Evidence for Extensive Duplication and Subfunctionalization of FCRL6 in Armadillo (Dasypus novemcinctus)

Abstract

The control of infections by the vertebrate adaptive immune system requires careful modulation to optimize defense and minimize harm to the host. The Fc receptor-like (FCRL) genes encode immunoregulatory molecules homologous to the receptors for the Fc portion of immunoglobulin (FCR). To date, nine different genes (FCRL1-6, FCRLA, FCRLB and FCRLS) have been identified in mammalian organisms. FCRL6 is located at a separate chromosomal position from the FCRL1-5 locus, has conserved synteny in mammals and is situated between the SLAMF8 and DUSP23 genes. Here, we show that this three gene block underwent repeated duplication in Dasypus novemcinctus (nine-banded armadillo) resulting in six FCRL6 copies, of which five appear functional. Among 21 mammalian genomes analyzed, this expansion was unique to D. novemcinctus. Ig-like domains that derive from the five clustered FCRL6 functional gene copies show high structural conservation and sequence identity. However, the presence of multiple non-synonymous amino acid changes that would diversify individual receptor function has led to the hypothesis that FCRL6 endured subfunctionalization during evolution in D. novemcinctus. Interestingly, D. novemcinctus is noteworthy for its natural resistance to the Mycobacterium leprae pathogen that causes leprosy. Because FCRL6 is chiefly expressed by cytotoxic T and NK cells, which are important in cellular defense responses against M. leprae, we speculate that FCRL6 subfunctionalization could be relevant for the adaptation of D. novemcinctus to leprosy. These findings highlight the species-specific diversification of FCRL family members and the genetic complexity underlying evolving multigene families critical for modulating adaptive immune protection.

Keywords: FCRL; FCRL6; armadillo; gene duplication; leprosy; subfunctionalization.

Figure 1

Structure of the FCRL6 locus among 11 mammalian genomes. Horizontal lines correspond to the chromosome on which FCRL6 and its flanking genes are located. Genes are color-coded: FCRL6 (red), SLAMF8 (purple), VSIG8 (blue), DUSP23 (yellow) and SH2B (green). Pseudogenes are labelled with an asterisk (*).

Figure 2

Phylogenetic analysis comparing representative mammalian and D. novemcinctus FCRL6 cDNA sequences. The maximum likelihood (ML) method and the T92 + G + I model of nucleotide substitution were used to assemble the phylogenetic tree. Some groups were collapsed for simplification. The tree includes FCRL6 genes from D. novemcinctus (n = 5) (red), FCRL6 genes from other indicated mammals (n = 20) (black), and was rooted with FCRL3 (H. sapiens, B. taurus, O. cuniculus, F. catus and M. monocero) (green). Bootstrap values are indicated to the left of each node.

Figure 3

Phylogenetic tree of the predicted amino acid sequences for FCRL6 Ig domains from D. novemcinctus and a panel of mammals (n = 20). The maximum likelihood (ML) method and the JTT + G + I model of amino acid substitution were used for analysis and assembly. D1 corresponds to the membrane-distal Ig-like domain, D2 to a middle Ig-like domain, D3 to a membrane-proximal Ig-like domain.

Figure 4

Multiple sequence alignment of the human and nine-band armadillo FCRL6 cytoplasmic tails. Marked are potential tyrosine-based activating and inhibitory sequences, along with cysteine, histidine and threonine residues. Alignment was performed using CLUSTALW in the BioEdit software package. An ITAM-like sequence (H. sapiens) is colored green and ITIM are red. Cysteines are in black, bold and highlighted grey. Histidine is highlighted green. Threonine is highlighted yellow.