Cytidine deaminase 2 is required for VLRB antibody gene assembly in lampreys

Abstract

The antibodies of jawless vertebrates consist of leucine-rich repeat arrays encoded by somatically assembled VLRB genes. It is unknown how the incomplete germline VLRB loci are converted into functional antibody genes during B lymphocyte development in lampreys. In Lampetra planeri larvae lacking the cytidine deaminase CDA2 gene, VLRB assembly fails, whereas the T lineage–associated VLRA and VLRC antigen receptor gene assemblies occur normally. Thus, CDA2 acts in a B cell lineage–specific fashion to support the somatic diversification of VLRB antibody genes. CDA2 is closely related to activation-induced cytidine deaminase (AID), which is essential for the elaboration of immunoglobulin gene repertoires in jawed vertebrates. Our results thus identify a convergent mechanism of antigen receptor gene assembly and diversification that independently evolved in the two sister branches of vertebrates.

Co-evolution of mutagenic genome editors and vertebrate adaptive immunity

“…The dashed box around proto-CDA2 and proto-VLR indicate that these were subsequently exapted to underpin the adaptive immune systems of jawless fishes, comprising hagfishes and lampreys (upper brown boxes). In lampreys, it has now been demonstrated that CDA2 is required for VLRB receptor gene assembly [ 20 •• ]; likewise, it is assumed but not yet proven that CDA1 assembles VLRA/C receptor genes. In hagfishes, the VLR/CDA system might operate in a similar fashion; however, since the repertoire of AAD genes in this group of animals remains uncharacterized, their potential roles in the assembly of VLRA/C are unknown.…”

“…Recently, following improvements in the methodology to rear in vitro -fertilized lampreys in the laboratory, in combination with highly efficient CRISPR/Cas9-mediated gene disruption, we have been able to directly examine a potential role of CDA2 in the VLRB assembly process in L. planeri [ 20 •• ]. We generated a large series of CDA2 -deficient L. planeri CRISPR/Cas9-induced mutants (crispants), exhibiting bi-allelic deleterious mutations of the CDA2 gene.…”

“…Interestingly, both CDA1 and CDA1L1_4 expression is found in VLRA + and VLRC + lymphocytes [ 24 •• , 25 • ], and in cells situated in the thymoid, representing the presumptive thymus equivalent of lampreys that is located at the tips of the larval gill filaments [ 83 •• ]. Based on the role of CDA2 in VLRB gene assembly [ 20 •• ], we hypothesize that CDA1 or CDA1L1_4 genes are required for the somatic assembly of VLRA and VLRC genes in developing T-like lymphocytes. Of note, the greater variability of CDA1 and CDA1L proteins (as compared to the highly conserved CDA2 sequences) may have precluded the identification of members of this clade of AADs in sequence collections of hagfishes; alternatively, the CDA2 gene might assume a more general role in VLR gene assembly in these species ( Figure 2 a).…”

“…A small subset of self-DNA mutating enzymes plays key roles in adaptive immune systems of vertebrates. RAG1/2 genes [ 15 •• , 16 •• , 17 ] and cytidine deaminases [ 18 •• , 19 •• , 20 •• ] are required for the somatic diversification of the two molecularly distinct classes of antigen receptors of vertebrates. Jawed vertebrates (ranging from cartilaginous fishes to humans) deploy immunoglobulin domain-based antigen receptors for self/nonself discrimination [ 21 •• ], whereas jawless vertebrates (lampreys, hagfishes) rely on leucine rich repeat-based multi-domain proteins for antigen-specific immune responses [ 22 •• , 23 • , 24 •• , 25 • ].…”

“…Jawed vertebrates (ranging from cartilaginous fishes to humans) deploy immunoglobulin domain-based antigen receptors for self/nonself discrimination [ 21 •• ], whereas jawless vertebrates (lampreys, hagfishes) rely on leucine rich repeat-based multi-domain proteins for antigen-specific immune responses [ 22 •• , 23 • , 24 •• , 25 • ]. While the Ig-type antigen receptors require the RAG proteins for their functional assembly in lymphocytes [ 26 ], evidence is mounting that the LRR-type of receptors are assembled by institutionalized cytidine deaminases [ 19 •• , 20 •• , 24 •• , 27 • ].…”