© 2003 by The Society for Integrative and Comparative Biology
Novel Immune-type Receptor Genes and the Origins of Adaptive and Innate Immune Recognition1
1 Children's Research Institute, University of South Florida/All Children's Hospital, 140 Seventh Avenue South, St. Petersburg, Florida 33701
2 H. Lee Moffitt Cancer Center and Research Institute, 12901 Magnolia Avenue, Tampa, Florida 33612
3 Department of Biology, University of South Florida, 4202 E. Fowler Avenue, Tampa, Florida 33620
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SYNOPSIS |
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 SYNOPSIS INTRODUCTIONEVOLUTION OF NK RECOGNITIONPHYLOGENY OF DIVERSIFIED...IDENTIFICATION OF NOVEL IMMUNE...NITR GENES IN ZEBRAFISHRECOGNITION SPECIFICITY OF NITRSVARIATIONS IN OVERALL STRUCTURE...POSSIBLE FUNCTIONS OF NITRSEVOLUTION OF NITRSCONCLUSIONSReferences |
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The prototypic forms of teleost novel immune-type receptors (NITRs) consist of a variable (V) region, a unique V-like C2 (V/C2) domain, a transmembrane region and a cytoplasmic tail containing immunoreceptor tyrosine-based inhibition motifs (ITIMs). NITRs encode diversified V regions in large multigene families but do not undergo somatic rearrangement. Studies in four different bony fish model systems have identified a number of different organizational forms of NITRs. Specifically, NITR genes encode N-terminal ectodomains of the V-type but otherwise vary in the: total number of extracellular immunoglobulin domains, number and location of joining (J) region-like motifs, presence of transmembrane regions, presence of charged residues within transmembrane regions, presence of cytoplasmic tails, and/or distribution of ITIM(s) within the cytoplasmic tails. V region-containing NITRs constitute a far more complex family than recognized originally and currently include individual members that potentially function through inhibitory as well as activating mechanisms. The genomic organization of the NITR gene cluster as well as the structural diversity and overall architecture of the NITR proteins is reminiscent of genes encoded at the mammalian leukocyte receptor cluster (LRC); however, there presently is no functional evidence to support an orthologous relationship between NITR and LRC gene products. Comparisons of the predicted structures of the NITRs have identified several short regions of sequence identity and a novel cloning strategy has been devised that selects for secretory and transmembrane proteins that encode these short motifs. Using this approach, related genes termed immune-type receptors (ITRs) have been identified in cartilaginous fish. Taken together, these studies indicate that leukocyte regulatory receptors, including those that mediate natural killer function, might have emerged early in vertebrate evolution and that the NITR/ITR genes represent a new and potentially highly significant link between innate and adaptive immune responses.
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INTRODUCTION |
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SYNOPSISINTRODUCTIONEVOLUTION OF NK RECOGNITIONPHYLOGENY OF DIVERSIFIED...IDENTIFICATION OF NOVEL IMMUNE...NITR GENES IN ZEBRAFISHRECOGNITION SPECIFICITY OF NITRSVARIATIONS IN OVERALL STRUCTURE...POSSIBLE FUNCTIONS OF NITRSEVOLUTION OF NITRSCONCLUSIONSReferences |
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The vertebrate immune system can be divided into adaptive and innate components. The adaptive immune system, which can be traced to the earliest jawed vertebrates (reviewed in Litman et al., 1999
), is associated with two major families of immune receptors, immunoglobulins (Ig) and T cell antigen receptors (TCRs). Both of these gene families diversify receptors through both segmental rearrangement of independent genetic elements and other forms of somatic change, including (in the case of antibodies) extensive somatic hypermutation. Adaptive immunity thus consists of inherited germline as well as somatic components. The "joined" Ig genes in cartilaginous fish, which likely effect innate function, represent a significant exception (Yoder and Litman, 2000). Despite fundamental differences in functions mediated by Ig and TCRs, both utilize diversified V regions to form active sites that recognize a seemingly limitless variety of antigenic structures. Notwithstanding this extensive variation, V regions of Ig and TCR share certain conserved core residues.
Increasing attention is being directed towards the role of innate mechanisms in host immunity, the variety and diversity of innate immune receptors, the interactions of these receptors with other surface molecules, and the mechanisms that innate immune receptors employ to transduce intracellular signals (Hoffmann et al., 1999). The common features of innate receptors and their signaling pathways in species as divergent as insects and mammals are providing important information about how infectious processes can be contained and eliminated within the time delays that are inherent in the clonal selection/expansion phase of the adaptive immune response. Of the various mechanisms of innate immune function in mammals, most attention has been directed to natural killer (NK) cells, their receptors, the interactions of these receptors with target cells and the mechanisms by which receptor binding transduces intracellular signals (reviewed in Lanier, 1998; Ravetch and Lanier, 2000; Lanier, 2001; McQueen and Parham, 2002).
In mammals, natural killer (NK) cells are large granular lymphocytes that differentiate from a common lymphoid progenitor cell. NK cells can kill tumor and virus-infected cells spontaneously in vitro, and are thought to function in surveillance of neoplasia and cellular infection in vivo. NK reactivity is triggered by the reduction or absence of major histocompatibility complex class I (MHC I), MHC I-related gene products, as well as viral components on the surface of a target cell, and ultimately leads to direct killing as well as the release of various immune modulators (Ravetch and Lanier, 2000; Cerwenka and Lanier, 2000). Despite the frequent designation of NK cells as a "primitive lymphoid lineage," their phylogenetic origin is not well understood in terms NK-specific receptors, owing in part to a lack of lower vertebrate models in which specific NK function can be studied. However, certain types of intercellular recognition in lower vertebrates have been interpreted as representing NK- or cytotoxic T lymphocyte interactions (Shen et al., 2001).
Before going on to describe a family of innate receptors that potentially effect NK reactivity in teleost fish, it is constructive to consider the foundations for our current understanding of the basic molecular biology of NK cell function in man and mouse. NK receptor gene families can be classified into four general categories. Three of these families share a common mode of intracellular regulation through activating/inhibitory signaling: 1) killer cell immunoglobulin (KIR)-type, which are present in man and recognize MHC I (Lanier, 1998); 2) lectin-type (Ly49), which are encoded at the NK complex (NKC) in mouse and also recognize MHC I (Lanier, 1998); and 3) lectin-type (NKG2D), which are found in both man and mouse and recognize the products of the stress-inducible, nonconventional MHC genes RAE-1 and H60 (Cerwenka and Lanier, 2000). The fourth category, represented by NKp44 and NKp46, are activating receptors that recognize viral hemagglutinins and are encoded by single copy genes (Mandelboim et al., 2001; Arnon et al., 2001).
The mechanism of NK cell activation is based on an interaction between an NK receptor and an adaptor protein that possesses a cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM); this interaction involves the association of a positively charged residue in the receptor transmembrane region with a negatively charged residue in the adaptor molecule (Ravetch and Lanier, 2000; Lanier, 2001; Cerwenka and Lanier, 2000). The mechanism of inhibitory signaling utilizes cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIMs), which are present in certain lectin and Ig-type NK receptors. Activating and inhibitory receptors are co-expressed on normal cells and engage different forms of MHC I, maintaining a signaling equilibrium (Fig. 1). The inhibitory mechanism is stronger but a shift towards the activating pathway can override the inhibitory pathway, ultimately resulting in target destruction (Ravetch and Lanier, 2000). Viral infection, malignancies, and stress, which suppress the expression of the ligand for the inhibitory receptor, activate NK cells to kill an infected, cancerous or stressed cell (Cerwenka and Lanier, 2000).
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EVOLUTION OF NK RECOGNITION |
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SYNOPSISINTRODUCTIONEVOLUTION OF NK RECOGNITIONPHYLOGENY OF DIVERSIFIED...IDENTIFICATION OF NOVEL IMMUNE...NITR GENES IN ZEBRAFISHRECOGNITION SPECIFICITY OF NITRSVARIATIONS IN OVERALL STRUCTURE...POSSIBLE FUNCTIONS OF NITRSEVOLUTION OF NITRSCONCLUSIONSReferences |
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NK function has long been hypothesized to have an early phylogenetic origin, predating that of adaptive immune mechanisms and their apparent obligatory requirements for somatic rearrangement (Litman et al., 1999
). In a general sense, certain mechanisms of innate recognition, even those identified in invertebrate species, demonstrate remarkable similarities to corresponding systems found in mammals (Lemaitre et al., 1996; Levashina et al., 2001). However, outside of studies in mammals, there is a paucity of molecular information about the gene products that effect NK function and even in cases where putative mediators of NK function have been identified, e.g., NCCRP-1 in bony fish, the relevant molecular structures are dissimilar to the mediators of NK function in mammals (reviewed in [Shen et al., 2001]). Furthermore, the absence of structural features such as ITIMs suggests that if indeed the functions that have been ascribed to these gene products are of the NK type, their signaling mechanisms likely differ from those associated with the products of the LRC and NKC loci in mammals. Notwithstanding the basic significance of understanding the evolutionary origins of the NK receptor families, it is important to note that the full range of molecules mediating NK function likely is not fully understood in any species, including mammals. Evolutionary studies can provide a relevant background for identifying additional, novel NK receptor genes in various species and may provide additional targets for cloning of novel mammalian NK receptor orthologs.
In order to understand the evolution of NK recognition, it is critical that genes corresponding to the LRC and/or NKC be identified in species outside of mammals. Functional evidence for NK reactivity has been described in avians (Gobel et al., 1996) as well as in bony fish (Shen et al., 2001); however, little detailed information relating to the structure of NK receptor genes is available for either of these groups. As with other studies that involve phylogenetically significant but experimentally challenging animal model systems (with their inherent limitations regarding in vitro cell viability, clonality, and well-defined lineage-specific markers), it has been more straightforward to identify homologous gene(s) at the molecular genetic level than to approach such targets from a functional standpoint. Recent advances in genomics have augmented molecular genetic approaches through the introduction of large insert cloning, microarray analysis, and high throughput sequencing. The dedication of considerable resources to resolving the complete genomes of nonmammalian vertebrates, such as zebrafish, ultimately will offer new avenues for addressing important evolutionary questions including the biology of NK receptors.
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PHYLOGENY OF DIVERSIFIED VARIABLE REGIONS |
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SYNOPSISINTRODUCTIONEVOLUTION OF NK RECOGNITIONPHYLOGENY OF DIVERSIFIED...IDENTIFICATION OF NOVEL IMMUNE...NITR GENES IN ZEBRAFISHRECOGNITION SPECIFICITY OF NITRSVARIATIONS IN OVERALL STRUCTURE...POSSIBLE FUNCTIONS OF NITRSEVOLUTION OF NITRSCONCLUSIONSReferences |
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The approach that we have taken to identify novel innate immune receptors originated with phylogenetic studies of the adaptive immune receptors, Ig and TCR, which are members of the immunoglobulin gene superfamily (IgSF) and consist of variable (V) and type 1 constant region (C1) domains. C1 domains also are found in MHC I and II molecules, as opposed to the structurally distinct C2 domains that are more broadly distributed throughout the IgSF. Despite significant variation between the V regions of Igs and TCRs, it is possible to discern localized regions of sequence identity. Such findings could be anticipated for genes that specify a general function (antigen binding) that is associated with a broad range of specificity. The presence of two highly conserved 3–4 amino acid motifs in the V region form the basis for a short primer PCR strategy (Rast and Litman, 1994
; Rast et al., 1995). This approach initially was employed in phylogenetically ancient species to identify orthologous forms of TCRs, which had eluded identification by conventional methods of cross-hybridization and degenerate PCR priming (Rast and Litman, 1994; Rast et al., 1997). The successful application of the short primer approach in studies of TCR genes in the most phylogenetically distant jawed vertebrates led us to hypothesize that this technology might identify genes possessing only nominal structural relatedness to the rearranging antigen binding receptors. |
IDENTIFICATION OF NOVEL IMMUNE-TYPE RECEPTORS |
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SYNOPSISINTRODUCTIONEVOLUTION OF NK RECOGNITIONPHYLOGENY OF DIVERSIFIED...IDENTIFICATION OF NOVEL IMMUNE...NITR GENES IN ZEBRAFISHRECOGNITION SPECIFICITY OF NITRSVARIATIONS IN OVERALL STRUCTURE...POSSIBLE FUNCTIONS OF NITRSEVOLUTION OF NITRSCONCLUSIONSReferences |
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Although there was no functional predicate for the existence of additional, diversified families of V region-containing genes other than Ig and TCR, we examined this possibility using the small genome model system, pufferfish (Spheroides nephelus). A unique, putative receptor was identified that had the characteristics of an Ig superfamily (IgSF) member. This particular novel immune-type receptor (NITR) gene was shown to encode two Ig ectodomains, a transmembrane region, and a cytoplasmic tail containing two potential ITIMs (Rast et al., 1995
); comparison of the structure of this NITR (SN193) with two different types of mammalian NK receptors and a TCR revealed general relatedness among these transmembrane molecules (Fig. 2). The N terminal ectodomain of SN193 is of the V-type and contains a contiguous joining (J) region motif (FGXG). The assignment of the V region is unequivocal; specifically, when the V region is used to scan the human genome, tBLASTN identifies the V domains of Ig
and TCR
/ß as being most similar. If the core amino acid residues of human Ig and TCR V regions (defined as those positions, that are 80–100% conserved), are compared with the corresponding positions in the large number of NITRs that have been sequenced, it is apparent that the NITR V regions are essentially as similar to Ig and TCR V regions as these regions are similar to one another.
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Several factors confound classification of the C terminal ectodomain of NITRs. Although somewhat shorter than typical V regions, the length difference between the N- and C-terminal ectodomains could be accounted for by shortened CDR1 and CDR2 loops. Alternatively, the loss of one V region strand would be consistent with an assignment of the C-terminal ectodomain to the broadly defined category of the C2 domain. Given this uncertainty and no available means to resolve the ambiguity, we have designated the C-terminal ectodomain as a V/C2-type (Strong et al., 1999
), which also is consistent with classification as an I type (Harpaz and Chothia, 1994). The V/C2 domain of SN193 encodes the core of a J motif (GXG).
The extent of NITR diversity was examined first in pufferfish, from which 26 NITR genes (including two pseudogenes) were identified in the complete sequence of a P1 artificial chromosome (PAC). The NITR V ectodomains in pufferfish can be classified into 13 distinct families, defined as possessing 70% or less amino acid identity. An NITR structural variant (e.g., SN6A, Fig. 3A) contains a core GXG motif C-terminal to the V ectodomain and a prototypic FGXG J motif C-terminal to the V/C2 domain. The consensus ITIM that is located in the N-terminal portion of the cytoplasmic tail of NITRs SN193 and SN6A is absent in 
40% of the pufferfish NITRs identified thus far (Strong et al., 1999), including SnNITR3 (Fig. 3A).
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NITR GENES IN ZEBRAFISH |
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SYNOPSISINTRODUCTIONEVOLUTION OF NK RECOGNITIONPHYLOGENY OF DIVERSIFIED...IDENTIFICATION OF NOVEL IMMUNE...NITR GENES IN ZEBRAFISHRECOGNITION SPECIFICITY OF NITRSVARIATIONS IN OVERALL STRUCTURE...POSSIBLE FUNCTIONS OF NITRSEVOLUTION OF NITRSCONCLUSIONSReferences |
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The complexity of the individual NITR gene families is being examined systematically in zebrafish. Sequence-tagged site (STS) markers have been used to map four families (defined by the sequences of V/C2 ectodomains) of nitr genes to a single,
1 megabase (Mb) chromosomal locus (Yoder et al., 2001). Putative orthologs of two forms of pufferfish NITRs have been described in zebrafish (DrNitr1.2 and DrNitr1.1, Fig. 3A). In addition, six other variants have been recognized in which differences are evident in J motifs, ITIMs and in the number of ectodomains (Fig. 3A). Collectively, these observations suggest that in addition to potential differences in ligand binding, variation likely occurs in intracellular signaling as well as in the overall function of these genes. The nitr gene cluster has been prioritized for complete sequencing in the Zebrafish Genome Project being conducted at Wellcome Trust Sanger Institute (UK); results to date suggest that the inhibitory subset of the zebrafish nitr genes, including polymorphic variants, consists of 80–100 different genes (see below). Furthermore, the genomic sequence analyses have revealed additional forms of nitr genes, including a nitr that encodes a putative ITAM in the cytoplasmic tail. It is likely that exon shuffling has factored in the evolution of the NITR gene complex (Litman et al., 2001) and accounts for the diversity of form seen in the negative inhibitory molecules. NITRs appear to be expressed in a number of different hematopoietic tissues as well as in peripheral blood. At this point, selective expression of specific NITRs has not been demonstrated in any tissue. |
RECOGNITION SPECIFICITY OF NITRS |
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SYNOPSISINTRODUCTIONEVOLUTION OF NK RECOGNITIONPHYLOGENY OF DIVERSIFIED...IDENTIFICATION OF NOVEL IMMUNE...NITR GENES IN ZEBRAFISHRECOGNITION SPECIFICITY OF NITRSVARIATIONS IN OVERALL STRUCTURE...POSSIBLE FUNCTIONS OF NITRSEVOLUTION OF NITRSCONCLUSIONSReferences |
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Although the nature of NITR ligands is not understood, it is likely that NITRs function as receptors and some information about their function can be inferred by examining their patterns of overall diversification. These comparisons are best restricted to pufferfish NITRs, specifically the single PAC sequence described above, in which complications arising from allelic differences are eliminated as only a single chromosomal segment is being considered. Furthermore, the number of NITR genes per unit genetic length in pufferfish is higher in this than in the other species in which NITRs have been identified. Pufferfish NITRs share a number of similarities with the Ig and TCR genes in terms of overall diversity. Specifically: 1) 13 different families of V regions, which are defined as exhibiting >70% predicted peptide sequence identity, are found in the 26 pufferfish NITR genes encoded in the single PAC; 2) the patterns of variation in their V regions, specifically the concentration of differences in CDR1 and CDR2, resemble patterns of sequence difference found in Ig and TCR; 3) a third region of sequence variation in NITRs possibly corresponds to hypervariable region 4 in TCRs (Wuilmart and Urbain, 1991
), and 4) variation in Ig and TCR structure is greater in the N- vs. C-terminal ectodomains. Some additional information about NITR function can be inferred from studies in which NITRs have been transfected into human NK cells. Specifically, epitope-tagged zebrafish NITRs can transduce negative regulatory signals, as evidenced by the inhibition of MAP kinase upon antibody-induced crosslinking; mutation of the N-terminal ITIM reduces this effect (Yoder et al., 2001). |
VARIATIONS IN OVERALL STRUCTURE OF NITRS |
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SYNOPSISINTRODUCTIONEVOLUTION OF NK RECOGNITIONPHYLOGENY OF DIVERSIFIED...IDENTIFICATION OF NOVEL IMMUNE...NITR GENES IN ZEBRAFISHRECOGNITION SPECIFICITY OF NITRSVARIATIONS IN OVERALL STRUCTURE...POSSIBLE FUNCTIONS OF NITRSEVOLUTION OF NITRSCONCLUSIONSReferences |
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Extraordinary variation in the overall structure (and presumably function) of the NITR genes has been observed in the NITR genes in a third bony fish model system (Ictalurus, catfish). Various assays of immunological function can be conducted in this species, making it potentially significant in terms of understanding NITR function. Eleven different structural forms of NITR (IpNITR) genes have been identified that vary in the number of Ig ectodomains (e.g., IpNITR1 vs. IpNITR5, Fig. 3A), presence or absence of charged groups in the transmembrane region (e.g., IpNITR7 vs. IpNITR10, Fig. 3B), presence or absence of ITIMs (e.g., IpNITR1 vs. IpNITR2, Fig. 3A and B), and presence or complete absence of transmembrane regions (e.g., IpNITR7 vs. IpNITR8, Fig. 3B and C) (Hawke et al., 2001
). Both inhibitory and activating forms of IpNITRs have been predicted (Hawke et al., 2001) and their general structural organizational features are compared with those of various NK receptors (Fig. 4A and B). Notably, closely related V regions are encoded by NITRs (IpNITRs 5–11) that otherwise exhibit markedly different structural organizations (Fig. 3A and B). Similarly, the ectodomains of certain mammalian NK receptors are related closely (Ryan et al., 2001), which is consistent with the recognition of similar ligands by molecules that effect different functions. As with NK receptors, the inhibitory forms of NITRs predominate over activating forms (McQueen and Parham, 2002). The nature of structural variation in NITRs can be examined in greater depth through the comprehensive interpretation of extensive genome sequence information, such as will be possible for zebrafish. Expansion of cell lineages expressing certain IpNITRs has been demonstrated during long-term mixed lymphocyte reactions in catfish; in addition, several different IpNITRs are expressed by individual hematopoietic cell lines in this species. NITR and IpNITR genes likely represent the single largest family of receptors that are predicted to associate with an activating as well as inhibitory signaling pathway in the innate immune response.
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and Fc receptor |
POSSIBLE FUNCTIONS OF NITRS |
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SYNOPSISINTRODUCTIONEVOLUTION OF NK RECOGNITIONPHYLOGENY OF DIVERSIFIED...IDENTIFICATION OF NOVEL IMMUNE...NITR GENES IN ZEBRAFISHRECOGNITION SPECIFICITY OF NITRSVARIATIONS IN OVERALL STRUCTURE...POSSIBLE FUNCTIONS OF NITRSEVOLUTION OF NITRSCONCLUSIONSReferences |
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As indicated above, only limited amounts of information are available regarding the function of NITR genes (Yoder et al., 2001
). The foremost consideration is that bony fish, in which NITRs have been characterized, possess diversified Ig and TCR gene families; thus, it is unlikely that NITRs supplant the functions of these receptors. No information has been obtained in either pufferfish or zebrafish that would suggest chromosomal linkage of NITRs (Strong et al., 1999) to either Ig or TCR genes. Linkage of nitrs with other genes, such as men 1 (Yoder et al., 2001), is likely not of functional significance; as of yet, no genes other than nitrs (and men I) have been identified using a variety of search parameters and available sequences of the zebrafish nitr gene cluster (J.A.Y., unpublished data). Gene families resembling the Ig-type of NK receptors, which are encoded at the LRC, have not been reported in species that occupy phylogenetic positions at or below the phylogenetic level of bony fish. However, we have identified and mapped an NITR locus in the rainbow trout (Oncorhynchus mykiss) (Yoder et al., 2002) and demonstrate its linkage to a single-copy ITIM-containing C-type lectin (Zhang et al., 2002). Our current working hypothesis, which is based on the patterns of sequence diversity in the V ectodomains of NITRs, as well as the overall organization of the gene complex and the presence of putative activating and inhibitory forms, is that NITRs effect innate immune functions through ligand binding and may represent the bony fish equivalent of the Ig-like NK receptors (Litman et al., 2001). The most immediate question to be resolved relates to the nature of the ligands that may be bound by these diversified receptors.
NITRs potentially could recognize ligands similar to those recognized by NK receptors. In this regard, it is important to note that certain members of the two major groups of NK receptors found in mammals, KIRs and the Ly49 gene products, recognize MHC I and are encoded in multigene families. Functional equivalents of L49 are not found in man (Barten et al., 2001; Lanier, 1998); however, several KIRs recently have been identified in rodents (Welch et al., 2003; Hoelsbrekken et al., 2003). Highly polymorphic MHC I has been identified in bony fish (reviewed in Flajnik and Kasahara, 2001). NK receptors in mammals do not encode diversified families of V regions and their genomic complexity is lower than that observed with any of the NITR gene families that have been identified thus far in bony fish (Barten et al., 2001). The ectodomains of NK receptors (e.g., NKp30, NKp44 [Moretta et al., 2000; Mandelboim et al., 2001]) that have been designated as V-type are typically single or limited copy number, lack the close similarity to Ig and TCR V regions that is demonstrated by NITRs, and do not possess J motifs (Mandelboim et al., 2001; Arnon et al., 2001). NITRs comprise a significantly larger family of diversified receptors than those that are encoded at the LRC or NKC. Alternatively, NITRs could recognize different ligands (e.g., nonconventional MHC I, viral proteins, etc.) or potentially function in some other manner.
Whether or not NITRs recognize the same ligands that are bound by receptors encoded at the LRC, it is reasonable to conclude that similarities exist between the transmembrane and negative intracellular signaling functions of the NITRs and various LRC gene products. It is likely that a wide range of receptor structures might associate with remarkably similar intracellular signaling pathways and, conversely, that closely related receptors might associate with very different intracellular signaling mechanisms.
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EVOLUTION OF NITRS |
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SYNOPSISINTRODUCTIONEVOLUTION OF NK RECOGNITIONPHYLOGENY OF DIVERSIFIED...IDENTIFICATION OF NOVEL IMMUNE...NITR GENES IN ZEBRAFISHRECOGNITION SPECIFICITY OF NITRSVARIATIONS IN OVERALL STRUCTURE...POSSIBLE FUNCTIONS OF NITRSEVOLUTION OF NITRSCONCLUSIONSReferences |
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Identifying and understanding the distribution of NITRs outside of the bony fish represents one means whereby the relatedness of these genes to other immune receptors could be examined. As indicated above, the intrinsic high degree of variation between NITR genes confounds the ability to isolate homologous forms in different species. The absolute identities between NITRs in different species often involve only one or two amino acids that are present at defined distances from other highly conserved residues, such relationships cannot be exploited in terms of current gene identification approaches. Furthermore, the lack of a clear understanding of NITR function precludes gene identification through either biological or biochemical means. In order to circumvent these difficulties we have developed a gene cloning strategy that requires knowledge of only a single conserved amino acid motif that can exhibit minor variation from conserved consensus sequences, thus circumventing the requirement of short primer PCR for accurately predicting two such motifs. The method is based on maximizing the recovery of intact 5' sequence, a directed RACE step and the selective cloning of signal peptide-containing amplicons, using rescue of ß-lactamase production for direct selection of candidate clones (Cannon et al., 2002). Using this approach, we have identified what appear to represent several classes of Ig-type receptors that potentially exhibit either inhibitory or activating function in Raja eglanteria (clearnose skate). A greater variation in the numbers of ectodomains and the presence of an N-terminal I-type domain distinguish the skate immune-type receptors (ITRs) from the NITRs. Certain ITR gene families exhibit extraordinarily complex patterns in Southern blot analyses, suggesting that they are encoded in diverse multigene families.
This selective cloning strategy also has identified putative secretory gene products in a protochordate (amphioxus), in which the N-terminal Ig-type domains also are of the V-type (Cannon et al., 2002). As the sequences of these and additional divergent molecules are resolved further, it could become possible for us to identify additional consensus sequences upon which amplification strategies can be based.
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CONCLUSIONS |
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SYNOPSISINTRODUCTIONEVOLUTION OF NK RECOGNITIONPHYLOGENY OF DIVERSIFIED...IDENTIFICATION OF NOVEL IMMUNE...NITR GENES IN ZEBRAFISHRECOGNITION SPECIFICITY OF NITRSVARIATIONS IN OVERALL STRUCTURE...POSSIBLE FUNCTIONS OF NITRSEVOLUTION OF NITRSCONCLUSIONSReferences |
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Further resolution of what appears to be a likely role of NITRs (and ITRs) in immune function and/or in other developmental processes will be facilitated by: identifying potential ligands, determining the expression patterns of NITRs/ITRs during development, characterizing hematopoietic mutant animals (zebrafish) in which expression of NITRs differs, refining electronic methods to search for short NITR/ITR-related sequence motifs (Hawke et al., 1999
) in the rapidly resolving genomic sequence databases of higher vertebrates, and characterizing these genes as well as related forms in species representing different levels of phylogenetic development. Notwithstanding their obvious significance from a phylogenetic perspective, the NITR/ITR genes may prove critical in understanding the most fundamental aspects of the divergence of adaptive and innate immune function.
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ACKNOWLEDGMENTS |
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This research was supported by grants AI23338 to GWL and GM20231 to JAY from the National Institutes of Health as well as a grant from The Pediatric Cancer Foundation, Inc. to GWL.
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FOOTNOTES |
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1 From the Symposium Comparative Immunology presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 2–6 January 2002, at Anaheim, California.
2 E-mail: litmang{at}allkids.org
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References |
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SYNOPSISINTRODUCTIONEVOLUTION OF NK RECOGNITIONPHYLOGENY OF DIVERSIFIED...IDENTIFICATION OF NOVEL IMMUNE...NITR GENES IN ZEBRAFISHRECOGNITION SPECIFICITY OF NITRSVARIATIONS IN OVERALL STRUCTURE...POSSIBLE FUNCTIONS OF NITRSEVOLUTION OF NITRSCONCLUSIONSReferences |
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Arnon, T. I., M. Lev, G. Katz, Y. Chernobrov, A. Porgador, and O. Mandelboim. 2001. Recognition of viral hemagglutinins by NKp44 but not by NKp30. Eur. J. Immunol, 31:2680-2689.[CrossRef][Medline]
Barten, R., M. Torkar, A. Haude, J. Trowsdale, and M. J. Wilson. 2001. Divergent and convergent evolution of NK-cell receptors. Trends Immunol, 22:52-57.[CrossRef][ISI][Medline]
Cannon, J. P., R. N. Haire, and G. W. Litman. 2002. Identification of diversified immunoglobulin-like variable region-containing genes in a protochordate. Nature Immunol, 3:1200-1207.[CrossRef][ISI][Medline]
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