Variable lymphocyte receptors in hagfish

Abstract

A previously uncharacterized type of variable lymphocyte receptors (VLR) was identified recently in the Sea lamprey. This jawless vertebrate generates an extensive VLR repertoire through differential insertion of neighboring diverse leucine-rich repeat (LRR) cassettes into an incomplete germ-line VLR gene. We report here VLR homologs from two additional lamprey species and the presence of two types of VLR genes in hagfish, the only other order of contemporary jawless vertebrates. As in the Sea lamprey, the incomplete hagfish germ-line VLR-A and -B genes are modified in lymphocyte-like cells to generate highly diverse repertoires of VLR-A and -B proteins via a presently undetermined mechanism. This jawless-fish mode of VLR diversification starkly contrasts with the rearrangement of Ig V(D)J gene segments used by all jawed vertebrates to produce diverse repertoires of T and B lymphocyte antigen receptors. The development of two very different strategies for receptor diversification at the dawn of vertebrate evolution approximately 500 million years ago attests to the fitness value of a lymphocyte-based system of anticipatory immunity.

Fig. 1.

Evolutionarily conserved agnathan VLRs. VLR amino acid sequences representing the Inshore hagfish (E. burgeri), Pacific hagfish (E. stoutii), Sea lamprey (P. marinus; GenBank accession no. AY577946), American brook lamprey (L. appendix), and Northern brook lamprey (I. fossor). Blue shade, 100% identity; yellow, 60-99%; green, 40-59%; and red, hydrophobic tail region.

Fig. 2.

Genetic distance among Pacific hagfish VLR diversity regions (LRRNT to LRRCT). Proteins predicted from PCR-amplified lymphocyte-like cDNA clones or blood genomic PCR amplicons from five animals. Bars represent 5% amino acid divergence. (A) VLR-A (n = 139); (B) VLR-B (n = 70). Green, unstimulated; red, immunostimulated; and blue, genomic mature VLR; asterisk-related sequences.

Fig. 3.

Hagfish VLR gene loci. (A) Pacific hagfish VLR-A.(B) Inshore hagfish VLR-A.(C) Pacific hagfish VLR-B.(D) Inshore hagfish VLR-B. Sequence of inserts from four BAC clones, with uncaptured gaps marked. Location of VLR germ-line genes and flanking cassettes, in reverse (indicated by inverted orientation) or forward orientation, is indicated in kilobases (graphics are out of scale). GenScan gene predictions are indicated in blue, an unrelated LRR gene upstream from the Pacific hagfish germ-line VLR-A gene and two flanking transposase ORFs in the Inshore hagfish VLR-A and Pacific hagfish VLR-B loci.

Fig. 4.

Agnathan VLR genes, transcripts and phylogeny. (A) Schematic presentation of germ-line and mature VLR genes of Pacific hagfish and Sea lamprey. Colored bars indicate coding regions; size in nucleotides; positions of PCR primers (Table 2) used to amplify hagfish VLR are indicated by arrows and labeled F (forward) R (reverse). (B) Pacific hagfish VLRs PCR amplified from lymphocyte-like transcripts (RT-PCR) or blood genomic DNA. Agarose gel image; molecular weight marker indicated on the left (kilobases); position of germ-line and mature VLR amplicons indicated on the right. (C) Phylogenetic analysis of agnathan VLRs. Neighbor-joining tree of hagfish and lamprey VLR proteins (same sequences as in Fig. 1); bootstrap values are indicated. Bar represents 10% amino acid divergence. (D) Hypothetical model for the evolution of agnathan VLR.