Structural analysis of the nurse shark (new) antigen receptor (NAR): Molecular convergence of NAR and unusual mammalian immunoglobulins

Abstract

We recently have identified an antigen receptor in sharks called NAR (new or nurse shark antigen receptor) that is secreted by splenocytes but does not associate with Ig light (L) chains. The NAR variable (V) region undergoes high levels of somatic mutation and is equally divergent from both Ig and T cell receptors (TCR). Here we show by electron microscopy that NAR V regions, unlike those of conventional Ig and TCR, do not form dimers but rather are independent, f lexible domains. This unusual feature is analogous to bona fide camelid IgG in which modifications of Ig heavy chain V (V H ) sequences prevent dimer formation with L chains. NAR also displays a uniquely f lexible constant (C) region. Sequence analysis and modeling show that there are only two types of expressed NAR genes, each having different combinations of noncanonical cysteine (Cys) residues in the V domains that likely form disulfide bonds to stabilize the single antigen-recognition unit. In one NAR class, rearrangement events result in mature genes encoding an even number of Cys (two or four) in complementarity-determining region 3 (CDR3), which is analogous to Cys codon expression in an unusual human diversity (D) segment family. The NAR CDR3 Cys generally are encoded by preferred reading frames of rearranging D segments, providing a clear design for use of preferred reading frame in antigen receptor D regions. These unusual characteristics shared by NAR and unconventional mammalian Ig are most likely the result of convergent evolution at the molecular level.At the heart of the adaptive immune system are the antigen receptors, Ig and T cell receptor (TCR), that are generated in anticipation of recognition of pathogens (1). The typical antigen receptor is composed of two polypeptide chains [heavy (H) and light (L) for Igs and ␣ and ␤ or ␥ and ␦ for TCRs]. Each chain, in turn, is composed of a single, variable (V) domain at the N-terminal end followed by one to seven constant (C) domains. C domains define the effector functions characteristic of a given class of Ig whereas V domains each display a unique sequence and structure defining antigen specificity. Igs can be subdivided further into Fab and Fc fragments, responsible for antigen binding and for effector function, respectively. Ig and TCR V regions are encoded by a mosaic of genes ligated together somatically during lymphocyte ontogeny (2). Specifically, single V and J elements are joined together at the DNA level for Ig L chain or TCR ␣ and ␥ V regions. In Ig H chains and TCR ␤ and ␦ chains, one or, occasionally, two D elements are joined between the V and J segments. Together, the V, (D), and J elements encode framework (FR, responsible for protein folding and structure) and complementarity-determining regions (CDR, responsible for antigen interactions) within the V domains.The evolutionary origin of antigen receptors is unknown, but the first indication of their emergence phylogenetically is in cartilaginous fish (sharks, skates, and rays), where at least three types of Ig (3-9)...

First molecular and biochemical analysis ofin vivoaffinity maturation in an ectothermic vertebrate

“…In contrast, the study of an IgNAR V-region family presented here demonstrated that high-affinity (nM) binders could be selected directly from the primary repertoire. The presence of these high-affinity binders is probably a consequence of the huge structural diversity imparted through the rearrangement of three D regions (the likes of which is not found in any other shark or nonshark Ig isotype) and the different CDR3 conformations forced by differential disulfide bonding in the two V-region types (13,15). After the in vivo introduction of somatic mutations, we see incremental increases in affinity, strongly indicative of affinity maturation, but these improvements are comparatively small (Ϸ10-fold).…”

“…IgNAR is a paucicopy (few-copy) gene family arranged in the cluster organization typical of elasmobranchs; of the four member genes, only two (type I and type II) are expressed in adult animals. Type I and type II V regions are highly similar in sequence but differ in the positions of their noncanonical Cys residues (13). The IgNAR V-region primary repertoire is entirely CDR3-based and is, subsequently, subject to high levels of apparent antigen-driven mutation (14).…”

“…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).…”

A naturally occurring NAR variable domain binds the Kgp protease from Porphyromonas gingivalis

“…With an a⁄nity for the target antigen of V130 nM, these single domains have antigen speci¢city comparable to recombinant forms of the camelid V H H single domain antibodies, where the a⁄nity varies between 2 and 300 nM [5,6]. However, NARs encompass this a⁄nity in two, rather than three CDR loops, as the CDR2 region is severely truncated [3,9]. The cysteine residues seen within many NAR (and camelid) CDR loops are conserved in both proteins 12A-9 and 12A-14, and probably contribute to the antigen-binding a⁄nity by disulphide bond formation and structural stability.…”

An evolutionarily mobile antigen receptor variable region gene: Doubly rearranging NAR-TcR genes in sharks

“…The poorly understood IgW isotype occurs in multiple forms (13) [as does IgM from elasmobranchs (14)]. IgNAR, which is apparently found only in cartilaginous fish, binds antigen by means of a single V domain (15), which is no more similar to IgV than to TcR V (16,17). The IgNARV gene undergoes extensive hypermutation resulting in affinity maturation (18).…”