Immunoglobulin variable-region diversity in the zebrafish.

As part of an investigation of the immune system in the developing zebrafish, Danio rerio, we cloned and characterized a genomic V(H) segment and a number of cDNAs encoding IgM heavy chains. The genomic V(H) has the characteristic features of V(H) in other vertebrates, including a leader segment interrupted by a short intron, and regulatory and recombination signal sequences. The V(H) sequences of 75 cDNA clones fell into four distinct groups or families. All of these families cluster most closely with other teleost V(H); one of the families, V(H)3, is also closely related to other vertebrate V(H). Analysis by Southern blotting suggests that there are a number of germline genes corresponding to each family. Most variability within the V(H)1 family is confined to codons 30-32, at the end of the first framework region and in the beginning of the first complementarity-determining region. The nucleotide sequence of the genomic V(H) in this region contains motifs associated with a relatively high frequency of somatic mutation. There is much variability in segments of the clones corresponding to the third complementarity-determining region. The cDNA sequences encoding the fourth framework region are consistent with the contribution of four distinct J(H) segments.

Antibodies of sharks: revolution and evolution.

The combinatorial immune response is restricted to jawed vertebrates with cartilaginous fishes being the lowest extant species to have the mechanism for diversification and an extensive panoply of immunoglobulins, T-cell receptors and MHC products. Here, we review the molecular events of the "big bang" or rapid evolutionary appearance of the functionally complete combinatorial immune system coincident with the appearance of ancestral jawed vertebrates, suggesting that this event was catalyzed by horizontal transfer of DNA processing systems. We analyze the nature and extent of variable and constant domain diversity among the distinct immunoglobulin sets of carcharhine sharks focusing upon the lambda-like light chains and the mu and omega heavy chains. The detection and isolation of natural antibodies from the blood of unimmunized sharks illustrates a surprising range of recognition specificities and the existence of polyspecificity suggests that the antibody-forming system of sharks offers unique opportunities for studies of immunological regulation. Although the homologies between shark and mammalian immunoglobulins are unequivocal, major differences in segmental gene organization present challenges to our understanding of basic immunological phenomena such as clonal restriction.

Sequence and expression pattern of J chain in the amphibian, Xenopus laevis.

We have determined the cDNA sequence encoding J chain, a polypeptide accessory molecule associated with polymeric Ig, from the anuran amphibian, Xenopus laevis (South African clawed frog). The translated polypeptide consists of 164 amino acid residues, including the signal sequence, and is somewhat longer than the corresponding sequence in mouse and cow, the two mammalian species in which the signal sequence of J chain has been determined. J chain in several mammalian species (human, mouse, cow and rabbit) has eight Cys residues. In the human chain, two of these Cys residues, the second and third in the sequence, have been shown to form disulfide bridges to heavy chains in IgM or IgA; the remaining Cys residues form intrachain disulfide bonds. The Xenopus J chain contains only seven of these Cys residues. Ser is found at the position corresponding to the third Cys in mammalian J chains. Northern blot analysis, performed on RNA isolated from various organs of 3-month old frogs, indicated that the highest level of expression was in the intestine. Transcripts corresponding to J chain were also detected in the spleen and at very low levels in the thymus.

Diversity of Ig light chain clusters in the sandbar shark (Carcharhinus plumbeus).

The genes encoding Ig chains in elasmobranchs are arranged in clusters or cassettes, an organizational pattern dramatically different from the mammalian translocon gene arrangement. Cluster gene arrangements, which have now been found in non-elasmobranchs as well, pose interesting dilemmas for understanding the mechanisms of Ig gene expression and regulation in terms of allelic exclusion and clonal selection. We have sequenced five lambda genomic clones encoding complete sandbar shark (Carcharhinus plumbeus) lambda L chain gene loci. While the coding regions among all five clones are highly homologous, the noncoding regions have significant differences that allowed us to identify two types of lambda L chain clusters. The noncoding regions are < 60% identical between groups, while the three clones belonging to the first group share > 95% identity in their noncoding regions. The second group is more diverse and may be comprised of several related subgroups. The two clones in this group share approximately 85% identity in the noncoding regions. Variations in the promoter region, including octamer and TATA box orientation and position, are identified between the two groups and may have implications for the molecular regulation of Ab production. Our results show the sandbar shark lambda L chain family to be a complex and diverse system.

Cell surface recognition and the immunoglobulin superfamily.

Immunoglobulins serve as humoral recognition and effector molecules and as antigen-specific cell surface receptors on B and T cells. These molecules are constructed according to a characteristic domain pattern. Variable and constant domains diverged from one another early in vertebrate evolution, and they are joined by a "switch peptide" specified by the joining gene segments. Peptides specified by J-gene segments are strongly conserved in evolution in comparison among Ig light chains and T-cell receptors. Molecules less strongly related to Ig domains have been assembled into an Ig "superfamily" where the identities to classical IgC or V domains are < or = 20%. Among these are cell surface adhesion molecules, receptors for cytokines, and Fc receptors. Moreover, MHC antigens have an Ig-like membrane-proximal domain significantly related to IgC regions. We will analyze putative evolutionary relationships among canonical Igs and members of the Ig superfamily using highly conserved sequences from light and heavy chains of primitive vertebrates (e.g., the sandbar shark) as prototypes to ascertain similarities between Ig-related molecules of vertebrates and invertebrates.

Genomic clone for sandbar shark lambda light chain: generation of diversity in the absence of gene rearrangement.

While the general structure of immunoglobulin chains has remained relatively unchanged throughout evolution, the organization of the genes encoding these molecules differs substantially. To understand how the rearranging immunoglobulin system arose, it is necessary to examine living representatives of the most early vertebrate phyla. Elasmo-branches, which include the sharks, skates, and rays, are the most primitive phylogenetic class of vertebrates from which immunoglobulin DNA sequences have been obtained. In the sandbar shark (Carcharhinus plumbeus), the genes are arranged in individual clusters in which a single variable (V), joining (J), and constant (C) region gene, along with upstream regulatory elements, span a distance of approximately 4.4 kb or approximately 5.8 kb. We report the complete sequence of a genomic clone encoding sandbar shark lambda light chain. A unique finding of our study is that the V and J genes are fused in the germ line. Three additional clones have been shown by DNA sequencing to also have fused V and J genes. The four clones have complementarity-determining regions 3 of various lengths and amino acid sequence variability similar to the products of rearranged genes. Furthermore, analysis by polymerase chain reaction technology revealed an additional 26 genomic clones demonstrating fusion of the V and J segments. Therefore, VJ fusion is the prominent organizational feature of sandbar shark immunoglobulin light chain genes. This finding raises questions concerning the necessity of recombination to produce an antibody repertoire capable of reacting against a diverse array of antigens.

Shared antigenic determinants of immunoglobulins in phylogeny and in comparison with T-cell receptors.

1. Immunoglobulins are a complex multigene family of proteins specified by genes encoding variable (V), sometimes diversity (D), joining (J), and constant (C) domains. 2. Cross-reactions involving conformational determinants related to the VHa system of rabbits occur on heavy chains of vertebrate species ranging from elasmobranchs to man. 3. Serological markers characteristic of mu chains, the heavy chain of the IgM macroglobulins, occur on homologous heavy chains of species representing all vertebrate classes. 4. Serological markers characteristic of gamma type heavy chains, the major isotype in man, are restricted to the mammals, but are found on representatives of even the most primitive mammals, the egg-laying monotremes. 5. Variable region markers characteristic of lambda light chains are shared by light chains of shark and man. 6. Certain idiotypic markers defined by combining site V region sequences are broadly distributed in evolution. 7. Use of synthetic peptides as antigens and in epitope mapping show that amino acid sequences from the third framework region of the variable domain are broadly shared among light chain in phylogeny and between light chains and T-cell receptor beta chains. 8. The "switch peptides" linking the V and C domains of light chains and T-cell receptors, specified by the C-terminal portion of the J segment and the N-terminus of the constant region, are exposed in the three-dimensional structure of immunoglobulin or Tcrs, show striking homology, and form broadly shared antigenic determinants characteristic of immunoglobulins. 9. Although the multigene nature of the immunoglobulins and the complexity of antigenic determinants expressed by these large proteins renders comparison among molecules difficult, serum immunoglobulins and the closely related T-cell receptors express numerous shared determinants defined on the basis of amino acid sequence homology and three-dimensional conformations.

Antibody production in sharks and humans: a role for natural antibodies.

Although gene segments specifying Igs of all vertebrates show clear homology, their arrangements differ markedly, thereby suggesting that the mechanisms for the generation of diversity and for the regulation of gene expression may be quite distinct. In the sandbar shark, light chain gene segments are distributed as apparently independent clusters consisting of V, J, and C elements that require rearrangement for expression. The usual distance between V and C in the clusters is 3 kb but larger clusters occur. The V, J, and C elements are clearly homologous to those of human lambda chains. Shark Igs resemble mammalian IgM in structure and gene similarity. IgM may comprise as much as 50% of serum proteins in the shark. By contrast, IgM in humans comprises less than 5%. Human autoantibodies usually are IgM. These show little dependence on thymic function for expression and tend to increase with age. We have carried out a study of the capacity of Igs of unimmunized sharks and people (normals and patients suffering from autoimmune diseases) to react against a panel of antigens, including those usually considered autoantibodies, such as thyroglobulin and single-stranded DNA. Sharks and humans possess IgM antibodies that react with thyroglobulin and ssDNA. Affinity-purified natural shark antibodies to thyroglobulin or ssDNA constitute small fractions of total IgM. They illustrate extensive cross-reactivity comparable to that shown by polyspecific IgM autoantibodies produced by human B cells (CD5+) that appear early in ontogeny.

Antigenic cross-reactions among immunoglobulin of diverse vertebrates (elasmobranchs to man) detected using xenoantisera.

1. Antisera raised in rabbits and goats against intact immunoglobulins or their constituent light and heavy chains from man, mouse and the galapagos shark (Carcharhinus galapagenesis) were tested for their reactivity with immunoglobulins of elasmobranchs, other lower vertebrates and eutherian and prototherian mammals. 2. Xenoantisera directed against human heavy chain isotypes allowed the serological identification of IgM and IgG immunoglobulins in the echidna (Tachyglossus aculeatus), a monotreme which is one of the most primitive species of extant mammals. 3. The antisera to heavy chains reacted to varying degrees with purified immunoglobulins of non-mammalian species, including the chicken, teleost fish and elasmobranchs in a fashion that was specific for immunoglobulins, but was not related to defined human isotypic markers. 4. The reactions of some antisera seem to skip species known to possess homologous immunoglobulins. 5. Antisera directed against isotypic markers of human kappa and lambda light chains reacted with shark light chains in a manner that was specific for light chain determinants but was not isotype-related. 6. Antisera directed against heavy chains of either sharks or mammals reacted with heavy chains, but not with light chains of diverse species. 7. A rabbit antiserum specific for shark light chain reacted with human and murine monoclonal lambda chains and with two synthetic peptides corresponding to human V lambda Fr3 and Fr4 sequences. 8. These results establish that a variety of antigenic markers including conformational and linear determinants can be shared among immunoglobulins of vertebrates species that had an ancestral divergence more than 400 million years ago.

Complete sequence of a cDNA clone specifying sandbar shark immunoglobulin light chain: gene organization and implications for the evolution of light chains.

A full-length cDNA clone specifying sandbar shark (Carcharhinus plumbeus) immunoglobulin light chain has been isolated and sequenced. By alignment with human lambda chains, the leader, framework, complementarity-determining, joining, and constant regions are clearly identified in the shark light chain. Approximately 40-50% identity is shared between the human and shark sequences in the variable and constant regions. We have performed sequence comparisons of the individual segments and constructed phylogenetic trees for the variable region. These studies identify the shark protein as a lambda chain. In addition, the sandbar shark light chain is only distantly related to that of horned shark (Heterodontus francisci) [Shamblott, M. J. & Litman, G. W. (1989) Proc. Natl. Acad. Sci. USA 86, 4684-4688], demonstrating that the long evolutionary time of divergence among shark species has led to the generation of substantial differences in sequence. The positions of the variable, joining, and constant gene segments in 14 genomic clones have been mapped. The segments are linked in individual clusters (variable, joining, constant) occupying 3-7 kilobases. Cluster arrangement can be grouped into two patterns based upon spacing between the genes in the individual clones. This arrangement is fundamentally different from that observed in higher vertebrates.

Evolution of immunoglobulin light chains: cDNA clones specifying sandbar shark constant regions.

Sharks are living fossils that are indistinguishable morphologically from their Devonian ancestors of approximately equal to 400 million years ago. If parallel conservatism characterizes their biochemical evolution, characterization of their immunoglobulin chains could provide information regarding the primordial features of these essential defense molecules. Shark immunoglobulins are polydisperse like those of mammals, but these species lack homogeneous myeloma proteins. This heterogeneity has precluded direct determination of the sequence of elasmobranch light-chain proteins. We have sequenced four cDNA clones that contain the constant-region sequence as well as varying degrees of variable- or joining-region segments. The sandbar shark (Carcharhinus plumbeus) has at least four distinct light-chain constant regions, and these can be considered homologs of mammalian lambda chains. Approximately 40% identity was found in comparison from sharks to mammals. Certain stretches of sequence were remarkably conserved, whereas others varied in a manner consistent with accepted concepts of speciation. One hexapeptide (Ala-Thr-Leu-Val-Cys-Leu) occurred in lambda constant regions of all vertebrate species. There was a universal conservation of certain cysteines, phenylalanines, tryptophans, and glycines and strong identities in the block of residues from Ser-176 to Trp-186. Comparison of the shark sequence with that of the characterized human lambda myeloma protein Mcg indicates a strong conservation of three-dimensional structure in this light-chain domain representing species whose ancestors diverged early in vertebrate evolution. The shark light-chain sequence contains primordial features shared by mammalian kappa and lambda chains and by T-cell receptor beta chains.