Shark class II invariant chain reveals ancient conserved relationships with cathepsins and MHC class II

The invariant chain (Ii) is the critical third chain required for the MHC class II heterodimer to be properly guided through the cell, loaded with peptide, and expressed on the surface of antigen presenting cells. Here, we report the isolation of the nurse shark Ii gene, and the comparative analysis of Ii splice variants, expression, genomic organization, predicted structure, and function throughout vertebrate evolution. Alternative splicing to yield Ii with and without the putative protease-protective, thyroglobulin-like domain is as ancient as the MHC-based adaptive immune system, as our analyses in shark and lizard further show conservation of this mechanism in all vertebrate classes except bony fish. Remarkable coordinate expression of Ii and class II was found in shark tissues. Conserved Ii residues and cathepsin L orthologs suggest their long co-evolution in the antigen presentation pathway, and genomic analyses suggest 450 million years of conserved Ii exon/intron structure. Other than an extended linker preceding the thyroglobulin-like domain in cartilaginous fish, the Ii gene and protein are predicted to have largely similar physiology from shark to man. Duplicated Ii genes found only in teleosts appear to have become sub-functionalized, as one form is predicted to play the same role as that mediated by Ii mRNA alternative splicing in all other vertebrate classes. No Ii homologs or potential ancestors of any of the functional Ii domains were found in the jawless fish or lower chordates.     

Diversity and repertoire of IgW and IgM <Emphasis Type="Italic">VH</Emphasis> families in the newborn nurse shark

Background

Adult cartilaginous fish express three immunoglobulin (Ig) isotypes, IgM, IgNAR and IgW. Newborn nurse sharks, Ginglymostoma cirratum, produce 19S (multimeric) IgM and monomeric/dimeric IgM_1gj, a germline-joined, IgM-related VH, and very low amounts of 7S (monomeric) IgM and IgNAR proteins. Newborn IgNAR VH mRNAs are diverse in the complementarity-determining region 3 (CDR3) with non-templated nucleotide (N-region) addition, which suggests that, unlike in many other vertebrates, terminal deoxynucleotidyl transferase (TdT) expressed at birth is functional. IgW is present in the lungfish, a bony fish sharing a common ancestor with sharks 460 million years ago, implying that the IgW VH family is as old as the IgM VH family. This nurse shark study examined the IgM and IgW VH repertoire from birth through adult life, and analyzed the phylogenetic relationships of these gene families.

Results

IgM and IgW VH cDNA clones isolated from newborn nurse shark primary and secondary lymphoid tissues had highly diverse and unique CDR3 with N-region addition and VDJ gene rearrangement, implicating functional TdT and RAG gene activity. Despite the clear presence of N-region additions, newborn CDR3 were significantly shorter than those of adults. The IgM clones are all included in a conventional VH family that can be classified into five discrete groups, none of which is orthologous to IgM VH genes in other elasmobranchs. In addition, a novel divergent VH family was orthologous to a published monotypic VH horn shark family. IgW VH genes have diverged sufficiently to form three families. IgM and IgW VH serine codons using the potential somatic hypermutation hotspot sequence occur mainly in VH framework 1 (FR1) and CDR1. Phylogenetic analysis of cartilaginous fish and lungfish IgM and IgW demonstrated they form two major ancient gene groups; furthermore, these VH genes generally diversify (duplicate and diverge) within a species.

Conclusion

As in ratfish, sandbar and horn sharks, most nurse shark IgM VH genes are from one family with multiple, heterogeneous loci. Their IgW VH genes have diversified, forming at least three families. The neonatal shark Ig VH CDR3 repertoire, diversified via N-region addition, is shorter than the adult VDJ junction, suggesting one means of postnatal repertoire diversification is expression of longer CDR3 junctions.

     

The evolutionary origin of the major histocompatibility complex: Polymorphism of class II α chain genes in the cartilaginous fish

T cells recognize antigen (Ag) in the form of peptides bound to the major histocompatibility complex (MHC) molecule. One of the important issues in evolutionary immunology is to identify the stage in phylogeny when this mode of Ag recognition emerged. At present, there is a considerable controversy as to whether the cartilaginous fish have the bona fide MHC. In our previous study, we showed that the nurse shark, a member of the cartilaginous fish, has (a) gene(s) capable of encoding MHC class II a chains. In the present study, we examined the polymorphism of nurse shark MHC class II a chain genes designated Gici-DAA and Gici-DBA using the polymerase chain reaction. The Gici-DAA and Gici-DBA genes had six and five alleles, respectively, and individual alleles usually differed by multiple nucleotides. In addition, most of the nucleotide substitutions were located at the putative Ag-binding sites, where non-synonymous substitutions occurred more frequently than synonymous substitutions. The fact that the Gici-DAA and Gici-DBA genes display a polymorphism pattern essentially similar to that of mammalian MHC genes playing a major role in Ag presentation suggests that the cartilaginous fish have the bona fide MHC. Thus, the MHC-peptide-based T cell recognition system appears to have arisen at or before the emergence of the cartilaginous fish.     

Complex expression patterns of lymphocyte-specific genes during the development of cartilaginous fish implicate unique lymphoid tissues in generating an immune repertoire

Cartilaginous fish express canonical B and T cell recognition genes, but their lymphoid organs and lymphocyte development have been poorly defined. Here, the expression of Ig, TCR, recombination-activating gene (Rag)-1 and terminal deoxynucleosidase (TdT) genes has been used to identify roles of various lymphoid tissues throughout development in the cartilaginous fish, Raja eglanteria (clearnose skate). In embryogenesis, Ig and TCR genes are sharply up-regulated at 8 weeks of development. At this stage TCR and TdT expression is limited to the thymus; later, TCR gene expression appears in peripheral sites in hatchlings and adults, suggesting that the thymus is a source of T cells as in mammals. B cell gene expression indicates more complex roles for the spleen and two special organs of cartilaginous fish-the Leydig and epigonal (gonad-associated) organs. In the adult, the Leydig organ is the site of the highest IgM and IgX expression. However, the spleen is the first site of IgM expression, while IgX is expressed first in gonad, liver, Leydig and even thymus. Distinctive spatiotemporal patterns of Ig light chain gene expression also are seen. A subset of Ig genes is pre-rearranged in the germline of the cartilaginous fish, making expression possible without rearrangement. To assess whether this allows differential developmental regulation, IgM and IgX heavy chain cDNA sequences from specific tissues and developmental stages have been compared with known germline-joined genomic sequences. Both non-productively rearranged genes and germline-joined genes are transcribed in the embryo and hatchling, but not in the adult.     

The second immunoglobulin class is commonly present in cartilaginous fish belonging to the order Rajiformes

Six species of cartilaginous fish distributed into four orders, Rajiformes (skates and guitarfishes), Myliobatiformes (rays), Heterodontiformes (sharks) and Carcharhiniformes (sharks), were investigated for the possible presence of a second class of immunoglobulin (Ig) other than IgM. Among those orders, fish belonging to the order Rajiformes were found to have a second Ig (IgR) with a non-covalently associated dimeric structure in which the H chain was different from that of IgM in mol. wt and antigenicity. Cartilaginous fish belonging to the other orders investigated had only one class of IgM.     

Isolation of the new antigen receptor from wobbegong sharks, and use as a scaffold for the display of protein loop libraries

The new antigen receptor (NAR) from nurse sharks consists of an immunoglobulin variable domain attached to five constant domains, and is hypothesised to function as an antigen-binding antibody-like molecule. To determine whether the NAR is present in other species we have isolated a number of new antigen receptor variable domains from the spotted wobbegong shark (Orectolobus maculatus) and compared their structure to that of the nurse shark protein. To determine whether these wNARs can function as antigen-binding proteins, we have used them as scaffolds for the construction of protein libraries in which the CDR3 loop was randomised, and displayed the resulting recombinant domains on the surface of fd bacteriophages. On selection against several protein antigens, the highest affinity wNAR proteins were generated against the Gingipain K protease from Porphyromonas gingivalis. One wNAR protein bound Gingipain K specifically by ELISA and BIAcore analysis and, when expressed in E. coli and purified by affinity chromatography, eluted from an FPLC column as a single peak consistent with folding into a monomeric protein. Naturally occurring nurse shark and wobbegong NAR variable domains exhibit conserved cysteine residues within the CDR1 and CDR3 loops which potentially form disulphide linkages and enhance protein stability; proteins isolated from the in vitro NAR wobbegong library showed similar selection for such paired cysteine residues. Thus, the New Antigen Receptor represents a protein scaffold with possible stability advantages over conventional antibodies when used in in vitro molecular libraries.     

Identification of diversified genes that contain immunoglobulin-like variable regions in a protochordate

The evolutionary origin of adaptive immune receptors is not understood below the phylogenetic level of the jawed vertebrates. We describe here a strategy for the selective cloning of cDNAs encoding secreted or transmembrane proteins that uses a bacterial plasmid (Amptrap) with a defective beta-lactamase gene. This method requires knowledge of only a single target motif that corresponds to as few as three amino acids; it was validated with major histocompatibility complex genes from a cartilaginous fish. Using this approach, we identified families of genes encoding secreted proteins with two diversified immunoglobulin-like variable (V) domains and a chitin-binding domain in amphioxus, a protochordate. Thus, multigenic families encoding diversified V regions exist in a species lacking an adaptive immune response.     

Insight into the primordial MHC from studies in ectothermic vertebrates

Summary: MHC classical class I and class II genes have been identified in representative species from all major jawed vertebrate taxa, the oldest group being the cartilaginous fish, whereas no class I/II genes of any type have been detected in animals from older taxa. Among ectothermic vertebrate classes, studies of MHC architecture have been done in cartilaginous fish (sharks), bony fish (several teleost species), and amphibians (the frog Xenopus). The Xenopus MHC contains class I, class II, and class III genes, demonstrating that all of these genes were linked in the ancestor of the tetrapods, but the gene order is not the same as that in mouse/man. Studies of poly-ploid Xenopus suggest that MHC genes can be differentially silenced when multiple copies are present; i.e. MHC 'subregions'can be silenced. Surprisingly, in all teleosts examined to date class I and class II genes are not linked. Likewise, class III genes like the complement genes factor B (Bf) and C4 are scattered throughout the genome of teleosts. However, the presumed classical class I genes are closely linked to the'immune'proteasome genes, LMP2 and LMP7, and to the peptide-transporter genes (TAP), implying that a true'class I region'exists in this group. A similar type of linkage group is found in chickens and perhaps Xenopus, and thus it may reveal die ancestral organization of class I-associated genes, In cartilaginous fish, classical and non-classical class I genes have been isolated from three shark species, and class II A and B chain genes from nurse sharks. Studies of MHC linkage in sharks are being carried out to provide further understanding of the putative primordial organization of MHC. Segregation studies in one shark family point to linkage of class I and class II genes, suggesting that the non-linkage of these genes in teleosts is a derived characteristic.     

The development of primary and secondary lymphoid tissues in the nurse shark Ginglymostoma cirratum: B-cell zones precede dendritic cell immigration and T-cell zone formation during ontogeny of the spleen

Secondary lymphoid tissue and immunoglobulin (Ig) production in mammals is not fully developed at birth, requiring time postnatally to attain all features required for adaptive immune responses. The immune system of newborn sharks - the oldest vertebrate group having adaptive immunity - also displays immature characteristics such as low serum IgM concentration and high levels of IgM1gj, an innate-like Ig. Primary and secondary lymphoid tissues in sharks and other cartilaginous fish were identified previously, but their cellular organization was not examined in detail. In this study of nurse shark lymphoid tissue, we demonstrate that the adult spleen contains well-defined, highly vascularized white pulp (WP) areas, composed of a central T-cell zone containing a major histocompatibility complex (MHC) class II+ dendritic cell (DC) network and a small number of Ig+ secretory cells, surrounded by smaller zones of surface Ig+ (sIg+) B cells. In neonates, splenic WPs are exclusively B-cell zones containing sIgM+-MHC class IIlow B cells; thus compartmentalized areas with T cells and DCs, as well as surface Ig novel antigen receptor (sIgNAR)-expressing B cells are absent at birth. Not until the pups are 5 months old do these WP areas become adult-like; concomitantly, sIgNAR+ B cells are readily detectable, indicating that this Ig class requires a 'mature immune-responsive environment'. The epigonal organ is the major site of neonatal B lymphopoiesis, based on the presence of developing B cells and recombination-activating gene 1 (RAG1)/terminal deoxynucleotidyl transferase (TdT) expression, indicative of antigen receptor rearrangement; such expression persists into adult life, whereas the spleen has negligible lymphopoietic activity. In adults but not neonates, many secretory B cells reside in the epigonal organ, suggesting, like in mammals, that B cells home to this primary lymphoid tissue after activation in other areas of the body.     

Processing of fish Ig heavy chain transcripts: diverse splicing patterns and unusual nonsense mediated decay

While the diversification of the antigen-binding sites is realized by genomic VDJ rearrangements during B cell differentiation, different forms of immunoglobulin (Ig) heavy (H) chains can be produced through multiple splicing pathways. In most vertebrates, the secreted (S) and membrane (Mb) forms of IgM chain are created by alternative splicing through usage of a cryptic splice site in Cμ4 allowing the junction to the TM exon. The processing pattern for Igμ is different in teleosts, which generally use the Cμ3 donor site instead. In ancient fish lineages, multiple unusual splicing patterns were found for Ig H chain, involving donor sites that do not always follow the classical consensus. The production of IgD versus IgM H chains seems to be generally realized by alternative splicing in all vertebrates, but typical teleost IgD H chains are chimeric and contains a Cμ1 domain. Together, these observations raise questions on how different fish regulate RNA splicing and if their splicing machinery is especially complex. A preliminary scan of the zebrafish and stickleback genomes provides evidence that gene orthologs to the mammalian main splice factors are highly conserved as single copy genes, while the snRNPs U repertoire may be different and may explain other particular features of RNA processing in fish.     

Four primordial immunoglobulin light chain isotypes, including lambda and kappa, identified in the most primitive living jawed vertebrates

The discovery of a fourth immunoglobulin (Ig) light (L) chain isotype in sharks has revealed the origins and natural history of all vertebrate L chains. Phylogenetic comparisons have established orthology between this new shark L chain and the unique Xenopus L chain isotype sigma. More importantly, inclusion of this new L chain family in phylogenetic analyses showed that all vertebrate L chains can be categorized into four ancestral clans originating prior to the emergence of cartilaginous fish: one restricted to elasmobranchs (sigma-cart/type I), one found in all cold-blooded vertebrates (sigma/teleost type 2/elasmobranch type IV), one in all groups except bony fish (lambda/elasmobranch type II), and one in all groups except birds (kappa/elasmobranch type III/teleost type 1 and 3). All four of these primordial L chain isotypes (sigma, sigma-cart, lambda and kappa) have maintained separate V region identities since their emergence at least 450 million years ago, suggestive of an ancient physiological distinction of the L chains. We suggest that, based upon unique, discrete sizes of complementarity determining regions 1 and 2 and other features of the V region sequences, the different L chain isotypes arose to provide different functional conformations in the Ig binding site when they pair with heavy chains.     

Selection of cholera toxin specific IgNAR single-domain antibodies from a naïve shark library

Shark immunoglobulin new antigen receptor (IgNAR, also referred to as NAR) variable domains (Vs) are single-domain antibody (sdAb) fragments containing only two hypervariable loop structures forming 3D topologies for a wide range of antigen recognition and binding. Their small size ( approximately 12kDa) and high solubility, thermostability and binding specificity make IgNARs an exceptional alternative source of engineered antibodies for sensor applications. Here, two new shark NAR V display libraries containing >10(7) unique clones from non-immunized (na?ve) adult spiny dogfish (Squalus acanthias) and smooth dogfish (Mustelus canis) sharks were constructed. The most conserved consensus sequences derived from random clone sequence were compared with published nurse shark (Ginglymostoma cirratum) sequences. Cholera toxin (CT) was chosen for panning one of the na?ve display libraries due to its severe pathogenicity and commercial availability. Three very similar CT binders were selected and purified soluble monomeric anti-CT sdAbs were characterized using Luminex(100) and traditional ELISA assays. These novel anti-CT sdAbs selected from our newly constructed shark NAR V sdAb library specifically bound to soluble antigen, without cross reacting with other irrelevant antigens. They also showed superior heat stability, exhibiting slow loss of activity over the course of one hour at high temperature (95 degrees C), while conventional antibodies lost all activity in the first 5-10min. The successful isolation of target specific sdAbs from one of our non-biased NAR libraries, demonstrate their ability to provide binders against an unacquainted antigen of interest.     

Nurse shark T-cell receptors employ somatic hypermutation preferentially to alter alpha/delta variable segments associated with alpha constant region

In addition to canonical TCR and BCR, cartilaginous fish assemble noncanonical TCR that employ various B-cell components. For example, shark T cells associate alpha (TCR-α) or delta (TCR-δ) constant (C) regions with Ig heavy chain (H) variable (V) segments or TCR-associated Ig-like V (TAILV) segments to form chimeric IgV-TCR, and combine TCRδC with both Ig-like and TCR-like V segments to form the doubly rearranging NAR-TCR. Activation-induced (cytidine) deaminase-catalyzed somatic hypermutation (SHM), typically used for B-cell affinity maturation, also is used by TCR-α during selection in the shark thymus presumably to salvage failing receptors. Here, we found that the use of SHM by nurse shark TCR varies depending on the particular V segment or C region used. First, SHM significantly alters alpha/delta V (TCRαδV) segments using TCR αC but not δC. Second, mutation to IgHV segments associated with TCR δC was reduced compared to mutation to TCR αδV associated with TCR αC. Mutation was present but limited in V segments of all other TCR chains including NAR-TCR. Unexpectedly, we found preferential rearrangement of the noncanonical IgHV-TCRδC over canonical TCR αδV-TCRδC receptors. The differential use of SHM may reveal how activation-induced (cytidine) deaminase targets V regions.     

Structural aspects of human IgM antibodies expressed in chronic B lymphocytic leukemia.

BACKGROUND: Malignant B cells from patients with chronic B lymphocytic leukemia (B CLL) generally express both surface IgM and the pan T cell antigen CD5, a characteristic of the B1 population of B lymphocytes. The IgM on the surface of these B CLL cells is frequently polyreactive with respect to its capacity to recognize multiple structurally dissimilar antigens (Ag). OBJECTIVES: To understand the structural characteristics of the polyreactive binding sites of human IgM molecules expressed on B CLL cells by: (1) analyzing the nucleotide and protein sequences of the variable (V) domains of five IgM molecules expressed in cases of B CLL and; (2) utilizing these sequences to generate three-dimensional (3D) models of Fv (VL - VH) molecules. STUDY DESIGN: Peripheral blood leukocytes obtained from five cases of B CLL were tested for polyreactive binding properties by assessing their capacity to bind mouse IgG by indirect immunofluorescence. The V region genes of light and heavy chains were amplified using the polymerase chain reaction, subsequently cloned and their nucleotide sequences obtained. Translated amino acid sequences of the V domains were used to generate homology models of the Fv molecules. RESULTS: Low affinity binding of mouse IgG was demonstrated for all B CLL samples examined, confirming the polyreactive nature of the IgM expressed on these cells. There was an absence or minimal mutation within V region genes when compared to germline Ig genes. Junctional diversity was not observed for VL regions, although truncations and insertions were frequent in D minigenes of VH regions. The binding sites were predicted to form either relatively flat surfaces with occasional protrusions or cavities at the VL - VH domain interface. Aromatic side chains covered a large proportion of the potential binding surfaces in the models of B CLL Fv components. DISCUSSION: Primary DNA sequences can be categorized as germline, suggesting that the B cells involved in B CLL are germline or naive in origin. The medium to large HCDR3s provide the majority of probable contact residues for antigens. While prominent aromatic residues are likely to engage in binding patterns which are conserved (e.g. mouse Ig reactivity), the diverse binding sites predicted for B CLL-derived IgMs also have properties which are conducive to polyreactive antigen binding.     

Expression and Secretion of T-Cell Receptor Vα and Vβ Domains using Escherichia coli as a Host

An expression system for the production of recombinant T-cell receptor (TCR) variable domains would, inter alia, allow structural studies to be carried out and provide protein for the generation of anti-clonotypic antibodies. In this report the V alpha and V beta domain genes have been isolated from a T-cell hybridoma which is associated with the pathogenesis of experimental allergic encephalomyelitis (EAE) in the H-2u mouse. These have been expressed as secreted domains in Escherichia coli, using secretion vectors previously used for the production of immunoglobulin fragments. Both V alpha and V beta domains are secreted in milligram quantities into the culture supernatant, although the levels of the V alpha domain are about 10-20 fold higher than those of the V beta domain. This expression system offers a rapid route for the production of recombinant TCRs in soluble form.     

Novel immune-type receptor genes

Summary: Novel immune-type receptor ( NITR ) genes, which initially were identified in the Southern pufferfish ( Spheroides nephelus ), encode products which consist of an extracellular variable (V) and V-like C2 (V/C2) domain, a transmembrane region, and a cytoplasmic tail, which typically possesses an immunoreceptor tyrosine-based inhibition motif (ITIM). Multiple NITR genes have been identified in close, contiguous chromosomal linkage. The V regions of NITRs resemble prototypic forms defined for immunoglobulin (Ig) and T-cell antigen receptor (TCR), are present in multiple families and exhibit regionalized variation in sequence, which also occurs in Ig and TCR. Comparisons of exons encoding transmembrane and cytoplasmic regions of multiple NITRs suggest that exon shuffling has factored in the diversification of the NITR gene complex. Zebrafish ( Danio rerio ) NITRs exhibit many of these characteristics. NITRs that have been identified in additional species of bony fish demonstrate additional variation in the number of extracellular domains as well as in the presence of intramembranous charged residues, cytoplasmic tails and ITIMs. The presence in NITRs of V regions that are related closely to those found in Ig and TCR, as well as regulatory motifs and other structural features that are characteristic of immune inhibitory receptors encoded at the leukocyte receptor cluster, suggests that the NITRs are representative of an integral stage in the evolution of innate and adaptive immune function.
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.     

Identification of a second immunoglobulin in the most primitive shark, the frill shark, Chlamydoselachus anguineus.

Three molecular forms of immunoglobulins: pentamer, dimer and monomer, were isolated from serum of the frill shark, Chlamydoselachus anguineus, the most primitive extant shark. A pentamer having an apparent mass of 900 kDa, consisting of 68 kDa heavy (H) chains and 22-24 kDa light (L) chains, was considered to be IgM because of its similarity to mammalian IgM in both molecular form and H chain molecular weight. The dimer and monomer with apparent masses of 300 kDa and 150 kDa, respectively, were composed of the same H chains of 45-50 kDa, a value smaller than that of the H chains from the pentamer, and light chains identical to those of the pentamer. The H chains of 68 kDa and 45-50 kDa represented individual epitopes and were synthesized by different plasma cells. We thus concluded that the frill shark has two distinct classes of immunoglobulins: one a pentameric IgM and the other a second class of immunoglobulin with dimeric and monomeric structure. The presence of the second Ig is of considerable interest with respect to antigen recognition and exclusion.