© 2003 by The Society for Integrative and Comparative Biology
Rapid Evolutionary Emergence of the Combinatorial Recognition Repertoire1
1 Department of Microbiology and Immunology,
2 Department of Biochemistry, University of Arizona, College of Medicine, Tucson, Arizona 85724
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Although the capacity of cells to respond to environmental challenges such as oxidative damage are ancient evolutionary developments that have been carried through to modern higher vertebrates as "innate" immunity, the characteristic immune response of vertebrates is a relatively recent evolutionary development that is present only in jawed vertebrates. The vertebrate "combinatorial" response is defined by the presence of lymphocytes as specific antigen recognition cells and by the complete panel of antibodies, T cell receptors, and major histocompatibility complex molecules all of which are members of the immunoglobulin family. Its emergence in evolution was an extremely rapid event (approximately 10 million years) that was catalyzed by the horizontal transfer of recombinase activator genes (RAG) from microbes to an ancestral jawed vertebrate. RAGs occur in jawed vertebrates, but have not been found in invertebrates and other intermediate species. We propose that antigen recognition capacity contributed by this novel combinatorial mechanism gave jawed vertebrates the ability to recognize the entire range of potential antigenic molecular structures, including self components and molecules of infectious microbes not shared with vertebrates. The contrast within the vertebrates is striking because the most ancient extant jawed vertebrates, sharks and their kin, have the complete panoply of T-cell receptors, antibodies, MHC products and RAG genes, whereas agnathans possess cells resembling lymphocytes but ostensibly lack all of the molecules definitive of combinatorial immunity. Another vertebrate innovation may have been the utilization of nuclear receptor superfamily, in the regulation of lymphocytes and other cells of the immune lineage. Unlike, RAG, however, this superfamily occurs in all metazoans with the exception of sponges.
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INTRODUCTION |
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Interest in the immune response arose historically because of its clinical relevance in vaccination against infectious diseases, but the applications of biochemistry and molecular biology disclosed that immunity was rooted in fundamental biological mechanisms as well as exhibiting novel and unique genetic innovations. The first of these was the finding that antibodies failed to obey the Beadle/Tatum dictum of "one gene-one enzyme" (protein) because at least three gene segments were required to form a functional antibody chain gene. Diversity in recognition was generated by recombination involving large numbers of variable, joining and diversity gene segments. Here, we consider several ancient defense mechanisms that can be considered to comprise part of the "innate" immune system in the light of their emergence and evolution. We analyze the possibility of horizontal, also termed lateral, transfer of genes for its contribution to the emergence of the combinatorial (also termed adaptive) immune response of jawed vertebrates. Finally, we compare the natural antibody recognition repertoires of sharks, the most anciently arisen vertebrates to have the complete combinatorial response (Marchalonis et al., 1998a
; DuPasquier and Flajnik, 1999; Litman et al., 1999) with that of humans, the most recent and "advanced" to share fundamental features of this response.
We marshall evidence to develop two hypotheses. First, that the combinatorial response arose rapidly in evolution because of the horizontal transfer of genes enabling site-specific recombination from microbes to ancestral jawed vertebrates (Bernstein et al., 1996; Agrawal et al., 1998; Hiom et al., 1998; Plasterk, 1998). Second, that the primordial combinatorial recognition repertoire in evolution and in individuals in ontogeny encompasses the entire range of specificities recognized by the innate system (Marchalonis et al., 2002).
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MILESTONES IN THE EVOLUTION OF ORGANISMS AND RECOGNITION |
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Figure 1 is a schematic diagram illustrating selected major events in evolution plotted on a time scale of billions of years (bya) starting with the origin of the earth at approximately 5 bya and ending at the present. The so-called RNA world arose more than 4 bya, with RNA serving as enzymes and the subsequent emergence of DNA polymerases and proteins. Retroposons capable of reproducing themselves most probably arose shortly after this period. The first evidence of life occurred approximately 3.8 bya with the earliest known microfossils dated at 3.5 bya. The first organisms most probably were anaerobes such as present-day archaea, but true bacteria arose shortly afterwards. It is difficult to construct a universal phylogenetic tree based upon RNA or protein because of extensive horizontal transfer of genes among bacteria and archaea, and to a significant but lesser degree among the later-arising eukaryotes (Doolittle, 1999
). The first oxygen producing organisms arose approximately 2.7 bya with atmospheric oxygen reaching approximately present levels 2.2 bya (Kerr, 1999). Proteins essential for defense against oxidative stress and other possible stress factors such as heat shock proteins and superoxide dismutase must have arisen early in evolution because they are present in archaebacteria as well as bacteria and eukaryotes (May and Dennis, 1987; Ayala, 1997). The nuclear transcription factor Nf
b and its associated mechanisms were first studied in B lymphocytes (Ghosh et al., 1998) and is often taken as a definitive characteristic of "innate immunity" (Medzhitov and Janeway, 1997). However, it is likely that this system arose to protect cells against oxidative damage (Baeuerle and Henkel, 1994). Proteins significantly related to Nfb and the "toll" receptor (Hoffman et al., 1999) and other elements of this regulatory system occur in plants as well as animals (Staskawicz et al., 1992). Thus, we speculate that this system emerged more anciently than the 1.2 bya at which recognizable plants such as higher algae were present. Fungi and animals diverged approximately 0.9 bya and the split between protostomes and deuterostomes occurred approximately 0.7 bya (Blackwell, 2000). The Cambrian explosion of animal diversity occurred 0.54 bya. This relatively rapid appearance in the fossil record of forms resembling virtually all of those extant today did not represent the initial appearance of these organisms. Rather, it represented the diversification and emergence of relatively large numbers of these species. We would stress that there was widespread horizontal transfer of genetic information across species, within phyla, and even across kingdoms during this time period (Woese, 1998), and extensive duplication of gene and genomes most probably also occurred (Ohno et al., 1968). Familiar vertebrates first appeared approximately 530 mya with cartilaginous fishes appearing approximately 450 mya. The Nfb system, particularly with respect to its appearance in insects, has been discussed at length by others (Medzhitov and Janeway, 1997; Hoffman et al., 1999) and we refer to it here only to illustrate its ancient evolutionary emergence. We proposed previously that the combinatorial system coopted existing recognition and cellular mechanisms of activation and differentiation (Marchalonis and Schluter, 1990), but incorporated a novel recognition system based upon horizontal transfer of genes allowing diversification to occur through recombination (Bernstein et al., 1996; Marchalonis and Schluter, 1998; Schluter et al., 1999). Examples of other ancient recognition mechanisms are those of acute phase proteins related to the pentraxins such as C-reactive protein, a molecule capable of binding DNA, phosphorylcholine, streptococcal carbohydrate and complement components that are present in recognizable forms in an ancient arachnoid, the horseshoe crab, and all vertebrates (Liu et al., 1994). Other examples of acute phase proteins are ferritin, which is needed by LPS activated leukocytes of starfish (Beck et al., 2002), as well as cells involved in inflammatory reactions of vertebrates. A recently appreciated contributor to the innate immune system consists of the Vitamin D receptor and its ligand Vitamin D3 (Nashold et al., 2001). However, as we will argue, the immunomodulatory role of the Vitamin D/VDR system may actually predate its more familiar role in calcium regulation.
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ANCIENT SYSTEMS PARTICIPATING IN INNATE OR COMBINATORIAL IMMUNITY |
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Table 1 gives a selection of ancient systems coopted by the combinatorial immune response of jawed vertebrates that participate either in recognition and defense reactions of non-lymphoid cells or in processes of cellular activation and differentiation. At least two types of acute phase proteins expressed in blood or hemolymph are shared between protostomes and jawed vertebrates; those of the pentraxin family that includes serum amyloid and C-reactive proteins (Liu et al., 1994
), and those of the NCAM (Neural Cell Adhesion Molecule) family of molecules that are expressed in insects (Sun et al., 1990), and molluscs (Hoek et al., 1996). The NCAM-related molecules are members of the immunoglobulin superfamily (IgSf) and consist of the so-called C2 domains that are distantly related to the bona fide immunoglobulin (Ig) constant domains, which are termed C1, of Igs, TCRs and MHC Ig modules. Antigenic challenge of insects tends to increase the levels of the circulating NCAM-like molecule termed hemolin, whereas challenge of molluscs tends to downregulate appearance of a related molecule. The complement system of jawed vertebrates comprises approximately 30 distinct proteins that form two enzyme cascades to bring about the destruction of foreign cells either through interaction with antibodies bound to the cells (the so-called classical pathway), or via the "alternative pathway" that does not involve antibodies. The lytic complement pathway can be activated through binding of lectins or of C-reactive protein to the target cells. Complement components are related to 
2-macroglobulin, a serum inhibitor of proteases that occurs in arthropods (Armstrong et al., 1991) as well as in deuterostomes. Molecules homologous to actual complement components are present in lower deuterostomes including the echinoderm, the sea urchin (Smith et al., 1998), and in agnathans (Hanley et al., 1992; Ishiguro et al., 1992). Complement molecules, particularly C3, can function as opsonins in the recognition of bacterial pathogens and in their elimination by phagocytosis of the bound complexes. Thus, recognizable elements of the complement system precede the appearance of jawed vertebrates but form a bridge between the combinatorial system and the innate recognition system.
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Molecules of the Ig superfamily (IgSf) comprise approximately 40% of protein domains described to date (Doolittle, 1995
), and arose early in evolution with representation in sponges (Blumbach et al., 1999), nematodes (Teichmann and Chothia, 2000), insects (Sun et al., 1990), molluscs (Hoek et al., 1996), and vertebrates (Williams and Barclay, 1988). Although the superfamily members can be distinguished from the bona fide Ig variable and constant domains based upon sequence homology and signature sequences, they still can be considered to be related to these domains. Initially, it was thought that Ig domains must of necessity be associated with cell membranes and function in recognition, but it was subsequently found that Ig domains, in covalent association with fibronectin 3 domains, form large contractile proteins in muscles (Teichmann and Chothia, 2000). Unlike Igs and T-cell receptors that essentially contain only Ig domains, membrane associated IgSf recognition/activation molecules are frequently mosaic structures containing Ig domains, fibronectin type 3 domains, transmembrane segments and internal tyrosine kinase units. Such a situation occurs with many cytokine receptors, (e.g., IL-1) (Sims, 1989), and also in receptors on natural killer cells (NK cells) (Lanier, 1998) as well as for surface receptors widely distributed in evolution such as receptor tyrosine kinases of the sponge Geodia cyndonium (Pancer et al., 1996). Interestingly, the IgSf domains can be either C2-like or V-like with the two Ig-like domains found in the sponge membrane-associated molecule being similar to Ig V domains. A parallel situation occurs for the membrane receptor-linked protein tyrosine phosphatases where homologous receptor units occur in both protostomes and deuterostomes. Thus, the IgSf represents a very ancient and diverse component, not only of the immune system, but of other functional systems as well.
One of the major destructive stresses faced by all cells was the problem of reactive oxygen following the achievement of high levels of atmospheric oxygen approximately 2.2 bya. One ancient molecule that functions in the elimination of toxic oxygen radicals is superoxide dismutase, an enzyme that occurs in Eubacteria, Archebacteria and most probably all living organisms (May and Dennis, 1987; Ayala, 1997). Although this molecule has no recognizable sequence identity to members of the IgSf, crystallographic analysis revealed that it contains the Ig fold. A second system that is induced by oxidative stress (Ghosh et al., 1998) and functions to protect diverse cells including colon cancer cells (Payne et al., 1998), and neuronal cells (Lezooualc'h et al., 1998) from apoptotic events includes the nuclear transcription factor Nfb, the interleukin-1 receptor and their well-characterized homologs in Drosophila (Hoffman and Reichhart, 1997). Defense systems containing homologous elements, in fact, are widely distributed among plants, protozoans, echinoderms, protostomes, lower vertebrates and mammals. Likewise, molecular chaperones and heat shock proteins arose early in evolution and are apparently universally distributed. These molecules protect against cellular stresses of oxidation and heat shock, and have been incorporated into cellular regulatory events involved in innate immunity.
Still another ancient defense against oxidative damage is the sequestering of iron inside the ferritin molecule. The synthesis of ferritin in vertebrates is dependent mainly on iron status, but the process can also be stimulated by activation with cytokines or LPS. Beck et al. (Beck et al., 2002) found that treatment of starfish (Asterias forbesi) by lipopolysaccharide caused an increased expression of ferritin mRNA and characterized cDNA encoding a ferritin molecule showing 64% identity to human ferritin H chain. Ferritin occurs in most eucaryotes, including insects (Pham et al., 1996), and in crayfish (Huang et al., 1996), with the starfish ferritin showing a greater similarity to vertebrate ferritins than it does to those of protostome invertebrates.
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EVOLUTION OF THE NUCLEAR RECEPTOR (NR) SUPERFAMILY WITH FOCUS UPON THE VITAMIN D RECEPTOR (VDR) |
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The nuclear receptor (NR) superfamily that occurs in all metazoans except sponges arose early in evolution (Escriva et al., 2000
). There are six subfamilies of nuclear receptors for steroids with members in C. elegans and Drosophila, as well as in higher vertebrates (Maglich et al., 2001). Some NR receptor groups such as the estrogen/progesterone subfamily occur only in vertebrates with representation in lampreys as the most primitive members (Thornton, 2001). An association between steroid receptors and the immune system has long been noted because T lymphocytes are exquisitely sensitive to killing by estrogen (Sternberg and Parker, 1988). Recent studies show that vitamin D3 can play a substantial role in regulating inflammatory processes and the inhibition of the secretion of TH1-type cytokines (Manolagas et al., 1994). Furthermore, RAG1-dependent cells were found to be necessary for prevention of experimental autoimmune encephalitis by 1,25-dihydroxyvitamin D3 (Nashold et al., 2001). The vitamin D receptor (VDR) of terrestrial vertebrates binds DNA as a heterodimer in association with a homolgous, but distinct, retinol receptor (RXR) and mediates the genomic actions of this vitamin. One of the prominent actions of VDR-regulated gene products is to maintain blood calcium allowing for the calcification of bones and teeth. We prepared a cDNA library from mRNA derived from the proto-spleen of larval lampreys and used evolutionary PCR to isolate clones homologous to the VDR of jawed vertebrates (Whitfield, Dang, Schluter, Bernstein, Marchalonis, unpublished data). Recombinant lamprey VDR expressed in COS-7 cells bound 1,25 dihydroxy D3 with a Kd of 0.7 nM which compared favorably with that of human VDR having a Kd of 0.3 nM. Initially, we found it surprising that VDR occurred in the lamprey because the major role of this hormone is thought to be in mobilization of calcium for bone and tooth mineralization, and lampreys lack both true bones and calcified teeth. However, the recently recognized regulatory role of this vitamin in inflammation and immune reactivity involving T cells suggests that the immunoregulatory function may have preceded the application of this system to bone formation. The link of the VDR receptor system to mechanisms of innate immunity is strengthened by the finding that vitamin D3 regulates expression of Nfb subunits (Manolages et al., 1994).
As shown in Figure 2, the lamprey VDR resembles VDRs of teleost fish, with an overall amino acid identity in the range of 59–62% to VDRs from higher vertebrates. The similarity of lamprey VDR to other VDRs can be seen most clearly in the functional domains for DNA binding and for ligand binding/heterodimerization. The DNA binding segment is especially highly conserved, with >80% amino acid identity to the corresponding domains in VDRs from higher vertebrates. There is a lesser degree of identity in the C-terminal ligand binding domain, which also contains residues that mediate dimerization of ligand VDR with the retinoid X receptor to create a transactivational complex (Haussler et al., 1998).
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A phylogenetic analysis of vertebrate VDR sequences was performed using the insect ecdysone receptor as an invertebrate outgroup (Fig. 3). This receptor is also from subfamily 1, but represents a different group from that of the VDRs. The lamprey VDR clustered unambiguously with other VDRs and thus does not appear to represent a common ancestor of VDRs (PXRs/CARs). However, as expected on phylogenetic grounds, lamprey VDL forms the basal member of known vertebrate VDRs (Fig. 3).
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THE RAPID EVOLUTIONARY EMERGENCE OF COMBINATORIAL IMMUNITY |
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SYNOPSISINTRODUCTIONMILESTONES IN THE EVOLUTION...ANCIENT SYSTEMS PARTICIPATING IN...EVOLUTION OF THE NUCLEAR...THE RAPID EVOLUTIONARY EMERGENCE...THE PRIMORDIAL REPERTOIRE AND...CONCLUSIONSReferences |
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Immunoglobulins
The preceding material illustrates that recognition and regulatory mechanisms that arose early in evolution and may be shared among all groups of multicellular animals can persist into the vertebrates where they become part of the complex series of events underpinning the adaptive or combinatorial, immune response. The degree of completeness of the combinatorial system in the most anciently arisen living vertebrates to possess the combinatorial immune response was surprising. Sharks, and their relatives the rays and chimeras, with an ancestral emergence approximately 450 mya have lymphocytes and plasma cells, MHC class I and class II antigens, TCRs of the
, ß, 
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types, Ig µ heavy chains, and a large array of Ig light chains including some identifiable as "primitive" relatives of and 
chains. A simplified diagram of the immunological status of sharks (Marchalonis and Schluter, 1998; Marchalonis et al., 1998b) is shown in Figure 4. Since the basic structures and evolution of Igs and TCRs have been reviewed in detail recently (Marchalonis et al., 1998a; DuPasquier and Flajnik, 1999; Litman et al., 1999), we will only point out that the combining sites for antigen are formed, with rare exceptions, by association of heterodimeric V-domains. The genes specifying V-domains contain Fr1, CDR1, Fr2, CDR2 and Fr3 segments, but the dominant complementarity determining region (CDR3) is formed by combinatorial rearrangement of V, J and sometimes D segments. The frameworks provide the scaffold, and the loops formed by the CDRs provide contact residues for binding of antigens. To illustrate the degree of evolutionary conservation of frameworks, there can be as much as 60% identity between V-domain frameworks of shark heavy and light chains and those of humans.
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Elements of the IgSf are widely distributed throughout living organisms (Williams and Barclay, 1988
). In addition, large families of molecules containing V region-like domains sometimes associated with C2-like extracellular domains and transmembrane regions containing tyrosine kinase motifs occur in teleost fish (Hawke et al., 2001). Moreover, similar molecules occur in animal groups as primitive as sponges (Pancer et al., 1996; Blumbach et al., 1999). The key issue is that even though large families have been found, these molecules do not generate diversity by recombination mechanisms like those found in antibodies and T-cell receptors of gnathostomes. The agnathan vertebrates lampreys and hagfish and the non-vertebrate deuterostomes such as tunicates and echinoderms are important to evolutionary considerations because these organisms possess elements of the innate system that have been retained in jawed vertebrates. However, many attempts by us and other workers have failed to disclose concrete evidence of Ig, TCRs or RAG genes in agnathans. The RAG genes are of particular interest because site-specific recombinases showing apparent homology occur in microbes (Bernstein et al., 1996; Fugmann et al., 2000) but do not exist in metazoans below the gnathostomes. Cyclostomes, however, possess cells identified histologically as lymphocytes and plasma cells and possible gut-associated lymphoid tissue (Zapata and Cooper, 1990) as well as nuclear transcription factors homologous to those found in vertebrates but not in invertebrates (Anderson et al., 2001). Although multiple gene duplications in the generation of Ig light and heavy chains and TCR domains make recognition of exact orthologous relationships uncertain if not impossible, sufficient degrees of identity were observed among these domains of jawed vertebrates to enable calculation of rates of evolution and estimation of time of appearance. According to these calculations, the TCR constant domains appeared approximately 500 mya which coincides roughly with the emergence of jawed vertebrates. The Ig constant domains, by contrast, have an estimated time of emergence of approximately one bya. Thus, the possibility exists that bona fide IgC (C1) domains arose prior to the appearance of jawed vertebrates, but representatives of the ancestral species expressing these are extinct. While models have been suggested for the nature and evolution of precursors of Ig domains (Du Pasquier et al., 1999), it is likely that we will never be able to identify the exact molecule. We (Marchalonis and Schluter, 1990; Bernstein et al., 1996; Schluter et al., 1999) and others (Agrawal et al., 1998; Hiom et al., 1998; Plasterk, 1998) have hypothesized that the event that initiated the explosive burst of the generation of combinatorial immunity was the horizontal transfer of genes from microbes and/or fungi enabling site-specific recombination of DNA.
Emergence and evolution of recombinase activator genes
Our studies, initially of the shark RAG1 gene (Bernstein et al., 1996) and more recently of the complete sequence (S.F. Schluter and J.J. Marchalonis, unpublished results) of the shark RAG1-spacer-RAG2 cluster (Fig. 5), support the hypothesis that a horizontal transfer of this cluster from microbes to an ancestral jawed vertebrates triggered the rapid evolutionary emergence within approximately 10 million years of the combinatorial immune response ("Big Bang"). Factors adding credence to this suggestion are as follows: first, molecules showing homology to RAG1 and RAG2 have not been found in multicellular organisms below the level of jawed vertebrates; second, microbial integrases and integration host factors show recognizable homology to RAG1 and RAG2, respectively; third, the genes encoding RAG1 and RAG2 resemble those of microbes in lacking introns; fourth, the spacer segment between RAG 1 and RAG 2 contains defective pieces of retroposons consisting of both SINE (short interspersed nuclear elements) and LINEs (long intersperse nuclear elements); and fifth, constructs consisting of RAG1 and RAG2 have been shown to act as transposons in transfection of mammalian cells (Agrawal et al., 1998; Hiom et al., 1998). It may prove significant that the 9 kb DNA spacer separating shark RAG1 and RAG2 contains retroposon LINE and SINE elements comprising about 40% of the sequence. These are homologous to the CR1 retroposons found in chickens (Burch et al., 1993) and turtles (Kajikawa et al., 1997), supporting an early emergence of these sequence elements in vertebrate evolution. Ohshima and his colleagues (Ogiwara et al., 1999) have reported that LINE and SINE elements are also widely dispersed among living sharks and rays, raising the possibility that this coevolving LINE and SINE family may be of ancient origin. Like RAG1/RAG2 clusters of higher gnathostomes (Fugmann et al., 2000), that of the bull shark has RAG genes lacking in introns arranged in opposite orientation and separated by DNA spacer. The DNA spacer is comparable in length to that in mammals, whereas that in teleost fish is considerably shorter (approximately 2 kb).
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Our present knowledge of the function of the RAG1/RAG2 complex in binding to the conserved recombination signal sequences (RSS) and introducing specific double-stranded breaks in chromosomal DNA by hydrolysis followed by transesterification was largely obtained by studies involving site-directed mutagenesis of the murine genes (Callebaut and Mornon, 1998
; Landree et al., 1999; Aidinis et al., 2000; Fugmann et al., 2000). However, evolution itself operates through natural site-directed mutagenesis with selection of functional variants. Since both RAG genes apparently occur only in gnathostomes, it is worthwhile to consider the degree to which functional regions identified in the mouse are conserved in the shark. RAG1 by itself can bind to RSS, but RAG2 is required for effective and stable binding, with the complex containing three to five molecules of RAG2 per RAG1. Although RAG1 residues appear to be critical for catalysis, both RAG molecules are needed for catalytic activity. The active functional core of RAG1 consists of residues 384–1008 (murine numbering system). This segment is readily identifiable in shark RAG1 as residues 393–1020. These segments show overall 80% identity, and contain elements critical for specific binding to DNA (the "nonamer binding domain," NBD; aa389–444), the recruitment of RAG2, and three acidic amino acid residues essential for catalysis and transesterification (DDE-motif; D600, D708 and E962). The residues comprising NBD are 86% identical between shark and mouse. Furthermore, the regions containing the crucial acidic residues are clearly identifiable in shark RAG1. Comparing nine residues to the left and right of the conserved amino acids, shark and mouse RAG1 molecules were identical in 17 of 19 residues around D600, 18 of 19 around D708, and 19 of 19 around E962. Another Zinc-finger motif, the "Zinc finger Ring" (aa290–329), that is found in the less essential N-terminal domain of RAG1 shows only 67% identity between sharks and mouse. A homologous Zn-finger ring motif is found in an excision-repair enzyme, RAD 18, of yeast that has 33% and 46% identities to shark and mouse, respectively.
The RAG2 protein consists of approximately 530 amino acids, with the active core of the murine molecule ranging from aa1–371. This segment consists of a six-fold repeat of a 50-residue motif corresponding to the kelch superfamily (Callebaut and Mornon, 1998) and a C-terminal segment of 57aa that provides a surface for interaction with RAG1 (Aidinis et al., 2000). The remaining C-terminal 25% of RAG2 resembles homeodomain finger-like motifs of plants, suggesting a possible interaction of this last domain with chromatin (Callebaut and Mornon, 1998). Individual Kelch domains form a four-stranded twisted antiparallel ß sheet with these organized in a circular arrangements resembling the blades of a propeller (Adams et al., 2000). The six-bladed propeller portion of the RAG2 active core could contribute to the formation of the tight complex of the RAG1 and RAG2 proteins in association with RSSs. Tryphophan W317 within the sixth kelch repeat was found to be critical for mediating contact with RAG1 and the concurrent stabilization of the complex and cleavage of the RSS (Aidinis et al., 2000). This critical residue, with the triplet WFG, is absolutely conserved in RAG2s of all vertebrate species. An unrooted dendrogram (Fig. 6) indicates that teleost fish (pufferfish, trout and zebrafish) cluster together as do mammals with Xenopus and chicken locating as expected for tetrapods. The shark RAG2 can be considered to be the basal member that separates the teleosts from the tetrapod groups. Similar results were obtained in phylogenetic analysis of RAG1 (Bernstein et al., 1996).
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THE PRIMORDIAL REPERTOIRE AND ITS CONSERVED DESCENDANTS |
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The antibodies and TCR remaining from the initial ancestral emergence of 450 mya have antigen binding sites constructed on the same principles as those of well-characterized mammalian homologs. Furthermore, they express similar profiles (spectratypes) in recognition of molecules implicated in regulatory or defense functions even though the germline organization of their IG gene segments is substantially different; i.e., compare the translocon arrangements of mammals with the individual cluster organization of sharks. Two crucial features of antibodies of all species as well as for mammalian TCR are epitope promiscuity and polyreactivity (Marchalonis et al., 2001
). Shark TCR have not been studied in sufficient detail to ascertain whether this property obtains for these molecules as well, but the clear homology suggests that it would. Another key property of receptors of the combinatorial system is degeneracy based upon two criteria. In the first place, individual antibodies, whether naturally-occurring (NAbs) or induced by specific immunization, as well as TCRs, can recognize separate and distinct antigenic epitopes. Second, the same epitope can be recognized by a variety of Abs or TCRs. The recognition repertoires of germfree mice are comparable to those of conventional animals, but the levels of Igs are lower (Coutinho et al., 1995). This is consistent with the expectation that the complete recognition repertoire must be present in all living vertebrates including "normal" individuals within species.
The original combinatorial repertoire to arise in evolution was the result of an accidental event independent of antigenic selection. It encompassed the entire repertoire of noncombinatorial recognition and regulatory molecules. The "antigen driven" compartment involving invasive pathogens and mutating or sequestered self antigens, by contrast, operated in "real time" showing inducibility and small (IgM/TCR) to great increases (IgG) increases in affinity as a consequence of clonal selection following immunization. Individuals within a species would be expected to differ in their repertoires generated under the influence of distinct antigenic challenges by pathogens and other possibly individual sources. The "homeostatic" compartment that recognizes invariant cell and serum components and functions in cellular regulation or removal of damaged elements should be fundamentally conserved in all individuals of a species. Since the combining sites of Abs and TCRs vary continually as part of the somatic combinatorial process, anti-Ab and TCR idiotypes arise continuously within the Ab and TCR components. Naturally-occurring antibodies (NAbs), and their T-cell homologs serve the essential role of lymphocyte receptors for antigen (Burnet, 1959) in the selective basis of combinatorial/adaptive vertebrate immunity. Comparisons between NAb repertoires of sharks and humans are of fundamental importance to an understanding of the operation of the CIR (combinatorial immune repertoire) because these species had the earliest ancestral divergence and are the most disparate extant vertebrates to express the combinatorial immune system. The potential to recapitulate the entire recognition spectrum was regenerated during the formation of new species in evolution. This was accomplished by various means because of the genetic organizational plasticity of the combinatorial system. The combinatorial immune systems of teleosts and other vertebrates "higher than" sharks occurred by inheritance of a small number of light and heavy chain gene cassettes out of the hundreds or thousands in the germlines of ancestral germlines of ancestral sharks and amplification of these by tandem duplication of the variable domains and sometimes the D and J minigenes.
Analyses of affinity-purified NAbs of both humans and sharks species for recognition repertoires and peptide-defined epitope-specificities elucidate conserved features that bear upon fundamental properties of the repertoire in all species. Persuasive evidence for the encompassing of the entire pre-existing repertoire by the combinatorial response is obtained in studies of the natural antibodies present in intravenous Ig (IVIG) preparations (Kazatchkine and Kaveri, 2001). These consist of highly purified IgG isolated from pools of serum collected from more than 10,000 normal individuals. Studies have also been carried out with natural IgM antibodies of humans (Hurez et al., 1997), mice (Avrameas et al., 1998), bony fishes (Gonzalez et al., 1988) and sharks (Leslie and Clem, 1970; Rudikoff et al., 1970; Marchalonis et al., 1993b, 1998b, 2000). The antibodies considered are termed "natural" (NAbs) because they arose in the absence of either deliberate immunization or, apparently, of exposure to foreign antigens.
Table 2 gives a simplified selection of natural antibodies identified in the sera of humans and sharks. Our studies focused upon NAbs of carcharhine sharks, the sandbar (Carcharhinus plumbeus) and tiger sharks (Galeocerdo curveri). The normal human Igs, like those of the shark contain subsets (usually less than 0.1%) of molecules binding to both foreign antigens and to those considered to be autoantigens. Bona fide epitopes expressed by infectious agents are phosphorylcholine, a membrane component of pathogenic bacteria and other organisms, and recombinant envelope proteins of retroviruses. NAbs against many types of antigens considered autologous and widely conserved in vertebrate evolution occur in both species. These include antibodies directed against surface components such as the senescent cell antigen, which is a marker of defective red cells that initiates removal by antibody-dependant phagocytosis (Kay et al., 1990), variable and constant epitopes of Igs (Kaymaz and Marchalonis, 1993) and TCR (Marchalonis et al., 1992), and autoantigens recognized in autoimmune diseases including thyroglobulins and denatured and native DNA (Avrameas, 1991). Shark NAbs bind to the human Igs, TCRs and mammalian thyroglobulins, human, porcine, bovine (Marchalonis et al., 1993a), most probably reflecting the strong homologies of these molecules among the vertebrates. The human pool, moreover, contains NAbs to regulatory molecules including cytokines and their receptors, markers of cell differentiation such as CD4 and CD5, and Fas (Kazatchkine and Kaveri, 2001). NAbs to heat shock proteins also occur in mammals (Menoret et al., 2000). Results in progress suggest that the NAb pools of carcharhine shark species contain antibodies representing the entire range of specificities.
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Although antibodies are considered to be exquisitely specific with monoclonal antibodies (mAbs) often thought to be monospecific, recent studies using peptide mimeotopes have established that both naturally-occurring and induced monoclonals show epitope recognition promiscuity in their capacity to bind peptides having little or no sequence identity to their combining sites (Marchalonis et al., 2001
). We have focused mainly upon human (Marchalonis et al., 1999; Robey et al., 2000) and murine (Dehghanpisheh and Marchalonis, 1997) IgM monoclonal NAAbs to TCR V-domain epitopes, but parallel findings obtain for human monoclonal IgG myeloma proteins that bind TCR-epitopes (Marchalonis et al., 1997) and for a deliberately-induced murine IgG mAb to HIV-p41 (Keitel et al., 1997; Kramer et al., 1997). This property was accepted for the combining sites of /ß TCR prior to its acceptance for antibodies (Bhardwaj et al., 1993; Brawley and Concannon, 1996). Furthermore, monoclonal Abs can be polyreactive in the sense that they bind complex molecules unrelated to the immunizing or selecting antigen (Martin et al., 1994; Diaw et al., 1997; Avrameas et al., 1998; Deng and Notkins, 2000). IgM molecules are usually considered to be polyreactive, but this property has been demonstrated for IgG and IgA mAbs as well. In all species studied, NAbs of the IgM class tend to use unmutated VH and VL genes corresponding to germline sequences with the promiscuity or polyreactivity dependant upon the sequence of the third complementarity determining segment (HCDR3) of the heavy chain (Deng and Notkins, 2000). Affinity-purified NAbs of both sharks and man illustrate these properties (Marchalonis et al., 2001). In particular, NAbs to DNA of both species show strong polyreactivity, binding to proteins such as thyroglobulin and ovalbumin as well as to the selecting antigen. Furthermore, affinity-purified NAbs to TCR-epitopes of both species (Marchalonis et al., 2000) as well as human mAbs (Marchalonis et al., 1992, 2001) bind to the shared idiotopes of Vß (CDR1 and the hypervariable region of the third framework) and show varying degrees of promiscuity in binding to unrelated peptides. In general, NAbs to DNA tend to be polyspecific, but NAbs to thyroglobulins, viral glps and TCR can show relative specificity for the cognate selecting antigens. Nonetheless, NAbs and induced, selected mAbs can show varying degrees of epitope recognition promiscuity based upon binding of peptide epitopes while binding specifically to the intact molecules used in the immunization or affinity-purification.
The universe of antigens recognized by the primordial antigen-independent combinatorial antibody repertoire is the set of all epitopes including both "self" and "non-self" because in principle there is no difference between them. All proteins can be antigenic. The ability of individuals to accept their own antigens is learned through mechanisms for the generation of tolerance. Overlap between the endogenous or self markers and the non-self or exogenous antigens exists; for example, cross reactions occur between retroviral glycoproteins and lymphocyte surface receptors (Lake et al., 1994). Even in the case of microbial antigens not found in vertebrates such as lipopolysaccharides (Valadon et al., 1998; Macpherson et al., 2000), it should be noted that peptide epitopes can mimic bacterial carbohydrates (Pincus et al., 1998) and DNA (Putterman et al., 2000). The subsequent inducible repertoire is conditioned by the genetics and antigenic experience of individuals. In the simplest case of the sharks, the predominant antibody is IgM which is of low affinity and does not show class switching or affinity maturation following multiple immunizations (Makela and Litman, 1980). The IgM component operates in a comparable manner in all vertebrates, but mammals switch predominantly to IgG molecules that show increased affinity following repeated immunizations due to somatic hypermutation and selection by antigen.
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SYNOPSISINTRODUCTIONMILESTONES IN THE EVOLUTION...ANCIENT SYSTEMS PARTICIPATING IN...EVOLUTION OF THE NUCLEAR...THE RAPID EVOLUTIONARY EMERGENCE...THE PRIMORDIAL REPERTOIRE AND...CONCLUSIONSReferences |
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Many anciently arisen molecules that functioned originally in cellular stress and recognition responses have been retained by the vertebrates as components of innate immunity and also serve as a groundwork for cellular reactivity and regulation in the combinatorial immune system. We mentioned as examples, the pentraxin-related acute phase proteins, complement components and their early relative
2-macroglobulin, and the NfB transcription factor family with its associated receptor molecules. Moreover, we considered ferritin which is not usually considered part of the innate system, but there is clearly a need for iron mobilization with macrophage activation. Recent evidence indicates that vitamin D, a steroid, can regulate the expression of NfB subunits via its receptor VDR, and can modulate cellular immune responses involved in inflammation including stimulation of TH1 type lymphocytes and their interaction with dendritic cells. Because of the usual association of vitamin D with calcium mobilization for bone and tooth formation, we were initially surprised to find that lampreys which lack these calcified structures and arose in evolution prior to vertebrates that can form these structures contained an expressed gene unequivocally homologous to VDRs of higher vertebrates. Thus, the gene for the VDR apparently arose in evolution prior to a need for calcium mobilization. We would venture to speculate that the original function of the vitamin D3/VDR system was to modulate cellular interactions in innate immunity, and that its crucial role in bone formation emerged subsequently.
It is evident that the natural antibody repertoires of sharks and humans share the propensity to recognize exogenous antigens of viral and bacterial pathogens as well as molecules considered to be autoantigens. Although it has yet to be established for shark T-cells, a parallel situation obtains for natural recognition by T cells of mammals, particularly those bearing the receptor (Spada et al., 2000). This molecule has been suggested to be the primordial Ig recognition unit (Richards and Nelson, 2000), and it binds antigen in a manner comparable to that of Igs, rather than in the indirect manner of ß TCRs that require presentation by the MHC complex (Schluter et al., 1989; Ramsland et al., 1997; Marchalonis et al., 1998a). Ig domains, as opposed to domains of the IgSf which are universally dispersed among the metazoans, occur only in gnathanstomes with the homologies in variable domain frameworks being sufficiently great that the 3-dimensional structure of shark mammalian Igs are predicted to be extremely similar. The recognition repertoires of sharks and man are also very comparable, although we would predict that the crucial third complementarity determining segments will be quite distinct in antibodies of the same nominal specificities. This prediction follows from our studies and those of others, who have investigated the structures of murine and human monoclonal antibodies directed against the same epitopes. There is great diversity both in length and in amino acid sequence of the heavy chain CDR3 segments of mammalian monoclonal natural antibodies directed against DNA and TCR epitopes. Furthermore, none of the available complete shark heavy chain VH/DH/JH sequences have HCDR3 sequences sufficiently similar to those of mammalian antibodies of known specificity to allow prediction of binding specificities of the molecules. Interestingly, Nuttal et al. (Nuttal et al., 2001) have selected individual V domains derived from a recombinant NAR (new antigen receptor), a mutant of 
heavy chain lacking the capacity to form LH dimers (Greenberg et al., 1995), for the capacity to bind nominal antigens. Affinity-purified shark natural antibodies to common autoantigens such as DNA resemble those of humans in showing polyreactive binding to other complex antigens including ovalbumin and thyroglobulins. Affinity-purified natural shark antibodies to TCR V domain epitopes resemble human natural IgM and IgG NAbs, both in peptide epitope recognition specificity and in showing various degrees of epitope recognition promiscuity by binding to peptides lacking in sequence homology to the cognate epitopes.
The emergence of the combinatorial immune response is considered to be an accidental phenomenon that was triggered by the horizontal transmission of genes that catalyze site specific recombination. Although the RAG genes do not occur in evolution below the level of jawed vertebrates, they share suggestive homologies with bacterial and retroviral integrases, and show remarkable conservation of sequences of functional units within the jawed vertebrates. Truncated RAG1/RAG2 complexes can act as transposons (Agrawal et al., 1998; Hiom et al., 1998). It is premature to propose an actual mechanism for the critical event underlying the "big bang" but it is obvious that the RAG gene complex must have been introduced into the germline of a primitive jawed vertebrate approximately 450 mya and passed on to its descendants.
Technically, it is much easier to focus on relationships among individual genes based upon sequence analysis and to deduce functional relationships from conserved sequences in critical sites. This approach has proven very useful previously when applied to Igs and TCRs and has been extended here to investigations of the nuclear receptor superfamily and the RAG genes of jawed vertebrates. We would emphasize, however, that it is the systems that are conserved; i.e., the "universal" recognition repertoire is expressed in all jawed vertebrates irrespective of antigenic challenge or sequence of HCDR3s, and is a consequence of the original accidental generation of diversity and degeneracy in the combinatorial system. Parallel conclusions can be drawn for the nuclear receptor superfamily that has more than 50 members clustered into subfamilies on the basis of the capacity to recognize particular steroids and function in restricted cellular systems. The NfB system, likewise, arose for one purpose but has been adapted to function both in embryonic differentiation and in cellular activation and differentiation involved with both innate and combinatorial immunity.
The emerging picture of the evolution of the immune system thus illustrates the refinement of preexisting components along with the introduction via horizontal transfer of transposon-like activities to generate the vast combinatorial repertoire of antibody diversity found in higher vertebrates.
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ACKNOWLEDGMENTS |
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This work was supported in part by National Science Foundation Grant #MCB 9906439 to JJM. We are grateful to Ms. Diana Humphreys for preparing the manuscript.
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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.
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