Immunoglobulin Superfamily Protein Research Articles

Discovery of receptor-ligand interfaces in the immunoglobulin superfamily.

Cell-surface-anchored immunoglobulin superfamily (IgSF) proteins are widespread throughout the human proteome, forming crucial components of diverse biological processes including immunity, cell-cell adhesion, and carcinogenesis. IgSF proteins generally function through protein-protein interactions carried out between extracellular, membrane-bound proteins on adjacent cells, known as trans-binding interfaces. These protein-protein interactions constitute a class of pharmaceutical targets important in the treatment of autoimmune diseases, chronic infections, and cancer. A molecular-level understanding of IgSF protein-protein interactions would greatly benefit further drug development. A critical step toward this goal is the reliable identification of IgSF trans-binding interfaces. We propose a novel combination of structure and sequence information to identify trans-binding interfaces in IgSF proteins. We developed a structure-based binding interface prediction approach that can identify broad regions of the protein surface that encompass the binding interfaces and suggests that IgSF proteins possess binding supersites. These interfaces could theoretically be pinpointed using sequence-based conservation analysis, with performance approaching the theoretical upper limit of binding interface prediction accuracy, but achieving this in practice is limited by the current ability to identify an appropriate multiple sequence alignment for conservation analysis. However, an important contribution of combining the two orthogonal methods is that agreement between these approaches can estimate the reliability of the predictions. This approach was benchmarked on the set of 22 IgSF proteins with experimentally solved structures in complex with their ligands. Additionally, we provide structure-based predictions and reliability scores for the 62 IgSF proteins with known structure but yet uncharacterized binding interfaces.

The HVEM-BTLA Axis Restrains T Cell Help to Germinal Center B Cells and Functions as a Cell-Extrinsic Suppressor in Lymphomagenesis

SummaryThe tumor necrosis factor receptor superfamily member HVEM is one of the most frequently mutated surface proteins in germinal center (GC)-derived B cell lymphomas. We found that HVEM deficiency increased B cell competitiveness during pre-GC and GC responses. The immunoglobulin (Ig) superfamily protein BTLA regulated HVEM-expressing B cell responses independently of B-cell-intrinsic signaling via HVEM or BTLA. BTLA signaling into T cells through the phosphatase SHP1 reduced T cell receptor (TCR) signaling and preformed CD40 ligand mobilization to the immunological synapse, thus diminishing the help delivered to B cells. Moreover, T cell deficiency in BTLA cooperated with B cell Bcl-2 overexpression, leading to GC B cell outgrowth. These results establish that HVEM restrains the T helper signals delivered to B cells to influence GC selection outcomes, and they suggest that BTLA functions as a cell-extrinsic suppressor of GC B cell lymphomagenesis.

VSTM4 is a novel negative regulator of T cell activation

Abstract The B7-like proteins play critical immunomodulatory roles and constitute attractive targets for the development of novel immunotherapies. In this study, we demonstrated that a fusion protein, containing the extracellular domain of V-set and transmembrane domain containing 4 fused to Fc (VSTM4-Fc), inhibited human T-cell activation. VSTM4 markedly suppressed human T cell proliferation in the presence of T-cell receptor signaling. Furthermore, VSTM4 significantly reduced IFN-γ, IL-2, and IL-17 cytokine production by human T cells. Moreover, VSTM4 profoundly decreased T cell activation marker CD69 expression on CD3+, CD4+, and CD8+ T cells. VSTM4 also inhibited naïve CD4+ T cell differentiation into Th1 cells. Our results showed strong binding of VSTM4-Fc to human activated T cells but weak binding to resting nonactivated T cells. Thus, we identify VSTM4 as a novel immunoglobulin superfamily (IgSF) protein with robust T cell inhibitory activity. VSTM4 represents a new negative regulator of T cells and may be a unique target for developing new immunotherapy strategies for the treatment of immune-related diseases, including autoimmunity and cancer.

Molecular basis of synaptic specificity by immunoglobulin superfamily receptors in Drosophila.

In stereotyped neuronal networks, synaptic connectivity is dictated by cell surface proteins, which assign unique identities to neurons, and physically mediate axon guidance and synapse targeting. We recently identified two groups of immunoglobulin superfamily proteins in Drosophila, Dprs and DIPs, as strong candidates for synapse targeting functions. Here, we uncover the molecular basis of specificity in Dpr-DIP mediated cellular adhesions and neuronal connectivity. First, we present five crystal structures of Dpr-DIP and DIP-DIP complexes, highlighting the evolutionary and structural origins of diversification in Dpr and DIP proteins and their interactions. We further show that structures can be used to rationally engineer receptors with novel specificities or modified affinities, which can be used to study specific circuits that require Dpr-DIP interactions to help establish connectivity. We investigate one pair, engineered Dpr10 and DIP-α, for function in the neuromuscular circuit in flies, and reveal roles for homophilic and heterophilic binding in wiring.

Generation of Fcabs targeting human and murine LAG-3 as building blocks for novel bispecific antibody therapeutics

The immunoglobulin superfamily protein lymphocyte-activation gene 3 (LAG-3) participates in immune suppression and has been identified as a suitable target for cancer therapies. In order to generate bispecific antibodies targeting LAG-3, Fcabs (Fc-region with antigen binding) targeting human and murine LAG-3 were generated from phage libraries. These Fcabs bind to LAG-3, inhibiting its interaction with MHC class II, and induce IL-2 production in a T cell assay. Bispecific antibodies, known as mAb2, were produced by replacing the Fc region of a monoclonal antibody with Fcab sequences in the CH3 domain. mAb2 containing anti-LAG-3 Fcabs have mAb-like biophysical characteristics and retain LAG-3 binding and functional activity. mAb2 can thus be generated using multiple Fabs to investigate bispecific parings and develop novel therapeutics.

Promotion of differentiation in developing mouse cerebellar granule cells by a cell adhesion molecule BT-IgSF

Brain- and testis-specific immunoglobulin superfamily (BT-IgSF) (also known as IgSF11), one of the immunoglobulin superfamily proteins, is a cell adhesion molecule, expressed in the developing cerebellum. We hypothesized that BT-IgSF might have some function in the development of cerebellum, although the physiological roles of BT-IgSF in the cerebellum remain unclear. To investigate the role of BT-IgSF in the development of mouse cerebellum, we first determined the presence of BT-IgSF in the newborn mouse cerebellum; its expression level was found to be much higher than that in the adults. BT-IgSF was abundantly expressed in the molecular layer, where cerebellar granule cell precursors (CGCPs) are in the differentiation stage during migration. We subsequently analyzed the effects of BT-IgSF-knockdown and -overexpression on the proliferation and differentiation of primary cultured CGCPs. BT-IgSF suppressed the proliferation of CGCPs, and promoted their differentiation into cerebellar granule cells. Taken together, our results suggested that BT-IgSF is one of the important cell adhesion molecules that regulate the developmentof mouse cerebellum.

VSIG-8 is a co-inhibitory ligand and an immune checkpoint molecule for human T cells

Abstract Cell surface-localized and secreted immunoglobulin superfamily (IgSF) proteins play central roles in regulating adaptive and innate immune responses, and are primary targets for the development of new immunotherapeutics. In this work we provide biologic-functional insight for VISG-8, one member of this important class of proteins. VSIG-8 inhibits the production of cytokines (IL-2, IFN-gamma, IL-17, IL-6, and IL-19), chemokines (MCP-1, MCP-10, and IP-10) and other proteins (IGFBP3 and RBP4) on anti-CD3 activated human CD3 T cells. Furthermore, VSIG-8 significantly reduces the production of IFN-gamma and IL-2 on both CD4 and CD8 T cells in the presence of T-cell receptor signaling. In addition, VSIG-8 markedly suppresses anti-CD3-induced human T cell proliferation. Moreover, VSIG-8 profoundly decreases the conversion of naïve CD4+ T cells into Th1 cells. Thus, we have identified VSIG-8 as a new immune checkpoint molecule that is able to inhibit human T-cell activation. This novel human T-cell co-inhibitory ligand may be a unique target for developing new immunotherapy strategies for the treatment of human cancers, autoimmune disorders, and infection diseases.

A Neutrophil Proteomic Signature in Surgical Trauma Wounds.

Non-healing wounds continue to be a clinical challenge for patients and medical staff. These wounds have a heterogeneous etiology, including diabetes and surgical trauma wounds. It is therefore important to decipher molecular signatures that reflect the macroscopic process of wound healing. To this end, we collected wound sponge dressings routinely used in vacuum assisted therapy after surgical trauma to generate wound-derived protein profiles via global mass spectrometry. We confidently identified 311 proteins in exudates. Among them were expected targets belonging to the immunoglobulin superfamily, complement, and skin-derived proteins, such as keratins. Next to several S100 proteins, chaperones, heat shock proteins, and immune modulators, the exudates presented a number of redox proteins as well as a discrete neutrophil proteomic signature, including for example cathepsin G, elastase, myeloperoxidase, CD66c, and lipocalin 2. We mapped over 200 post-translational modifications (PTMs; cysteine/methionine oxidation, tyrosine nitration, cysteine trioxidation) to the proteomic profile, for example, in peroxiredoxin 1. Investigating manually collected exudates, we confirmed presence of neutrophils and their products, such as microparticles and fragments containing myeloperoxidase and DNA. These data confirmed known and identified less known wound proteins and their PTMs, which may serve as resource for future studies on human wound healing.

A complex of Neuroplastin and Plasma Membrane Ca2+ ATPase controls T cell activation

The outcome of T cell activation is determined by mechanisms that balance Ca2+ influx and clearance. Here we report that murine CD4 T cells lacking Neuroplastin (Nptn−/−), an immunoglobulin superfamily protein, display elevated cytosolic Ca2+ and impaired post-stimulation Ca2+ clearance, along with increased nuclear levels of NFAT transcription factor and enhanced T cell receptor-induced cytokine production. On the molecular level, we identified plasma membrane Ca2+ ATPases (PMCAs) as the main interaction partners of Neuroplastin. PMCA levels were reduced by over 70% in Nptn−/− T cells, suggesting an explanation for altered Ca2+ handling. Supporting this, Ca2+ extrusion was impaired while Ca2+ levels in internal stores were increased. T cells heterozygous for PMCA1 mimicked the phenotype of Nptn−/− T cells. Consistent with sustained Ca2+ levels, differentiation of Nptn−/− T helper cells was biased towards the Th1 versus Th2 subset. Our study thus establishes Neuroplastin-PMCA modules as important regulators of T cell activation.

Prognostic role of Nectin-4 expression in luminal B (HER2 negative) breast cancer

Nectins are Ca2+-independent immunoglobulin (Ig) superfamily proteins that participate in the organization of epithelial and endothelial junctions and regulate several cellular activities including the entry of some viruses. Nectin-4 has recently been shown as a metastasis-associated protein in several cancers. In the following study, we have evaluated the expression of Nectin-4 inthe luminal B HER2 negative subtype breast cancer. The study group consisted of 147 patients presenting with primary unilateral breast carcinoma with no evidence of distant metastases. Nectin-4 protein expression was assessed by immunohistochemistry and the results were correlated with the clinical data using Kaplan–Meier curves, univariate and multivariate stepwise proportional-hazard analysis (Cox model). Nectin-4 overexpression was significantly correlated with the tumour size (p<0.05; Fisher's Exact Test), also Nectin-4 expression was negatively associated with overall survival, disease free survival and distant relapse free survival with the same significance (p<0,001; Kaplan-Meier, Cox model). We did not find statistically significant correlation between Nectin-4 and age, ER, PR, age, lymph node metastasis, tumour differentiation, histologicalsubtype and Ki-67proliferation index.We suggest that Nectin-4 is a relevant prognostic factor and a therapeutic target in luminalB (HER2 negative) breast cancer.

Regulation of WNT Signaling at the Neuromuscular Junction by the Immunoglobulin Superfamily Protein RIG-3 inCaenorhabditis elegans

Perturbations in synaptic function could affect the normal behavior of an animal, making it important to understand the regulatory mechanisms of synaptic signaling. Previous work has shown that in Caenorhabditis elegans an immunoglobulin superfamily protein, RIG-3, functions in presynaptic neurons to maintain normal acetylcholine receptor levels at the neuromuscular junction (NMJ). In this study, we elucidate the molecular and functional mechanism of RIG-3. We demonstrate by genetic and BiFC (Bi-molecular Fluorescence Complementation) assays that presynaptic RIG-3 functions by directly interacting with the immunoglobulin domain of the nonconventional Wnt receptor, ROR receptor tyrosine kinase (RTK), CAM-1, which functions in postsynaptic body-wall muscles. This interaction in turn inhibits Wnt/LIN-44 signaling through the ROR/CAM-1 receptor, and allows for maintenance of normal acetylcholine receptor, AChR/ACR-16, levels at the neuromuscular synapse. Further, this work reveals that RIG-3 and ROR/CAM-1 function through the β-catenin/HMP-2 at the NMJ. Taken together, our results demonstrate that RIG-3 functions as an inhibitory molecule of the Wnt/LIN-44 signaling pathway through the RTK, CAM-1.

S-Palmitoylation of Junctional Adhesion Molecule C Regulates Its Tight Junction Localization and Cell Migration

Junctional adhesion molecule C (JAM-C) is an immunoglobulin superfamily protein expressed in epithelial cells, endothelial cells, and leukocytes. JAM-C has been implicated in leukocyte transendothelial migration, angiogenesis, cell adhesion, cell polarity, spermatogenesis, and metastasis. Here, we show that JAM-C undergoes S-palmitoylation on two juxtamembrane cysteine residues, Cys-264 and Cys-265. We have identified DHHC7 as a JAM-C palmitoylating enzyme by screening all known palmitoyltransferases (DHHCs). Ectopic expression of DHHC7, but not a DHHC7 catalytic mutant, enhances JAM-C S-palmitoylation. Moreover, DHHC7 knockdown decreases the S-palmitoylation level of JAM-C. Palmitoylation of JAM-C promotes its localization to tight junctions and inhibits transwell migration of A549 lung cancer cells. These results suggest that S-palmitoylation of JAM-C can be potentially targeted to control cancer metastasis.

Molecular Determinants of Specificity in the Dpr-DIP Interaction Network

Understanding the physical and chemical principles governing specificity in protein—protein binding is important both for revealing mechanisms of molecular recognition and for designing novel biomolecular systems. The protein families Dprs and DIPs, comprised of 21 and 9 immunoglobulin superfamily (IgSF) proteins, respectively, mediate neural cell adhesions and are critical for synapse formation specificity during development in arthropods. Previous research has recently characterized the Dpr-DIP interaction network and found that each Dpr selectively interacts with specific DIPs; the synaptic connectivity pattern in the fly brain is encoded by this molecular interaction network. The goal of this project is to determine the molecular basis of the specificity of these interactions. To achieve this goal, we are computing the binding free energies of each possible Dpr-DIP interaction using both the Molecular Mechanics-Poisson Boltzmann Surface Area (MM-PBSA) approach and potential of mean force (PMF) based framework, constructed from all atom molecular dynamics (MD) simulations. We are also employing a new near-atomic level MD program, Upside, to rapidly sample possible poses. Due to the lack of high-resolution crystal structures for most of these interactions, homology modeling was used in order to generate the initial structures. The calculated binding free energies will be used to predict possible Dpr-DIP interactions. Ultimately, we hope to use our current approach to predict potential protein—protein interactions between other IgSF proteins, which make up the second largest protein family in the human proteome, and are crucial for the development and function of the nervous and immune systems.

ATP-Binding-Cassette A1 Regulates Extracellular Isopentenyl Pyrophosphate Release and Vγ9Vδ2 T-Cell Activation By Dendritic Cells

IntroductionPhosphorylated metabolites of mevalonate (Mev) pathway like isopentenyl-pyrophosphate (IPP) induce proliferation of Vγ9Vδ2 T cells. Endogenous IPP levels can be lowered or increased by aminobisphosphonates (NBPs) such as zoledronic acid (ZA). We have previously shown that ZA-treated dendritic cells (DC) produce and release high amounts of soluble IPP and are strong Vγ9Vδ2 T-cell activators. However, the mechanisms regulating IPP release in DC and other cells are still unknown.Methods"DC generation": purified CD14+ cells were cultured in standard culture medium at 0.5-1.5 x 106cells/ml supplemented with GM-CSF (1000 U/ml) and IL-4 (500 U/ml) in flat-bottomed 24-well plates for 24 h. After 24 h, DC were left untreated (ZA-) or treated for further 24 h with 1 µM ZA (ZA+). DC were washed after incubation with ZA and re-plated with fresh medium for an additional 24 and 48 h (washed DC [w-DC]). In selected experiments, supernatants were collected for quantification of extracellular IPP levels."Vγ9Vδ2 T-cell proliferation":on day 7, total counts of viable T cells were calculated with the trypan blue staining assay, and by flow cytometry. In selected experiments, PBMC from CTRL were stimulated for 7 days with supernatants obtained from autologous DCZA- and DCZA+and 10 IU/ml IL-2 to evaluate Vγ9Vδ2 T-cell proliferation."IPP efflux":cells were radiolabelled with 1 microCi/ml [3H]-acetate; after 24 h, cell supernatants were collected, [3H]-IPP was extracted and resolved by thin layer chromatography. After separation, the spot corresponding to IPP was cut and solubilized, and the radioactivity was quantified by liquid scintillation count."siRNA ABCA1": cells were incubated 96 h with a 20-25 nucleotide non targeting scrambled siRNA or specific ABCA1 siRNA (Accell Thermo Scientific), then lysed and subjected to the Western blot analysis of ABCA1 expression."Proximity ligation assay (PLA)":the ABCA1-BTN3A1 interaction was measured by the PLA method with the DuoLink In Situ kit (Sigma Chemicals Co.), using a mouse anti-human ABCA1 (Abcam) and a rabbit anti-human BTN3A1 (Sigma Chemicals Co.) antibody, respectively, as per the manufacturer's instructions. Nuclei were counterstained with 4',5-diamidino-2 phenylindole dyhydrochloride (DAPI). Cells were examined using a Leica TCS SP2 AOP confocal laser-scanning microscope (Leica Microsystem, Wetzlar, Germany).ResultsWe tested a variety of tumoral and non-tumoral cells for their ability to activate Vγ9Vδ2 T cells and correlated this ability with the release of extracellular IPP. We used samples from healthy donors included peripheral blood (PB) monocytes, monocyte-derived DCs, and bone marrow (BM) stromal cells; samples from cancer patients included myeloma cells, chronic lymphocytic leukemia (CLL) cells, and BM stromal cells from multiple myeloma (MM) and CLL patients; tumor cell lines included the monocytic THP1 and histiocytic U937 cells and the myeloma cell line SKMMI. We found that the activation of Vγ9Vδ2 T cells was proportional to the simultaneous efflux of IPP and to the activity/expression of ATP-binding cassette transporter A1 (ABCA1), a membrane transporter involved in the delivery of intracellular cholesterol to apolipoprotein A-I.We have demonstrated that the most potent IPP releaser cells had also the highest expression of ABCA1. Functional ABCA1 inhibition with probucol slightly increased intracellular IPP accumulation, but abrogated extracellular IPP release and Vγ9Vδ2 T-cell activation. Abca1 silencing experiments confirmed the key role played by ABCA1. ABCA1 is up-regulated by ZA viaLXRα transcriptional activation induced by intracellular IPP accumulation and inhibition of the PI3K/Akt/mTOR signaling pathway.Butyrophilin-3A1 (BTN3A1) is an immunoglobulin superfamily protein reported to be required for phosphoantigen presentation and target recognition by Vγ9Vδ2 T cells. We have also demonstrated that BTN3A1 is physically associated with ABCA1 and cooperates with ABCA1 in the activation of Vγ9Vδ2 T cells.ConclusionsThese results suggest that ABCA1 is involved in extracellular IPP release and Vγ9Vδ2 T-cell activation induced by ZA-treated DC and therefore play a crucial role as mediator of the immune response. DisclosuresBoccadoro:SANOFI: Honoraria, Research Funding; CELGENE: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Abbivie: Honoraria; Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Mundipharma: Research Funding; Amgen: Honoraria, Research Funding. Massaia:Roche: Other: advisory board, research support; Gilead: Other: advisory board; Janssen: Other: advisory board.

ProtLID, a Residue-Based Pharmacophore Approach to Identify Cognate Protein Ligands in the Immunoglobulin Superfamily

Members of the extracellular immunoglobulin superfamily (IgSF) play a key role in immune regulation through the control of the co-stimulatory pathway, and have emerged as potent drug targets in cancers, infectious diseases, and autoimmunity. Despite the difficult experimental access to this class of proteins, single structures of ectodomains of IgSF proteins are solved with regularity. However, the most biologically critical challenge for this class of proteins isthe identification of their cognate ligands that communicate intercellular signals. We describe a conceptually novel method, protein-ligand interface design (ProtLID), to identify cognate ligands from asubproteome for a given target receptor protein. ProtLID designs an optimal protein interface for agiven receptor by running extensive molecular dynamics simulations of single-residue probes. The type and location of residue preferences establish aresidue-based pharmacophore, which is subsequently used to find potential matches among candidate ligands within a subproteome.

Regulation of GABAergic synapse development by postsynaptic membrane proteins.

In the adult mammalian brain, GABAergic neurotransmission provides the majority of synaptic inhibition that balances glutamatergic excitatory drive and thereby controls neuronal output. It is generally accepted that synaptogenesis is initiated through highly specific protein-protein interactions mediated by membrane proteins expressed in developing presynaptic terminals and postsynaptic membranes. Accumulating studies have uncovered a number of membrane proteins that regulate different aspects of GABAergic synapse development. In this review, we summarize recent advances in understanding of GABAergic synapse development with a focus on postsynaptic membrane molecules, including receptors, synaptogenic cell adhesion molecules and immunoglobulin superfamily proteins.

Marriage shrines and worms impacting our understanding of mammalian fertilization.

ABSTRACTGenetic approaches in C. elegans are complementing the biochemical and antibody based strategies traditionally used to study the molecular underpinnings of fertilization in other organisms. A pair of worm studies, one based on forward genetics and one based on reverse genetics, converge on the sperm immunoglobulin superfamily molecule SPE-45. Loss of spe-45 function leads to the production of sperm that cannot fertilize wild-type eggs. This is a strikingly similar phenotype as those seen in mice lacking the immunoglobulin superfamily protein Izumo1. This work sets the stage for leveraging the power of the C. elegans model system to learn more about Izumo-like molecular function but also for the discovery of additional deeply conserved components of fertility pathways.

Are the extracellular IgV and IgC domains of butyrophilin 3A1 required for prenyl pyrophosphate stimulation of human Vγ2Vδ2 T cells?

Abstract Vγ2Vδ2 T cells play important roles in human immunity to pathogens and in cancer immunotherapy by responding to isoprenoid metabolites such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) and isopentenyl pyrophosphate (IPP). The immunoglobulin superfamily protein, butyrophilin 3A1 (BTN3A1), is required for prenyl pyrophosphate stimulation. Our mutational studies and the work of others has shown that HMBPP and IPP compounds are not antigens but instead are sensed molecules that bind to the intracellular B30.2 domain of BTN3A1. How this binding is detected by the Vγ2Vδ2 TCR is unclear. One possibility is that HMBPP binding alters the conformation and/or distribution of the extracellular domains (ECD) of the BTN3A1 dimer allowing direct binding to the Vγ2Vδ2 TCR or to another molecule that binds the Vγ2Vδ2 TCR. Alternatively, the Vγ2Vδ2 TCR could bind to a molecule that is recruited by the intracellular B30.2 domain upon HMBPP binding. To distinguish between these mechanisms, we mutagenized 32 residues on the surfaces of the IgV and IgC domains of BTN3A1 to alanine and tested the mutant proteins for their ability to mediate prenyl pyrophosphate stimulation of Vγ2Vδ2 T cells. Mutagenesis of residues in the IgV and IgC domains had no effect whereas mutagenesis of the B30.2 binding site abrogated Vγ2Vδ2 T cell responses to HMBPP. Therefore, these findings do not support direct binding to the Vγ2Vδ2 TCR (or to another protein) by the ECD of BTN3A1. Instead, Vγ2Vδ2 TCR recognition may be to a protein recruited to the B30.2 domain upon HMBPP binding. Identification of this second protein will help clarify the puzzling process of human Vγ2Vδ2 T cell receptor recognition.

Forward Genetics Identifies a Requirement for the Izumo-like Immunoglobulin Superfamily spe-45 Gene in Caenorhabditis elegans Fertilization

Fertilization is a conserved process in all sexually reproducing organisms whereby sperm bind and fuse with oocytes. Despite the importance of sperm-oocyte interactions in fertilization, the molecular underpinnings of this process are still not well understood. The only cognate ligand-receptor pair identified in the context of fertilization is sperm-surface Izumo and egg-surface Juno in the mouse [1]. Here we describe a genetic screening strategy to isolate fertilization mutants in Caenorhabditis elegans in order to generate a more complete inventory of molecules required for gamete interactions. From this screening strategy, we identified, cloned, and characterized spe-45, a gene that encodes an Izumo-like immunoglobulin superfamily protein. Mammalian Izumo is required for male fertility and has the same basic mutant phenotype as spe-45. Worms lacking spe-45 function produce morphologically normal and motile sperm that cannot fuse with oocytes despite direct contact in the reproductive tract. The power of this screen to identify proteins with ancient sperm functions suggests that characterization of additional mutants from our screen may reveal other deeply conserved components in fertility pathways and complement studies in other organisms.

The role of ICAM-2 in neuroblastoma

The role of immunoglobulin superfamily cell adhesion molecules (CAMs) in facilitating immune responses in normal and tumor cells is well established. Cell adhesion molecule-1 (CADM1), for example, suppresses development of mouse mammary tumor cell metastasis by interacting with CD8+ T cells in immune-competent hosts [1]. Similarly, co-expression of Intercellular Adhesion Molecule-2 (ICAM-2) and chemokine C-X-C motif ligand 17 (CXCL17) elicits anti-tumor immune responses and suppresses tumor growth [2]. Although controversial, current literature suggests that proteins of the immunoglobulin superfamily CAMs also have functions that may well be distinct from their roles in mediating immune responses. Junctional adhesion molecule-A (JAM-A), for example, negatively regulates breast cancer cell invasiveness by disrupting tight junctions [3]; and a member of the B7 family of the immunoglobulin superfamily proteins, B7-H3, impairs osteogenic differentiation in vitro and in vivo [4]. Our lab demonstrated that ICAM-2 inhibited the development of disseminated neuroblastoma tumors in a preclinical model of metastatic neuroblastoma [5-7]. This inhibition depended on the interaction of ICAM-2 with the actin cytoskeletal linker protein α-actinin, an interaction that inhibited cell motility [7]. Ectopic expression of ICAM-2 did not affect the tumorigenic potential of neuroblastoma cells [7]. Importantly, immunohistochemical analyses of primary neuroblastoma tumor specimens demonstrated that neuroblastoma cells expressing ICAM-2 are phenotypically and histologically those recognized clinically to have limited metastatic potential [5]. Since metastatic disease is responsible for >90% of cancer-related deaths for multiple types of solid tumors, we suggest that elucidation of the molecular mechanism by which ICAM-2 suppresses the metastatic potential of neuroblastoma cells would identify proteins or pathways that might be exploited therapeutically to prevent metastatic disease progression. In normal tissues ICAM-2 is expressed predominantly by neovascular endothelial cells, and at lower levels by established vasculature and some leukocytes. The 202 amino acids comprising its extracellular domain mediate binding of ICAM-2 on endothelial cells to β2-integrins on the surface of leukocytes, to facilitate migration of neutrophils through the vascular endothelium as a component of immune reactions [8]. In neuroblastoma cells ICAM-2 inhibits cell motility independent of immune response, as we observed this inhibition in wound healing and modified Boyden chamber assays in vitro, assays that clearly lack an immune component [5,7]. In silico modeling indicated that ICAM-2 with mutations in the proposed α-actinin binding domain had a more ‘closed’ configuration than the wild type protein, and predicted that these ICAM-2 mutants would not interact with α-actinin [7]. Co-immunoprecipitation experiments confirmed in silico predictions [7]. In support of these findings, the interaction of ICAM-2 with α-actinin was essential to inhibit development of disseminated tumors in vivo [7]. As mentioned, ICAM-2 did not affect tumorigenic potential, and unpublished data showed no effect of ICAM-2 on expression of epithelial-mesenchymal transition (EMT) or stemness markers. Although we regard the inhibition of tumor cell motility by ICAM-2 in vitro to be compelling evidence that ICAM-2 participates in cell functions distinct from immune responses, we acknowledge the complex nature of metastatic tumor progression and propose that our data suggest multiple hypotheses with respect to the mechanism by which ICAM-2-inhibits neuroblastoma cell motility. The first hypothesis is that the intracellular interaction of ICAM-2 with α-actinin initiates ‘inside out’ signaling, and causes conformational changes in the extracellular domain of ICAM-2 that facilitate (or inhibit) interactions with specific extracellular matrix proteins that play an integral role in cell motility. The second is that the interaction of ICAM-2 with α-actinin alters the conformation of α-actinin to facilitate (or inhibit) the association of this actin cytoskeletal protein with alternative binding partners, with ICAM-2 acting as an ‘activator’ (or inhibitor) of α-actinin rather than as a membrane anchor protein. The third hypothesis, based on unpublished microarray data, is that ICAM-2 expression indirectly up-regulates protein tyrosine phosphatases (PTPs) involved in the formation and maintenance of focal adhesions such as focal adhesion kinase (FAK), Src, or Rac and Rho GTPases, each of which has crucial role in tumor cell motility. Our data show that ICAM-2 inhibits tumor cell motility and suppresses the metastatic potential of neuroblastoma cells. We propose that elucidating molecular events associated with ICAM-2 expression will identify key protein interactions that regulate the metastatic process in this cell type.