ITSM) in the encoded cytoplasmic domains. In horse, two genes with intact ITIM/ITSM were identified and named arbitrarily CEACAM1 and CEACAM43. For abbreviation of species names see Table 1. The bar below the phylogenetic tree in (A) shows the scale for the number of substitutions per site. ^{a, b}, allelic CEACAM1 variants. Multiple B domains present in a CEACAM are indicated by numbers.

Figure 2

Domain organization of mammalian carcinoembryonic antigen (CEA) family members. The domain organization of CEA family members from selected species was predicted by gene analysis and confirmed, where available, by EST (cattle) and cDNA sequences [4,5,27,35]. The conserved members are shown in green, members expressed predominantly in trophoblast cells of the placenta in grey boxes. The predicted signaling motifs in the cytoplasmic domains are schematically shown as green (immunoreceptor tyrosine-based activation motifs; ITAM), blue (ITAM-like motif, no acidic amino acid present at position -1 to -3 from first Y in consensus motif E/Dx_0-2YxxL/Ix_6-8YxxL/I), red (immunoreceptor tyrosine-based inhibition motifs) and yellow boxes (immunoreceptor tyrosine-based switch motif). Due to gaps in the genomic sequences, some domains could not be clearly identified for a few proteins which are indicated by a question mark. C, CEA-related cell adhesion molecule.

Figure 3

Syntenic relationship of the carcinoembryonic antigen related cell adhesion molecule (CEACAM) gene loci in platypus, opossum, human, mouse, cattle and dog genomes. The syntenic chromosome regions from six mammalian species are shown except for platypus where only contig information is available. Arrowheads represent genes with their transcriptional orientation. CEACAM1-related genes are indicated in yellow or red when predominantly expressed in trophoblast cells, the orthologous CEACAM genes in blue and marker genes in black. Names of CEACAM1-like genes with immunoreceptor tyrosine-based inhibition motif/immunoreceptor tyrosine-based switch motif encoding exons are shown in red and with immunoreceptor tyrosine-based activation motif (ITAM) and ITAM-like motif-encoding exons in green and blue, respectively. Clockwise oriented arrows symbolize inversion events of the regions between the two CEACAM1-like gene clusters which have taken place independently in two different clades. Note that, in general, the genes in the same subcluster show the same transcriptional orientation, between the two subclusters opposite transcriptional orientation. The ancestral gene arrangement is unknown. Therefore, indication of species with inversion events is arbitrary; inversions relative to the human gene order are shown. The nucleotide numbering of the chromosomes starts at the telomere of the short arms. The scale indicated by dots is 1 Mbp unless interrupted by slanted lines. Note the inverse orientation of the opossum chromosome 4, cattle chromosome 18 and human chromosome 19 regions. C, CEACAM/Ceacam; chr, chromosome; P, PSG/Psg.

Figure 4

Identification of functionally important extracellular regions of carcinoembryonic antigen related cell adhesion molecules (CEACAMs) by multispecies sequence comparisons. N domain exon nucleotide sequences from orthologous CEACAMs of a selected set of mammalian species were aligned using the program ClustalW and the results were displayed as unrooted dendrogram (A). Note the high and low conservation of the CEACAM16 N2 and CEACAM1 N nucleotide sequences, respectively, as reflected by the size of the colored area. In order to determine the selective pressure on the maintenance of the nucleotide sequences, the number of nonsynonymous nucleotide substitution per nonsynonymous site (dN) and the number of synonymous substitutions per synonymous site (dS; B) as well as the accumulation of nonsynonymous and synonymous substitutions along immunoglobulin (Ig) variable- and constant-like domain exons (C) were determined after manual removal of sequence gaps for the following species: cattle, dog, human, mouse, opossum, rat and rhesus macaque (due to missing sequence information for the rhesus macaque CEACAM1 A2 exon, the corresponding exon from cat was used instead). The dN/dS ratios were calculated after manual editing of sequence gaps or insertions guided by the amino acid sequences for all branches of the resulting phylogenetic trees and displayed as mean values and standard deviations. Whole N domain exons were used for analysis including the regions (the first 12 codons) which encode part of the leader. Due to the variable truncation of the CEACAM20 N domain exons, the first common 27 codons were analysed. The mean dN/dS ratios were calculated and displayed as red (N domain exons) or blue columns (Ig constant-like domain exons); standard deviations are shown as bars. dN/dS values significantly greater than 1 (above the red line) indicate selective pressure for variability, a ratio less than 1 indicates pressures to conserve the protein sequence. Regions with high and low accumulation rates of nonsynonymous substitutions have been marked exemplarily in some graphs by black and red dotted lines. Note that the synonymous substitutions accumulate at the same rate for all exons.

Figure 5

Identification of functionally important intracellular sequence motifs of carcinoembryonic antigen related cell adhesion molecule (CEACAM1) and CEACAM1-related proteins by multispecies sequence comparisons. The amino acid sequences encoded by cytoplasmic domain exons of CEACAM1 (A), CEACAM1-like members containing an immunoreceptor tyrosine-based activation motif (ITAM)-like motif from eutherians (B) and marsupials (C) from the indicated species were aligned. The sequences were separated according to exon borders. The names of the cytoplasmic domain encoding exons (Cyt) and the intron types (0, xxx-intron-xxx; 1, x-intron-xx; 2, xx-intron-x; xxx = codon) are indicated. The following potential motifs could be detected where x represents any amino acid, and slashes separate alternative amino acids (in one letter code) that may occupy a given position and are indicated below the corresponding sequences: an immunoreceptor tyrosine-based inhibition motif (ITIM) is defined by the sequence (I/L/V/S)xYxx(L/V), an immunoreceptor tyrosine-based switch motif (ITSM) by TxYxx(V/I), an ITAM by (D/E)xxYxxX(L/I)x_6-8Yxx(L/I) and an endocytic, ITAM-like motif by Yxx(L/M/V/I/F). In catarrhinian primates and in platypus the ITSM has been switched to an ITIM. The opossum has two CEACAM1-like proteins one with two ITIM, and one with an ITIM and an ITSM. Note the characteristic split of the YxxL motif in the ITAM and ITAM-like motifs by phase 0 introns. The opossum ITAM domains could be predicted using an EST sequence [GenBank: EX196902] from the marsupial Macropus eugenii (tammar wallaby). An additional highly conserved motif (TEHKxS) and a conserved serine in the cytoplasmic domain of CEACAM1 proteins are boxed. For abbreviation of species names see Table 1.

Figure 6

Coevolution of carcinoembryonic antigen related cell adhesion molecule (CEACAM)1-like members with immunoreceptor tyrosine-based inhibition motif (ITIM) and immunoreceptor tyrosine-based activation motif (ITAM)-like signalling motifs. The phylogeny of CEACAM1-like CEA family members from opossum (A), cattle (B), dogs (C) and humans (D) was reconstructed based on the amino acid sequences differences of mature N domains (minus leader signal peptide sequence) using the neighbour-joining method. The statistical support for each node is expressed as bootstrap values. Genes containing ITIM/immunoreceptor tyrosine-based switch motif encoding exons are marked with red boxes, such with ITAM and ITAM-like motif-encoding exons green and blue, respectively. Note, genes with ITIM or ITAM/ITAM-like signalling motif exons are either the closest relatives (B, C) or belong to the same subgroup of closely related members (A, D). Both opossum and humans contain a group of highly similar genes which code for secreted (human pregnancy-specific glycoproteins) or apparently secreted proteins. Bars below the phylogenetic trees indicate the scale for the number of substitutions per site.a, b, pseudogene

Figure 7

Evidence for gene conversion/recombination between putative paired carcinoembryonic antigen related cell adhesion molecule (CEACAM) receptor genes. The accumulation of nonsynonymous (red curve) and synonymous substitutions (green curve) along immunoglobulin (Ig) variable- and Ig constant-like domain exons of putative paired CEACAM1-like receptors were determined after manual removal of sequence gaps. The type of encoded signalling motif is indicated by the colour of the gene name: red, immunoreceptor tyrosine-based inhibition motif/immunoreceptor tyrosine-based signal motif; green, immunoreceptor tyrosine-based activation motif (ITAM); blue, ITAM-like motif. Stretches of codons with no or minimal accumulation of synonymous substitutions suggest recent recombination events. No such recent events are evident for the putative opossum paired receptors CEACAM114 and CEACAM111 and CEACAM112 and CEACAM111. For abbreviation of species names see Table 1.

Figure 8

Delineation of regions with gene conversion/recombination events between putative paired carcinoembryonic antigen related cell adhesion molecule (CEACAM) receptor genes. Nucleotide sequences of immunoreceptor tyrosine-based inhibition motif (ITIM)/immunoreceptor tyrosine-based switch motif (ITSM)-encoding CEACAM genes from humans (A), cattle (B), opossum (C, D) and dogs (E) were compared with the sequences of CEACAM genes coding for their most closely related, putative paired receptor (gene names are shown in the lower right corner of the plot). For contiguous stretches of nucleotides conserved between gene pairs, the degree of identity was calculated and displayed as horizontal lines. The location of exons is indicated by numbered boxes. Genomic regions involved in gene conversion/recombination are marked with red boxes, involved N exons are shown in red. Note that these regions exhibit the highest degree of conservation. Repeat sequences indicated by differently shaped forms (see box for detailed explanation) have preferentially accumulated in most ITIM/ITSM-encoding genes between the immunoglobulin constant-like domain and the transmembrane domain exons. N, N domain exon; TM, transmembrane domain exon.

Figure 9

Gain and loss of carcinoembryonic antigen related cell adhesion molecule (CEACAM) family features in mammals during evolution. The phylogenetic and taxonomic relationship of selected mammalian species is shown schematically. Major radiation events with estimated radiation times in million years (Myr) are indicated [55]. The appearance of various features during radiation is marked by coloured dots. Linear and circular arrows indicate the absence or presence of an inversion event between the two main CEACAM1-like gene loci (see Figure 3 for details). V, B (boxed) indicate that CEACAM1 serves as bacterial (B) or viral (V) pathogen receptor in the marked lineage. Question marks in the pregnancy-specific glycoproteins dots indicate that the assignment to this CEACAM1-like group is still unclear due to a lack of expression data. For abbreviation of species names see Table 1.