Complexity in cattle <Emphasis Type="Italic">KIR</Emphasis> genes: transcription and genome analysis

Killer immunoglobulin (Ig)-like receptors (KIRs) are the major functional natural killer (NK) cell receptors in human. The presence of KIR genes has only recently been demonstrated in other (non-primate) species, and their expression, genomic arrangement, and function in these species have yet to be investigated. In this study, we describe the KIR gene family in cattle. KIR sequences were amplified from cDNA derived from four animals. Seventeen new sequences were identified in total. Some are alleles of two previously described genes, and the remainder are representative of at least four additional genes. These cDNA data, together with analysis of the cattle genome sequence, confirm that, as in humans, cattle have multiple inhibitory and activating KIR genes, with variable haplotype composition, and putative framework genes. In contrast to human, the majority of the cattle KIR genes encode three Ig-domain KIRs; most of the inhibitory genes encode only one immunoreceptor tyrosine-based inhibitory motif (ITIM), and the activating genes encode molecules with arginine rather than the more usual lysine in the transmembrane domain. A divergent gene, 2DL1, encodes a two Ig-domain KIR with an unusual D0-D2 structure, and a distinct signaling domain with two ITIMs. Similarity to pig and human two Ig-domain (D0-D2) KIRs suggest these may be more related to an ancestral gene than the other cattle KIR genes. Cattle have multiple NKG2A-related genes and at least one Ly49 gene; thus, the data presented here suggest that they have the potential to express more major histocompatibility complex-binding NK receptors than other species.     

Genomic analysis of immunity in a Urochordate and the emergence of the vertebrate immune system: “waiting for Godot”

Genome-wide sequence analysis in the invertebrate chordate, Ciona intestinalis, has provided a comprehensive picture of immune-related genes in an organism that occupies a key phylogenetic position in vertebrate evolution. The pivotal genes for adaptive immunity, such as the major histocompatibility complex (MHC) class I and II genes, T-cell receptors, or dimeric immunoglobulin molecules, have not been identified in the Ciona genome. Many genes involved in innate immunity have been identified, including complement components, Toll-like receptors, and the genes involved in intracellular signal transduction of immune responses, and show both expansion and unexpected diversity in comparison with the vertebrates. In addition, a number of genes were identified which predicted integral membrane proteins with extracellular C-type lectin or immunoglobulin domains and intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and immunoreceptor tyrosine-based activation motifs (ITAMs) (plus their associated signal transduction molecules), suggesting that activating and inhibitory receptors have an MHC-independent function and an early evolutionary origin. A crucial component of vertebrate adaptive immunity is somatic diversification, and the recombination activating genes (RAG) and activation-induced cytidine deaminase (AID) genes responsible for the Generation of diversity are not present in Ciona. However, there are key V regions, the essential feature of an immunoglobulin superfamily VC1-like core, and possible proto-MHC regions scattered throughout the genome waiting for Godot.     

Novel Immune-type Receptor Genes and the Origins of Adaptive and Innate Immune Recognition

The prototypic forms of teleost novel immune-type receptors(NITRs) consist of a variable (V) region, a unique V-like C2(V/C2) domain, a transmembrane region and a cytoplasmic tailcontaining immunoreceptor tyrosine-based inhibition motifs (ITIMs).NITRs encode diversified V regions in large multigene familiesbut do not undergo somatic rearrangement. Studies in four differentbony fish model systems have identified a number of differentorganizational forms of NITRs. Specifically, NITR genes encodeN-terminal ectodomains of the V-type but otherwise vary in the:total number of extracellular immunoglobulin domains, numberand location of joining (J) region-like motifs, presence oftransmembrane regions, presence of charged residues within transmembraneregions, presence of cytoplasmic tails, and/or distributionof ITIM(s) within the cytoplasmic tails. V region-containingNITRs constitute a far more complex family than recognized originallyand currently include individual members that potentially functionthrough inhibitory as well as activating mechanisms. The genomicorganization of the NITR gene cluster as well as the structuraldiversity and overall architecture of the NITR proteins is reminiscentof genes encoded at the mammalian leukocyte receptor cluster(LRC); however, there presently is no functional evidence tosupport an orthologous relationship between NITR and LRC geneproducts. Comparisons of the predicted structures of the NITRshave identified several short regions of sequence identity anda novel cloning strategy has been devised that selects for secretoryand transmembrane proteins that encode these short motifs. Usingthis approach, related genes termed immune-type receptors (ITRs)have been identified in cartilaginous fish. Taken together,these studies indicate that leukocyte regulatory receptors,including those that mediate natural killer function, mighthave emerged early in vertebrate evolution and that the NITR/ITRgenes represent a new and potentially highly significant linkbetween innate and adaptive immune responses.     

Novel immunoglobulin-like transcripts in teleost fish encode polymorphic receptors with cytoplasmic ITAM or ITIM and a new structural Ig domain similar to the natural cytotoxicity receptor NKp44

Members of the immunoglobulin superfamily (IgSF) include a group of innate immune receptors located in the leukocyte receptor complex (LRC) and other small clusters such as the TREM/NKp44 cluster. These receptors are characterised by the presence of immunoglobulin domains, a stalk, a transmembrane domain, and a cytoplasmic region containing either an immunoreceptor tyrosine-based inhibitory motif (ITIM) or are linked to an adapter molecule with an activation motif (ITAM) for downstream signalling. We have isolated two carp cDNA sequences encoding receptors in which the extracellular Ig domain structurally resembles the novel V-type Ig domain of NKp44. This is supported by a homology model. The cytoplasmic regions contain either an ITAM (Cyca-NILT1) or ITIMs (Cyca-NILT2). The tissue expression of these receptors is nearly identical, with the highest expression in the immunological organs. Peripheral blood leucocytes showed no detectable expression, but upon in vitro culture expressed NILT1, the activating receptor, and not the inhibitory NILT2 receptor. Southern blot analysis indicated that the NILT1 and NILT2 sequences belong to a multigene family. Analysis of the NILT Ig domain-encoding sequences amplified from both genomic DNA and cDNA revealed extensive haplotypic and allelic polymorphism. Database mining of the zebrafish genome identified several homologs on Chromosome 1, which also contains a cluster of class I major histocompatibility genes. This constellation is reminiscent of the TREM/NKp44 gene cluster and the HLA complex located on human Chromosome 6. The carp NILT genes form a unique cluster of innate immune receptors, which are highly polymorphic, and characterised by a new Ig structural subfamily and are distinct from the novel immune-type receptors (Nitrs) found in other fish species.     

Expression pattern of immunoglobulin superfamily members in the silkworm, Bombyx mori

Immunoglobulin superfamily (IgSF) proteins are involved in cell adhesion, cell communication and immune functions. In this study, 152 IgSF genes containing at least one immunoglobulin (Ig) domain were predicted in the Bombyx mori silkworm genome. Of these, 145 were distributed on 25 chromosomes with no genes on chromosomes 16, 18 and 26. Multiple sequence alignments and phylogenetic evolution analysis indicated that IgSFs evolved rapidly. Gene ontology (GO) annotation indicated that IgSF members functioned as cellular components and in molecular functions and biological processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis suggested that IgSF proteins were involved in signal transduction, signaling molecules and interaction, and cell communication. Microarray-based expression data showed tissue expression for 136 genes in anterior silkgland, middle silkgland, posterior silkgland, testis, ovary, fat body, midgut, integument, hemocyte, malpighian tubule and head. Expression pattern of IgSF genes in the silkworm ovary and midgut was analyzed by RNA-Seq. Expression of 105 genes was detected in the ovary in strain Dazao. Expression in the midgut was detected for 74 genes in strain Lan5 and 75 genes in strain Ou17. Expression of 34 IgSF genes in the midgut relative to the actin A3 gene was significantly different between strains Lan5 and Ou17. Furthermore, 1 IgSF gene was upregulated and 1 IgSF gene was downregulated in strain Lan5, and 4 IgSF genes were upregulated and 2 IgSF genes were downregulated in strain Ou17 after silkworms were challenged with B. mori cypovirus (BmCPV), indicating potential involvement in the response to BmCPV-infection. These results provide an overview of IgSF family members in silkworms, and lay the foundation for further functional studies.     

Hagfish leukocytes express a paired receptor family with a variable domain resembling those of antigen receptors

Jawed vertebrates are equipped with TCR and BCR with the capacity to rearrange their V domains. By contrast, jawless vertebrates, represented by hagfish and lampreys, apparently lack such receptors. We describe in this study a family of hagfish genes carrying a single V-type domain resembling those of TCR/BCR. This multigene family, which we call agnathan paired receptors resembling Ag receptors (APAR), is expressed in leukocytes and predicted to encode a group of membrane glycoproteins with organizations characteristic of paired Ig-like receptors, consisting of activating and inhibitory forms. APAR has a J region in its V-type domain, and its V and J regions are encoded in a single exon. Thus, APAR is a member of the emerging families of diversified, innate immune-type receptors with TCR/BCR-like V-type domains and has many of the features expected for a primordial TCR/BCR-like receptor. The extracellular domain of APAR may be descended from a V-type domain postulated to have acquired recombination signal sequences in a jawed vertebrate lineage.     

Expanding our understanding of immunoglobulin, T-cell antigen receptor, and novel immune-type receptor genes: a subset of the immunoglobulin gene superfamily

 The immunoglobulin superfamily (IgSF) is an extensively diversified multigene family whose members share a common structural feature, the Ig fold. Members of the Ig/T-cell antigen receptor (TCR) subset of the IgSF mediate antigen-specific recognition in adaptive immune responses. Antigen-binding receptors belonging to this subset are present in all species of jawed vertebrates. To explore whether there are additional structurally related but otherwise distinct members of this subset, we have developed a technique termed the short-primer polymerase chain reaction (PCR) that targets structurally conserved short motifs in the Ig fold. Large-scale sequencing efforts and recent advances in information biotechnology, including "electronic PCR," provide additional computational means to implement similarly directed searches within databases. The use of these approaches has led to the discoveries of Ig/TCR homologues in a variety of phylogenetically diverse organisms, a diversified family of novel immune-type receptor genes, as well as a novel human IgSF member. The potential of random sequencing efforts and virtual screening of databases is described in the context of two novel genes in bony fish. The various methodologies that are discussed and the examples shown provide means for further investigating, and/or elucidating novel, IgSF receptors as well as components of pathways that are involved in immune responses in both traditional and nontraditional model systems.     

On the origin of the olfactory receptor family: receptor genes of the jawless fish (Lampetra fluviatilis).

In vertebrates, recognition of odorous compounds is based on a large repertoire of receptor subtypes encoded by a multigene family. Towards an understanding of the phylogenetic origin of the vertebrate olfactory receptor family, attempts have been made to identify related receptor genes in the river lampreys (Lampetra fluviatilis), which are descendants of the earliest craniates and living representatives of the most ancient vertebrates. Employing molecular cloning approaches led to the discovery of four genes encoding heptahelical receptors, which share only a rather low overall sequence identity but several of the characteristic structural hallmarks with vertebrate olfactory receptors. Furthermore, in situ hybridization studies demonstrated that the identified genes are expressed in chemosensory cells of the singular lamprey olfactory organ. Molecular phylogenetic analysis confirmed a close relationship of the lamprey receptors to vertebrate olfactory receptors and in addition demonstrated that olfactory genes of the agnathostomes diverged from the gnathostome receptor genes before those split into class I and class II receptors. The data indicate that the lamprey receptors represent the most ancient family of the hitherto identified vertebrate olfactory receptors.     

Heterogeneity in the CD200R paired receptor family

Paired receptors are groups of closely related membrane proteins that have the potential to either inhibit or activate. The CD200R family consists of one inhibitory member, CD200R and various numbers of activating genes according to species with three defined in C57BL/6 mice. A genomic PCR strategy was used to examine the repertoire of genes in both laboratory and wild-derived mice. Most mouse strains tested (18/22) had three activating genes, and 16 of these had either the combination of CD200RLa, Lb, and Lc or CD200RLa, Lb, and Le. The Lc and Le genes were mutually exclusive and were equally common (10 strains). Wild-derived mice varied more with one example of strains with one, two, and four activating genes. An inhibitory CD200R gene was detected in each mouse strain, although two slightly different sequences were found in both laboratory and wild-derived mice. This diversity is probably being driven by pathogens but is less extensive than for many NK paired receptors such as KIR and Ly49. It is possible that myeloid paired receptors are involved in immune regulation of responses against pathogens rather than directly killing infected cells as for NK cells and, hence, under less intense evolutionary pressure.     

Genomic and functional characterization of the diverse immunoglobulin domain-containing protein (DICP) family

A heretofore-unrecognized multigene family encoding diverse immunoglobulin (Ig) domain-containing proteins (DICPs) was identified in the zebrafish genome. Twenty-nine distinct loci mapping to three chromosomal regions encode receptor-type structures possessing two classes of Ig ectodomains (D1 and D2). The sequence and number of Ig domains, transmembrane regions and signaling motifs vary between DICPs. Interindividual polymorphism and alternative RNA processing contribute to DICP diversity. Molecular models indicate that most D1 domains are of the variable (V) type; D2 domains are Ig-like. Sequence differences between D1 domains are concentrated in hypervariable regions on the front sheet strands of the Ig fold. Recombinant DICP Ig domains bind lipids, a property shared by mammalian CD300 and TREM family members. These findings suggest that novel multigene families encoding diversified immune receptors have arisen in different vertebrate lineages and affect parallel patterns of ligand recognition that potentially impact species-specific advantages.     

The chicken immunoregulatory receptor families SIRP, TREM, and CMRF35/CD300L

Mammalian immunoregulatory families of genes encoding activating and inhibitory Ig-like receptor pairs have been located on distinct chromosomes. In chicken, a single Ig-like receptor family with many members had been described so far. By looking at sequence similarity and synteny conservations in the chicken genome, the signal-regulatory protein (SIRP), triggering receptor expressed on myeloid cells (TREM), and CMRF35/CD300L Ig-like gene families were identified on chromosomes 20, 26, and 3, respectively. Further analysis of the three corresponding genomic regions and partial bacterial artificial chromosome sequencing were used to identify more members and to realign several contigs. All putative genomic sequences were monitored by investigating existing expressed sequence tag and cloning cDNA. This approach yielded a single pair of activating and inhibitory SIRP, two inhibitory, and one activating TREM as well as one inhibitory CMRF35/CD300L with a potentially soluble variant and an additional member lacking categorizing motifs. The CMRF35/CD300L and TREM receptors were composed of one or two V-set Ig domains, whereas in SIRP, either a single Ig V domain was present or a combination of a V and C1 domains. Like in many Ig superfamily members, separate exons encode individual Ig domains. However, in two CMRF35/CD300L genes, the signal peptide and the distal Ig domain were encoded by a single exon. In conclusion, the mammalian diversity of immunoregulatory molecules is present the chicken suggesting an important role for TREM, SIRP, and CMRF35/CD300L in a functionally conserved network.     

Evidence for Conservation of the Calcitonin Superfamily and Activity-regulating Mechanisms in the Basal Chordate Branchiostoma floridae: INSIGHTS INTO THE MOLECULAR AND FUNCTIONAL EVOLUTION IN CHORDATES*

The calcitonin (CT)/CT gene-related peptide (CGRP) family is conserved in vertebrates. The activities of this peptide family are regulated by a combination of two receptors, namely the calcitonin receptor (CTR) and the CTR-like receptor (CLR), and three receptor activity-modifying proteins (RAMPs). Furthermore, RAMPs act as escort proteins by translocating CLR to the cell membrane. Recently, CT/CGRP family peptides have been identified or inferred in several invertebrates. However, the molecular characteristics and relevant functions of the CTR/CLR and RAMPs in invertebrates remain unclear. In this study, we identified three CT/CGRP family peptides (Bf-CTFPs), one CTR/CLR-like receptor (Bf-CTFP-R), and three RAMP-like proteins (Bf-RAMP-LPs) in the basal chordate amphioxus (Branchiostoma floridae). The Bf-CTFPs were shown to possess an N-terminal circular region typical of the CT/CGRP family and a C-terminal Pro-NH₂. The Bf-CTFP genes were expressed in the central nervous system and in endocrine cells of the midgut, indicating that Bf-CTFPs serve as brain and/or gut peptides. Cell surface expression of the Bf-CTFP-R was enhanced by co-expression with each Bf-RAMP-LP. Furthermore, Bf-CTFPs activated Bf-CTFP-R·Bf-RAMP-LP complexes, resulting in cAMP accumulation. These results confirmed that Bf-RAMP-LPs, like vertebrate RAMPs, are prerequisites for the function and translocation of the Bf-CTFP-R. The relative potencies of the three peptides at each receptor were similar. Bf-CTFP2 was a potent ligand at all receptors in cAMP assays. Bf-RAMP-LP effects on ligand potency order were distinct to vertebrate CGRP/adrenomedullin/amylin receptors. To the best of our knowledge, this is the first molecular and functional characterization of an authentic invertebrate CT/CGRP family receptor and RAMPs.     

Functional Clustering of Immunoglobulin Superfamily Proteins with Protein–Protein Interaction Information Calibrated Hidden Markov Model Sequence Profiles

Secreted and cell-surface-localized members of the immunoglobulin superfamily (IgSF) play central roles in regulating adaptive and innate immune responses and are prime targets for the development of protein-based therapeutics. An essential activity of the ectodomains of these proteins is the specific recognition of cognate ligands, which are often other members of the IgSF. In this work, we provide functional insight for this important class of proteins through the development of a clustering algorithm that groups together extracellular domains of the IgSF with similar binding preferences. Information from hidden Markov model-based sequence profiles and domain architecture is calibrated against manually curated protein interaction data to define functional families of IgSF proteins. The method is able to assign 82% of the 477 extracellular IgSF protein to a functional family, while the rest are either single proteins with unique function or proteins that could not be assigned with the current technology. The functional clustering of IgSF proteins generates hypotheses regarding the identification of new cognate receptor–ligand pairs and reduces the pool of possible interacting partners to a manageable level for experimental validation.     

Parasite-responsive IgSF members in the snail Biomphalaria glabrata: characterization of novel genes with tandemly arranged IgSF domains and a fibrinogen domain

Two novel genes of the immunoglobulin superfamily (IgSF), FREP3 and FREP7, are reported from the snail Biomphalaria glabrata, a prominent intermediate host of the human parasite Schistosoma mansoni. They resemble other B. glabrata genes that encode fibrinogen-related proteins (FREPs), but differ in that they encode proteins with two tandemly arranged IgSF domains followed by a C-terminal fibrinogen domain. FREPs are hemolymph proteins that increase in abundance following exposure to a digenetic trematode, Echinostoma paraensei, and that bind to and precipitate parasite antigens. Within each gene, the two IgSF-coding regions are dissimilar from one another: the N-terminal IgSF1 domain is encoded by a single exon whereas the downstream IgSF2 domain is encoded by three exons. For both FREPs 3 and 7, the IgSF2 domain belongs to the variable (V) type, whereas the IgSF1 domain is not easily classified with respect to IgSF type. The fibrinogen-encoding region in both genes is relatively conserved and lacks introns. FREP3 exhibits extensive variation in the IgSF1 region. A ratio of nonsynonymous versus synonymous substitutions of 2.56 suggests that this region is under positive selection. A genomic fragment identifiable as FREP7 but lacking an exon was also found, further suggestive of variability within FREP IgSF-encoding regions. Insofar as FREPs are hypothesized to function in nonself recognition, the identification of additional novel FREP genes as part of a growing gene family in B. glabrata is of interest. Such genes, particularly given their variable nature, serve as a model to study the complexity of invertebrate defense responses.     

The adhesion protein IgSF9b is coupled to neuroligin 2 via S-SCAM to promote inhibitory synapse development

Synaptic adhesion molecules regulate diverse aspects of synapse formation and maintenance. Many known synaptic adhesion molecules localize at excitatory synapses, whereas relatively little is known about inhibitory synaptic adhesion molecules. Here we report that IgSF9b is a novel, brain-specific, homophilic adhesion molecule that is strongly expressed in GABAergic interneurons. IgSF9b was preferentially localized at inhibitory synapses in cultured rat hippocampal and cortical interneurons and was required for the development of inhibitory synapses onto interneurons. IgSF9b formed a subsynaptic domain distinct from the GABA_A receptor– and gephyrin-containing domain, as indicated by super-resolution imaging. IgSF9b was linked to neuroligin 2, an inhibitory synaptic adhesion molecule coupled to gephyrin, via the multi-PDZ protein S-SCAM. IgSF9b and neuroligin 2 could reciprocally cluster each other. These results suggest a novel mode of inhibitory synaptic organization in which two subsynaptic domains, one containing IgSF9b for synaptic adhesion and the other containing gephyrin and GABA_A receptors for synaptic transmission, are interconnected through S-SCAM and neuroligin 2.     

IgSF13, a novel human inhibitory receptor of the immunoglobulin superfamily, is preferentially expressed in dendritic cells and monocytes

A novel inhibitory receptor of immunoglobin superfamily (IgSF), IgSF member 13 (IgSF13), has been identified from human dendritic cells (DC). IgSF13 is a type I transmembrane protein containing an N-terminal signal peptide, a extracellular region with a single Ig V-like domain, a transmembrane region, and a cytoplasmic tail with two classical immunoreceptor tyrosine-based inhibitory motifs (ITIM), suggesting its inhibitory function. IgSF13 shows significant homology to human CMRF35 and pIgR. IgSF13 gene is mapped to chromosome 17q25.2, very close to that of CMRF35. IgSF13 is preferentially expressed in myelo-monocytic cells, including monocytes, monocyte-derived DC, and monocyte-related cell lines. Upon pervanadate treatment, IgSF13 was hyper-phosphorylated and associated with Src homology-2 domain-containing phosphatases SHP-1 and SHIP, but not SHP-2. The identification of IgSF13 as a novel ITIM-bearing receptor selectively expressed by DC and monocytes suggests that it may be potentially involved in the negative regulation of specific leukocyte population.     

Evolutionary Development of the Immunoglobulin Family

The origin of the ability of immunoglobulins (Ig) and T-cell receptors (TCRs) to specifically recognize antigens is related to the evolutionary development of proteins of the immunoglobulin superfamily (IgSF). The IgSF proteins are characterized by specific domain organization of molecules and statistically significant homology with known Ig. Four types of Ig domains (V1, V2, C1, and C2), differing from one another both in variations of their spatial organization and in the number of amino acid residues have been distinguished. Immunoglobulin superfamily comprises Ig; TCRs; class I and II major histocompatibility complex (MHC) molecules; one-domain proteins of thymocytes and T-cells (Thy-1); myelin protein P₀; ₂-microglobulin; two-domain proteins, such as sponge receptor tyrosine kinase (RTK), sponge adhesive protein (SAP), Drosophila tyrosine-kinase receptor (DTCR), Xenopus and human cortical-thymocyte receptors (CTX and CTH), etc.; and a large group of adhesins, coreceptors, and Ig receptors with varying number of domains. Evolutionary development of IgSF began with the evolvement of chaperones, Thy-1, and P₀ of prokaryotes and unicellular eukaryotes. Mutations, duplications, and translocations of the genes controlling both V and C domains yielded proteins with different numbers and combinations of these domains. All IgSF proteins are divided into two groups. The first group includes the proteins with nonrearranging V2 domains and homophilic mode of interaction; the second, the proteins with rearranging V1 domains and heterophilic mode of interaction (Ig, TCRs). The ability for heterophilic antigen-binding mode of interaction was apparently acquired due to the introduction of recombination-activating retroviral genes (RAG1 and RAG2) into the genome of Gnathostomata ancestors.