Structural analysis, selection, and ontogeny of the shark new antigen receptor (IgNAR): identification of a new locus preferentially expressed in early development
Abstract:The new antigen receptor (IgNAR) family has been detected in all elasmobranch species so far studied and has several intriguing structural and functional features. IgNAR protein, found in both transmembrane and secretory forms, is a dimer of heavy chains with no associated light chains, with each chain of the dimer having a single free and flexible V region. Four rearrangement events (among 1V, 3D, and 1J germline genes) generate an expressed NAR V gene, resulting in long and diverse CDR3 regions that contain … Show more
“…The IgNAR V-region primary repertoire is entirely CDR3-based and is, subsequently, subject to high levels of apparent antigen-driven mutation (14). However, because the IgNAR V region utilizes three diversity (D) regions, requiring four rearrangement events, the primary repertoire contains clones with an unprecedented diversity of CDR3 length and amino acid composition (13,15).…”
Section: First Molecular and Biochemical Analysis Of In Vivo Affinitymentioning
The cartilaginous fish are the oldest phylogenetic group in which Igs have been found. Sharks produce a unique Ig isotype, IgNAR, a heavy-chain homodimer that does not associate with light chains. Instead, the variable (V) regions of IgNAR bind antigen as soluble single domains. Our group has shown that IgNAR plays an integral part in the humoral response of nurse sharks (
Ginglymostoma cirratum
) upon antigen challenge. Here, we generated phage-displayed libraries of IgNAR V regions from an immunized animal and found a family of clones derived from the same rearrangement event but differentially mutated during expansion. Because of the cluster organization of shark Ig genes and the paucicopy nature of IgNAR, we were able to construct the putative ancestor of this family. By studying mutations in the context of clone affinities, we found evidence that affinity maturation occurs for this isotype. Subsequently, we were able to identify mutations important in the affinity improvement of this family. Because the family clones were all obtained after immunization, they provide insight into the
in vivo
maturation mechanisms, in general, and for single-domain antibody fragments.
“…The IgNAR V-region primary repertoire is entirely CDR3-based and is, subsequently, subject to high levels of apparent antigen-driven mutation (14). However, because the IgNAR V region utilizes three diversity (D) regions, requiring four rearrangement events, the primary repertoire contains clones with an unprecedented diversity of CDR3 length and amino acid composition (13,15).…”
Section: First Molecular and Biochemical Analysis Of In Vivo Affinitymentioning
The cartilaginous fish are the oldest phylogenetic group in which Igs have been found. Sharks produce a unique Ig isotype, IgNAR, a heavy-chain homodimer that does not associate with light chains. Instead, the variable (V) regions of IgNAR bind antigen as soluble single domains. Our group has shown that IgNAR plays an integral part in the humoral response of nurse sharks (
Ginglymostoma cirratum
) upon antigen challenge. Here, we generated phage-displayed libraries of IgNAR V regions from an immunized animal and found a family of clones derived from the same rearrangement event but differentially mutated during expansion. Because of the cluster organization of shark Ig genes and the paucicopy nature of IgNAR, we were able to construct the putative ancestor of this family. By studying mutations in the context of clone affinities, we found evidence that affinity maturation occurs for this isotype. Subsequently, we were able to identify mutations important in the affinity improvement of this family. Because the family clones were all obtained after immunization, they provide insight into the
in vivo
maturation mechanisms, in general, and for single-domain antibody fragments.
“…110 Most cartilaginous fish V NAR s bear an additional non-canonical disulfide linkage spanning either FR2-CDR3 (type I) or CDR1-CDR3 (types II and III 16 ). In addition, type I V NAR s also bear a CDR3-FR4 disulfide linkage and, sometimes, an intra-CDR3 disulfide linkage (three or four intradomain disulfide linkages in total 27 ). A minority of V NAR s (type IV) bear only the single canonical disulfide linkage.…”
“…A minority of V NAR s (type IV) bear only the single canonical disulfide linkage. As for V H Hs, most V NAR Cys residues in CDR1, FR2 and FR4 are probably encoded in the germline and non-canonical disulfide linkages are formed during primary repertoire development 4,27,28 Although the precise roles of non-canonical disulfide linkages in sdAb structure and function remain unclear, these linkages very likely influence sdAb paratope structure, since patterns of antigen-driven somatic hypermutation appear to vary depending on their presence and location. 27 10.1080/19420862.2018.1489633-F0002Fig 2.Properties of sdAb vs .…”
“…Each locus contains one V gene, two or three D genes and one J gene and thus primary repertoire diversity is almost entirely CDR3-based: 4 since V NAR CDR3 loops are formed through either three or four independent rearrangement events, these tend to be long. 27 Unlike the V H domains of IgMs and IgWs, V NAR s accrue somatic hypermutations upon encounter with antigen primarily in CDR1 and CDR3 but also in HV2 and HV4. 27,28
…”
Section: Introductionmentioning
confidence: 99%
“…27 Unlike the V H domains of IgMs and IgWs, V NAR s accrue somatic hypermutations upon encounter with antigen primarily in CDR1 and CDR3 but also in HV2 and HV4. 27,28
…”
Single-domain antibodies (sdAbs), the autonomous variable domains of heavy chain-only antibodies produced naturally by camelid ungulates and cartilaginous fishes, have evolved to bind antigen using only three complementarity-determining region (CDR) loops rather than the six present in conventional VH:VL antibodies. It has been suggested, based on limited evidence, that sdAbs may adopt paratope structures that predispose them to preferential recognition of recessed protein epitopes, but poor or non-recognition of protuberant epitopes and small molecules. Here, we comprehensively surveyed the evidence in support of this hypothesis. We found some support for a global structural difference in the paratope shapes of sdAbs compared with those of conventional antibodies: sdAb paratopes have smaller molecular surface areas and diameters, more commonly have non-canonical CDR1 and CDR2 structures, and have elongated CDR3 length distributions, but have similar amino acid compositions and are no more extended (interatomic distance measured from CDR base to tip) than conventional antibody paratopes. Comparison of X-ray crystal structures of sdAbs and conventional antibodies in complex with cognate antigens showed that sdAbs and conventional antibodies bury similar solvent-exposed surface areas on proteins and form similar types of non-covalent interactions, although these are more concentrated in the compact sdAb paratope. Thus, sdAbs likely have privileged access to distinct antigenic regions on proteins, but only owing to their small molecular size and not to general differences in molecular recognition mechanism. The evidence surrounding the purported inability of sdAbs to bind small molecules was less clear. The available data provide a structural framework for understanding the evolutionary emergence and function of autonomous heavy chain-only antibodies.
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