Overview & Discovery of VLRs in Lamprey
Discovery of VLRs
The primitive jawless vertebrates (lampreys and hagfish), the most phylogenetically distant vertebrates from mammals, possess an alternative form of the adaptive immune system that is regarded as counterparts of the immunoglobulin-based T and B cells of jawed vertebrates. The capability of mounting specific immune responses and recognizing foreign antigens is mediated by antigen receptors that are composed of tandem arrays leucine-rich repeat (LRR) cassettes; these somatically diversified receptors termed variable lymphocyte receptors (VLRs).
The VLRs appear to be diversified in three types (VLRA, VLRB, and VLRC) by a gene conversion mechanism involving the lymphocyte lineage-specific cytosine deaminases. VLRA is expressed on the surface of T-like lymphocytes that appear to develop in a thymus-equivalent region of the gills termed the thymoid. Besides, VLRC is more similar to VLRA than to VLRB and expressed by a distinct lymphocyte lineage. Conversely, VLRB is expressed on the surface of B-like lymphocytes in hematopoietic tissues and secreted as a multivalent antibody molecule. Although both the VLRA and the VLRB cells proliferate in response to antigenic stimulation, only the VLRB lymphocytes bind native antigens and further undergo differentiation and secretion into antigen-specific VLR antibody. VLRA and VLRB maintain cell surface expression of their receptors, while preferentially increasing the expression of proinflammatory cytokine interleukin-17 (IL-17), and macrophage migration inhibitory factor (MIF).
Fig.1 Three-dimensional structures of VLRs. (Boehm, 2012)
Structure of VLRs
Among the three receptors, VLRA and VLRC occur only as membrane-bound proteins. On the other hand, the VLRB receptor is a glycosylphosphatidylinositol-anchored (GPI) membrane protein that occurs in both membrane-bound and secretory forms. The domain organization of VLRA, VLRB, and VLRC receptors is identical, and cardinal features are conserved. The VLRs are expressed as membrane-bound proteins in which the N-terminal is a capping module known as an N-terminal LRR (LRRNT). This is followed by an N-terminal LRR module (LRR1), multiple residue variable LRRs (LRRV), a 13-residue truncated LRR called the connecting peptide (CP), a C-terminal LRR capping region (LRRCT), and an invariant stalk region rich in threonine and proline residues.
The LRRV module is composed of 24 amino acids with the consensus sequence. The most N-terminal LRR module, designated LRR1, is shorter than other LRR modules and is composed of 18 residues; the most C-terminal LRR module, known as LRRVe, is also unique with a distinct sequence signature. Sequence diversity is primarily found in the 3’-LRRNT (3’-part of LRRNT), LRR1, LRRV, LRRVe, CP, and the 5' LRRCT (5’-part of LRRCT) portions of the VLRs. In the case of VLRB, the stalk region has a GPI cleavage site and is anchored to the cell membrane by GPI linkage.
The Organization and Assembly of VLRs
The germline VLR genes lack sequences coding for a contiguous variable region and hence are incomplete to encode any proteins. However, each germline VLR gene is flanked by hundreds of different LRR-encoding sequences, encoding several kinds of LRR cassettes (LRRNT, LRR1, LRRV, CP, and LRRCT). During the development of lymphocyte-like cells, these LRR-encoding cassettes are chosen randomly and sequentially incorporated into the VLR gene by a gene conversion-like mechanism, eventually forming a completely assembled VLR gene. The combinatorial VLR assembly can generate a vast repertoire of anticipatory receptors comparable in diversity to the repertoire of Ig-domain-based antigen receptors in jawed vertebrates. The diversity is enough to identify any antigen outside and is generated through not only the sequence of each LRR cassette diversified, but also the number of cassettes inserted into the VLR gene. Thus, a conservative estimate of the combinatorial diversity has been predicted almost as up to ~1014.
Fig.2 Organization of the germline VLR and gene assembly. (Kishishita, 2014)
Antigen Binding by VLRs
VLRs use amino acid variations on their concave surfaces, which are composed of the LRRNT, LRR1, LRRVs, and CP motifs, along with the highly variable LRRCT loops to bind antigens. Sequence diversity of LRRNT is much less pronounced in VLRA and VLRC than in VLRB, compatible with the possibility that this module forms part of the antigen-binding surface only in VLRB. The VLRA and VLRC have a higher average number of LRRV modules than the VLRBs, indicating they have a larger concave surface for antigen-binding sites.
Another structural difference in the three VLR types relates to the highly variable LRRCT. The VLRAs and VLRBs typically have a protruding loop because of their relatively large LRRCT inserts, whereas the LRRCT inserted in VLRCs is too short of forming this loop. Furthermore, the length variation of LRRCT inserted into VLRAs is less pronounced than in the VLRBs. VLRA and VLRB bind antigens through their concave surface and a unique hypervariable loop in LRRCT, with the latter exhibiting sizable protrusions and playing a significant role in contributing to antigen binding. This protruding loop is more flexible and shares the unique feature of being able to bind to residues in antigen clefts with unusual heavy-chain antibodies made by camels and sharks. These structural differences suggest different antigen-binding modes for the three VLR isotypes. In addition to the remarkable avidity attributable to their antigen-binding sites, the VLRB antibodies are relatively resistant to denaturation by heat as well as very acidic and alkaline conditions.
Application Potential of Engineering VLRs
The discovery of VLRs affords a new and alternative perspective for the development of antibody reagents or drugs, and VLRs have the multifaceted advantages and uniqueness. The molecular weights of VLRs are small, and the molecular structure of VLRs is relatively simple, with only a single peptide chain. Production of VLRs is easier to enable one-step cloning/screening using any available high throughput surface expression technology, which makes it convenient for largescale industrial production. In addition, the evolutionary distance between lampreys and humans is not closed; thus, VLRs are suitable as diagnostic reagents to eliminate cross-reactivities. Because of their unique structures, VLRs have been shown to bind to different antigens with different binding affinities compared with conventional antibodies. Collectively, these properties provide a VLRs platform that is more applicable to the development of antibody drugs.
References
- Boehm, T.; et al. VLR-based adaptive immunity. Annual review of immunology. 2012, 30: 203-220.
- Kishishita, N.; Nagawa, F. Evolution of adaptive immunity: implications of a third lymphocyte lineage in lampreys. Bioessays. 2014, 36(3): 244-250.
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