The B. glabrata genome contains many copies of FREP genes that can be divided into approximately 13 subfamilies. A study of the sequences of expressed FREP3 subfamily members has revealed that the FREPs expressed in an individual organism are extensively diversified compared with the germline genes. There are fewer than five genes in the FREP3 subfamily, but an individual snail was found to generate more than 45 distinct FREP3 proteins, all with slightly different sequences. An analysis of the protein sequences suggested that this diversification was due to the accumulation of point mutations in one of the germline FREP3 genes. Although the precise mechanism of this diversification, and the cell type in which it occurs, are not yet known, it does suggest some similarity to somatic hypermutation that occurs in the immunoglobulins. Both the insect and Biomphalaria examples seem to represent a way of diversifying molecules involved in immune defense, but although they resemble in some ways the strategy of an adaptive immune response, there is no evidence of clonal selection—the cornerstone of true adaptive immunity.
somatic gene rearrangement to diversify receptors built from LRR domains.
Since the early 1960s it has been known that certain jawless fishes, the hagfish and the lamprey, could mount a form of accelerated rejection of transplanted skin grafts and exhibit a kind of immunological delayed-type hypersensitivity. Their serum also seemed to contain an activity that behaved as a specific agglutinin, increasing in titer after secondary immunizations, in a similar way to an antibody response in higher vertebrates. Although these phenomena seemed reminiscent of adaptive immunity, there was no evidence of a thymus or of immunoglobulins, but these animals did have cells that could be considered to be genuine lymphocytes on the basis of morphological and molecular analysis. Analysis of the genes expressed by lymphocytes of the sea lamprey Petromyzon marinus revealed none related to T-cell receptor or immunoglobulin genes. However, these cells expressed large amounts of mRNAs from genes encoding proteins with multiple LRR domains, the same protein domain from which the pathogen-recognizing Toll-like receptors (TLRs) are built (see Section 3-5).
This might simply have meant that these cells are specialized for recognizing and reacting to pathogens, but the LRR proteins expressed had some surprises in store. Instead of being present in a relatively few forms (like the invariant TLRs), they were found to have highly variable amino acid sequences, with a large number of variable LRR units placed between less variable amino-terminal and carboxy-terminal LRR units. These LRR-containing proteins, called variable lymphocyte receptors (VLRs), have an invariant stalk region connecting them to the plasma membrane by a glycosylphosphatidylinositol linkage, and they can either be tethered to the cell or, at other times, like antibodies, be secreted into the blood.
Analysis of the expressed lamprey VLR genes indicates that they are assembled by a process of somatic gene rearrangement (Fig. 5.25). In the germline configuration, there are three incomplete VLR genes, VLRA, VLRB, and VLRC, each encoding a signal peptide, a partial amino-terminal LRR unit, and a partial carboxy-terminal LRR unit, but these three blocks of coding sequence are separated by noncoding DNA that contains neither typical signals for RNA splicing nor the RSSs present in immunoglobulin genes (see Section 5-4). Instead, the regions flanking the incomplete VLR genes include a large number of DNA ‘cassettes’ that contain LRR units—one, two, or three LRR domains at a time.
Each mature lamprey lymphocyte expresses a complete and unique VLR gene, either VLRA, VLRB, or VLRC, which has undergone recombination of these flanking regions with the germline VLR gene.