Platypus TCRμ provides insight into the origins and evolution of a uniquely mammalian TCR locus

Abstract

TCRμ is an unconventional TCR that was first discovered in marsupials and appears to be absent from placental mammals and nonmammals. In this study, we show that TCRμ is also present in the duckbill platypus, an egg-laying monotreme, consistent with TCRμ being ancient and present in the last common ancestor of all extant mammals. As in marsupials, platypus TCRμ is expressed in a form containing double V domains. These V domains more closely resemble Ab V than that of conventional TCR. Platypus TCRμ differs from its marsupial homolog by requiring two rounds of somatic DNA recombination to assemble both V exons and has a genomic organization resembling the likely ancestral form of the receptor genes. These results demonstrate that the ancestors of placental mammals would have had TCRμ but it has been lost from this lineage.

FIGURE 1

Phylogenetic analyses of platypus and marsupial Cμ and C regions from conventional TCR chains. Phylogenetic relationship between Cμ and other conventional TCRs are simplified according to the phylogenetic trees constructed using different methods: (A) Neighbor-Joining (NJ); (B) Maximum Parsimony (MP); (C) Unweighted Pair Group Method with Arithmetic mean (UPGMA); (D) Minimum Evolution (ME). All phylogenetic analyses are based on nucleotide alignments and branch support is indicated as the percentage of out of 1000 bootstrap replicates.

FIGURE 2

Predicted amino acid alignment of full-length platypus TCRμ cDNA clones. Dashes indicate identity and gaps introduced to the alignment are shown as dots. The sequences were divided into the Leader, V1, V2 and C domains. The FR and CDR of the V domains along with the Cμ, CP, and TM-CT of C domain were shown above the sequence alignment. Conserved cysteines are shaded. Conserved lysines and arginines are shaded and indicated by *. Conserved residues YGXG and FXXG in FR4 of the V1 and V2 domains, respectively are noted. The borders of CDR and FR were indicated above the sequences.

FIGURE 3

Nucleotide sequence and translations of the 3’ end of Vμ1 and Vμ2 gene segments and complete Jμ1 and Jμ2 gene segments. RSS flanking V and J gene segments in platypus genome are indicated. The scaffolds on which V and J sequences were identified are shown on the left. Pseudogenes are indicated by ψ. Stop codons are indicated as *. Nucleotide sequences of V and J genes are shown in lowercase with amino acid sequences underneath, whereas the RSS sequences are shown in uppercase. Heptamers and nonamers are in bold, and 12 bp or 23 bp spacers are indicated. The YGXG and FXXG conserved motif corresponding to FR4 are shaded.

FIGURE 4

Sequences corresponding to the CDR3 of (A) V1 and (B) V2 domains from full-length and partial platypus splenic TCRμ cDNAs.

FIGURE 5

Phylogenetic analysis of platypus and marsupial Vμ including V genes from conventional TCR, shark NAR and NAR-TCR and Ig VH. This neighbor-joining tree is based on nucleotide alignments and branch support is indicated as the percentage of out of 1000 bootstrap replicates. Only those nodes with greater than 50% support are indicated. The three major clans of vertebrate VH are indicated by Roman numerals.

FIGURE 6

Diagrams of the predicted platypus TCRμ gene organization, transcripts, and protein structure. (A) Representative TCRμ scaffolds containing TCRμ coding sequences. Closed or open triangles flanking the Vμ, Dμ and Jμ gene segments indicate the presence of 23- or 12-bp spacer RSS, respectively. The L sequence, CP, TM-CT, and 3’ UTR exons are indicated. (B) Predicted TCRμ germ-line DNA and rearranged DNA structure. (C) Primary TCRμ mRNA transcript structure. (D) Predicted TCRμ protein organization. Conserved R and K residues in the TM region are indicated. (E) Predicted platypus TCRμ cluster and representative opossum TCRμ cluster.