Identification of a third variable lymphocyte receptor in the lamprey

Abstract

Jawless vertebrates such as lamprey and hagfish lack T-cell and B-cell receptors; instead, they have unique antigen receptors known as variable lymphocyte receptors (VLRs). VLRs generate diversity by recombining highly diverse leucine-rich repeat modules and are expressed clonally on lymphocyte-like cells (LLCs). Thus far, two types of receptors, VLRA and VLRB, have been identified in lampreys and hagfish. Recent evidence indicates that VLRA and VLRB are expressed on distinct populations of LLCs that resemble T cells and B cells of jawed vertebrates, respectively. Here we identified a third VLR, designated VLRC, in the lamprey. None of the approximately 100 VLRC cDNA clones subjected to sequencing had an identical sequence, indicating that VLRC can generate sufficient diversity to function as antigen receptors. Notably, the C-terminal cap of VLRC exhibits only limited diversity and has important structural differences relative to VLRA and VLRB. Single-cell PCR analysis identified LLCs that rearranged VLRC but not VLRA or VLRB, suggesting the presence of a unique population of LLCs that express only VLRC.

Fig. 1.

VLRC is a member of the VLR family. Deduced amino acid sequences of sea lamprey (Pm, P. marinus), Japanese lamprey (Lj, L. japonicum), inshore hagfish (Eb, E. burgeri), and Pacific hagfish (Es, Eptatretus stoutii) VLRs. C, conserved cysteine residues in LRRNT and LRRCT; #, highly variable inserts (23). Conserved residues are shaded: light-blue, ≥90% identity; green, 70–89% identity; and yellow, 69–50% identity. “-” indicates a gap. Accession nos. are as follows: LjVLRA, AB507269; PmVLRA, EF094821; EbVLRA, AY964726; EsVLRA, AY964825; LjVLRB, AB507270; PmVLRB, AY577957; EbVLRB, AY965530; EsVLRB, AY965561; and LjVLRC, AB507271.

Fig. 2.

Organization of the germline VLRC locus. (A) Structure of the germline VLRC gene of the Japanese lamprey (drawn to scale) and the schematic germline structures of other known VLR genes (not drawn to scale). (B) Organization of the sea lamprey VLRC locus. Two 5′-LRRCT-encoding modules, designated modules 1 and 2, are located downstream of the germline VLRC gene. This information is based on the analysis of contig 377 in the Pre Ensembl genome database. (C) The 5′-LRRCT of VLRC shows only limited diversity; basically only two major types of sequences were observed. One type represented by Lj1VLRC2 has a sequence identical or nearly identical to the 5′-LRRCT sequence located in the germline VLRC gene (LjgVLRC). The other type falls into two closely related subtypes represented by Lj1VLRC4 and Lj1VLRC16; the sequences of these clones are very similar to those of modules 1 and 2, respectively. “.” and “-” indicate identity with the top sequence and absence of residues, respectively. Clones are identified by the species name, followed by the animal number, gene name, and clone number. Thus, Lj1VLRC2 indicates a VLRC clone 2 isolated from Japanese lamprey individual 1.

Fig. 3.

Phylogenetic relationship of VLR genes. A neighbor-joining tree was constructed using the amino acid sequences of the diversity region that could be reliably aligned (LRRNT, LRR1, LRRVe, and LRRCT). Nodes that received bootstrap support of <70% were collapsed. Bootstrap values >90% are shown. For abbreviations of species’ names, see the legend to Fig. 1.

Fig. 4.

Tissue distribution of VLRC transcripts in Japanese lampreys. (A) Tissue distribution of VLRA, VLRB, and VLRC transcripts. PBL, peripheral blood leukocytes. (B) Real-time RT-PCR analysis. Two animals were subjected to experiments.

Fig. 5.

Identification of LLCs that rearrange only the VLRC gene. Single-cell genomic PCR identified LLCs that rearranged only the VLRA (Left), VLRB (Center), or VLRC (Right) gene. Bands marked with asterisks represent rearranged products, whereas unmarked bands represent germline products.