Kirrel2, a novel immunoglobulin superfamily gene expressed primarily in beta cells of the pancreatic islets

A novel immunoglobulin superfamily (Igsf) protein gene was discovered by computational analysis of human draft genomic DNA, and multiple cDNA clones were obtained. The protein encoded by this gene contains five Ig domains, one transmembrane domain, and an intracellular domain. It has significant similarity with several known Igsf proteins, including Drosophila RST (irregular chiasm C-roughest) protein and mammalian KIRREL (kin of irregular chiasm C-roughest), NEPH1, and NPHS1 (nephrin) proteins. All these proteins have multiple Ig domains, possess properties of cell adhesion molecules, and play important roles in organ development. RT-PCR and Northern blot results indicate this gene is predominantly expressed in pancreas, and public sequence databases indicate there is also expression in the nervous system. We have named this gene Kirrel2 (kin of irregular chiasm-like 2), to reflect its similarity to irregular chiasm C-roughest and Kirrel. Four splice forms of Kirrel2 were observed, including two that we cloned from pancreas mRNA as well as two GenBank entries, one from the brain and one from a retinoblastoma cell line. A partial cDNA clone of the mouse orthologue was obtained by RT-PCR from mouse brain, and the inferred protein sequence has 85% sequence identity to the human protein. Immunohistochemical staining results indicate that the KIRREL2 protein is conserved from rodents to primates, and it is highly expressed in pancreatic islets. RT-PCR results on mouse pancreatic cell lines indicate that expression in the pancreas is restricted to beta cells. Thus, KIRREL2 protein is a beta-cell-expressed Ig domain protein and may be involved in pancreas development or beta cell function.     

Molecular evolution of immunoglobulin superfamily genes in primates

Genes of the immunoglobulin superfamily (IgSF) have a wide variety of cellular activities. In this study, we investigated molecular evolution of IgSF genes in primates by comparing orthologous sequences of 249 IgSF genes among human, chimpanzee, orangutan, rhesus macaque, and common marmoset. To evaluate the non-synonymous/synonymous substitution ratio (ω), we applied Bn-Bs program and PAML program. IgSF genes were classified into 11 functional categories based on the Gene Ontology (GO) database. Among them, IgSF genes in three functional categories, immune system process (GO:0002376), defense response (GO:0006952), and multi-organism process (GO:0051704), which are tightly linked to the regulation of immune system had much higher values of ω than genes in the other GO categories. In addition, we estimated the average values of ω for each primate lineage. Although each primate lineage had comparable average values of ω, the human lineage showed the lowest ω value for the immune-related genes. Furthermore, 11 IgSF genes, SIGLEC5, SLAMF6, CD33, CD3E, CEACAM8, CD3G, FCER1A, CD48, CD4, TIM4, and FCGR2A, were implied to have been under positive selective pressure during the course of primate evolution. Further sequence analyses of CD3E and CD3G from 23 primate species suggested that the Ig domains of CD3E and CD3G underwent the positive Darwinian selection.     

Profiling of bacterial adhesin--host receptor recognition by soluble immunoglobulin superfamily domains

Several gram-negative human pathogens recognize members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family. Pathogenic Neisseriae employ distinct isoforms of the colony opacity-associated proteins (Opa(CEA) proteins) to bind to the amino-terminal domains of CEACAMs. Here we present a novel approach to rapidly determine the CEACAM-binding properties of single bacteria. Expression of the isolated amino-terminal domains of various CEACAMs in eukaryotic cells yields soluble probes that selectively recognize Opa(CEA)-expressing bacteria in a pull-down assay format. Furthermore, by expressing soluble CEACAMs as fusions to green-fluorescent protein (CEACAM-N-GFP), CEACAM-binding bacteria can be decorated with a fluorescent label and analysed by flow cytometry allowing the specific detection of receptor binding events on the level of single bacteria. Besides its potential for rapid and quantitative analysis of pathogen-receptor interactions, this novel approach allows the detection of receptor recognition in heterogeneous bacterial populations and might represent a valuable tool for profiling the host binding capabilities of various microorganisms.     

Widespread expressions of immunoglobulin superfamily proteins in cancer cells

RP215 monoclonal antibody (Mab) was shown to recognize a carbohydrate-associated epitope of cancer cell–expressed glycoproteins, known as CA215. Extensive MALDI-TOF MS analysis was performed to search for the molecular identity of CA215. Besides immunoglobulin (Ig) heavy chains, homology to human T-cell receptors (TCR) and Ig-like cell adhesion molecules was also detected. By using RT-PCR and cDNA sequencing, it was observed that as many as 80% of cancer cell lines showed significant levels of gene expressions of TCR-α and TCR-β. Selected Ig-like cell adhesion molecules such as CD47, CD54, CD58 and CD 147 were also highly expressed among all the cell lines tested. In contrast, co-receptors and co-stimulators of TCR such as CD3, CD4 and CD8 were rarely expressed demonstrating the non-functional nature of TCR in cancer cells. Results of immunohistochemical staining and Western blot assays of cancer cell lines as well as cancerous tissue sections were consistent with these observations. Anti-TCR and anti-human IgG antibodies were shown to induce complement-dependent cytotoxicity and apoptosis of cultured cancer cells indicating the surface nature of Ig-like proteins. Based on these experimental observations, it was hypothesized that the expressions of these immunoglobulin superfamily (IgSF) proteins may be relevant to the immune protection and proliferations of cancer cells during carcinogenesis or cancer progression. Surface-bound TCR-like proteins as well as immunoglobulins may be the potential targets for RP215-based anti-cancer drugs.     

The peroxidase-cyclooxygenase superfamily: Reconstructed evolution of critical enzymes of the innate immune system

The authors have reconstructed the phylogenetic relationships of the main evolutionary lines of mammalian heme containing peroxidases. The sequences of intensively investigated human myeloperoxidase, eosinophil peroxidase, and lactoperoxidase, which participate in host defence against infections, were aligned together with newly found open reading frames coding for highly similar putative peroxidase domains in all kingdoms of life. The evolutionary relationships were reconstructed using neighbor-joining, maximum parsimony, and maximum likelihood methods. It is demonstrated that this enzyme superfamily obeys the rules of birth-and-death model of multigene family evolution and contains proteins with a variety of function that could be grouped in seven subfamilies. On the basis of occurrence and the fact that two main enzymatic activities are related with these metalloproteins, they propose the name peroxidase-cyclooxygenase superfamily for this widely spread group of heme-containing oxidoreductases. Well known structure-function relationships in mammalian peroxidases formed the basis for the critical inspection of all subfamilies. The presented data unequivocally suggest that predecessor genes of mammalian heme peroxidases have segregated very early in evolution. Before organisms developed an acquired immunity, their antimicrobial defence depended on enzymes that were recruited upon pathogen invasion and could produce antimicrobial reaction products. Thus, these peroxidatic heme proteins evolved to important components in the innate immune defence system. This work shows that even in certain prokaryotic organisms, genes encoding putative antimicrobial enzymes are found providing a group of bacteria with an evolutionary advantage over the others.     

MHC superfamily structure and the immune system

During the past year, a plethora of structural information has provided detailed insights into the interactions between classical MHC class I molecules and their cognate receptors on T cells. Likewise, there have been major advances in our knowledge of the structures and functions of five nonclassical MHC-like molecules: HLA-DM (murine H2-M), HLA-E, HFE, ZAG and MIC-A.     

DIgR1, a novel membrane receptor of the immunoglobulin gene superfamily, is preferentially expressed by antigen-presenting cells

A novel membrane receptor of immunoglobulin gene superfamily (IgSF) has been identified from mouse dendritic cells (DC) and designated as DC-derived Ig-like receptor 1 (DIgR1). It encodes a 228-amino-acid (aa) residue polypeptide with a 21-aa signal peptide, a 20-aa transmembrane region, a 189-aa extracellular region, and a 19 aa intracellular region. Its extracellular region contains a single V domain of Ig. So it is a novel type I transmembrane glycoprotein of IgSF. DIgR1 shows significant homologies to human CMRF-35 antigens and polymeric immunoglobulin receptors (pIgR). The mRNA expression of DIgR1 was highly abundant in mouse spleen. The preferential expression of DIgR1 mRNA is observed in the known antigen-presenting cells (APC) including DC, monocytes/macrophages, and B lymphocytes. A 40 kDa of protein in NIH/3T3 cells transfected with the DIgR1 cDNA was detected by Western blot analysis using anti-DIgR1 polyclonal antibodies. The expression of DIgR1 protein on DC is not regulated by LPS stimulation. Further study should be conducted to investigate what were biological functions of DIgR1 in the immunobiology of APC.     

Molecular cloning of a novel immunoglobulin superfamily gene preferentially expressed by brain and testis

We have cloned and characterized a novel gene from both human and mouse that encodes a new member of the immunoglobulin superfamily. The gene is preferentially expressed in both brain and testis, and hence, termed BT-IgSF (brain- and testis-specific immunoglobulin superfamily). The predicted protein consists of V-type and C2-type immunoglobulin domains as well as a hydrophobic signal sequence, a single transmembrane region, and a cytoplasmic domain. Human BT-IgSF protein (431 amino acids) is 88% identical to the mouse protein (428 amino acids) and both show significant homology to coxsackie and adenovirus receptor (CAR) and endothelial cell-selective adhesion molecule (ESAM). We examined the expression of BT-IgSF with various cultured cells and found that the gene was expressed in both neurons and glial cells in vitro. Furthermore, the expression was preferentially detected in pyramidal cell layers of the dentate gyrus and hippocampus and in commissure fibers of the corpus callosum, in brain tissue sections examined. These findings suggest that BT-IgSF plays a role in the development or function of the central nervous system.     

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.     

The immunoglobulin superfamily in Drosophila melanogaster and Caenorhabditis elegans and the evolution of complexity

Drosophila melanogaster is an arthropod with a much more complex anatomy and physiology than the nematode Caenorhabditis elegans. We investigated one of the protein superfamilies in the two organisms that plays a major role in development and function of cell-cell communication: the immunoglobulin superfamily (IgSF). Using hidden Markov models, we identified 142 IgSF proteins in Drosophila and 80 in C. elegans. Of these, 58 and 22, respectively, have been previously identified by experiments. On the basis of homology and the structural characterisation of the proteins, we can suggest probable types of function for most of the novel proteins. Though overall Drosophila has fewer genes than C. elegans, it has many more IgSF cell-surface and secreted proteins. Half the IgSF proteins in C. elegans and three quarters of those in Drosophila have evolved subsequent to the divergence of the two organisms. These results suggest that the expansion of this protein superfamily is one of the factors that have contributed to the formation of the more complex physiological features that are found in Drosophila.     

FDF03, a novel inhibitory receptor of the immunoglobulin superfamily, is expressed by human dendritic and myeloid cells

In this study, we describe human FDF03, a novel member of the Ig superfamily expressed as a monomeric 44-kDa transmembrane glycoprotein and containing a single extracellular V-set Ig-like domain. Two potential secreted isoforms were also identified. The gene encoding FDF03 mapped to chromosome 7q22. FDF03 was mostly detected in hemopoietic tissues and was expressed by monocytes, macrophages, and granulocytes, but not by lymphocytes (B, T, and NK cells), indicating an expression restricted to cells of the myelomonocytic lineage. FDF03 was also strongly expressed by monocyte-derived dendritic cells (DC) and preferentially by CD14+/CD1a- DC derived from CD34+ progenitors. Moreover, flow cytometric analysis showed FDF03 expression by CD11c+ blood and tonsil DC, but not by CD11c- DC precursors. The FDF03 cytoplasmic tail contained two immunoreceptor tyrosine-based inhibitory motif (ITIM)-like sequences. When overexpressed in pervanadate-treated U937 cells, FDF03 was tyrosine-phosphorylated and recruited Src homology-2 (SH2) domain-containing protein tyrosine phosphatase (SHP)-2 and to a lesser extent SHP-1. Like engagement of the ITIM-bearing receptor LAIR-1/p40, cross-linking of FDF03 inhibited calcium mobilization in response to CD32/FcgammaRII aggregation in transfected U937 cells, thus demonstrating that FDF03 can function as an inhibitory receptor. However, in contrast to LAIR-1/p40, cross-linking of FDF03 did not inhibit GM-CSF-induced monocyte differentiation into DC. Thus, FDF03 is a novel ITIM-bearing receptor selectively expressed by cells of myeloid origin, including DC, that may regulate functions other than that of the broadly distributed LAIR-1/p40 molecule.     

A novel ITAM-containing immunoglobulin superfamily receptor involved in lamprey VLRB+lymphocyte immune response

Adaptive immunity is a specific response of lymphocytes under the stimulation of pathogens,which plays a key role in eliminating pathogens and can produce immun...     

Beta-strand recombination in tricalbin evolution and the origin of synaptotagmin-like C2 domains

Two protein families involved in membrane traffic, tricalbins and synaptotagmins, contain several copies of C2 domains and are related based on their sequence and domain architecture. Paradoxically, tricalbin and synaptotagmin C2 domains belong to different structural types with apparent circular permutation of terminal beta-strands. To understand whether a topological switch took place, we analyzed tricalbin and synaptotagmin-like C2 domains using two-dimensional structural analysis. We found that yeast tricalbins contain five to six C2 domains. One of these C2 domains possesses many features of synaptotagmin-like C2 domains and also carries a conserved C-terminal strand that is similar to its structural equivalent in synaptotagmin-like C2 domains, suggesting a structural permutation event. Indeed, among higher eukaryotes, animal tricalbins have evolved a C2 domain with synaptotagmin-like topology indicating that the structural conversion has taken place. Investigation of plant synaptotagmins, however, proves that they are direct tricalbin orthologs. Our analysis shows that beta-strand recombination is a possible evolutionary mechanism to generate new structural topologies with altered functional properties.     

Polymeric immunoglobulin receptor expressed in MDCK cells transcytoses IgA

We expressed cDNA for the rabbit polymeric immunoglobulin receptor in polarized Madin-Darby Canine Kidney epithelial cells, which normally do not produce this receptor. The receptor appeared to function as in vivo; dimeric IgA was transported from the basolateral to the apical surface and released into the apical medium, together with the cleaved fragment of the receptor, known as secretory component. This system enabled us, for the first time, to study quantitatively IgA transcytosis in vitro and thus make the following observations. First, greater than 90% of the newly made receptor that is ultimately cleaved to secretory component and released into the apical medium goes first to the basolateral surface. Second, transport of the receptor does not depend on ligand binding. Third, transcytosis of bound ligand has a t 1/2 of 30 min.