Glycosylation status of the membrane protein CD9P-1

The membrane protein CD9P-1 is a major component of the tetraspanin web, a network of molecular interactions in the plasma membrane, in which it specifically associates with tetraspanins CD9 and CD81. The various functional effects of CD9 and CD81 may be related to their partners. Thus, we have addressed the characterization of the CD9P-1 glycosylation using stably transfected HEK-293 cells. After immunoprecipitation, CD9P-1 was subjected to enzymatic PNGase F cleavage of N-glycans, resulting in Asn to Asp conversion and increase in 1 mass unit. Thus, following protease digestion, deglycosylated peptides were selectively identified by high mass accuracy FTICR-MS, using this conversion as a signature. This has demonstrated that all nine potential N-glycosylation sites were actually engaged. On the other hand, the N-glycan structures were determined combining chemical derivatization and exoglycosidase digestions followed by MALDI-TOF MS, ESI-MS/MS, and GC-MS analysis. CD9P-1 was shown to exhibit more than 40 different N-glycans, essentially composed of complex and high mannose-type structures. Finally, 2-D PAGE and lectino-blot analyses have revealed the presence of at least 17 glycosylated isoforms of CD9P-1 at cell surface. All CD9P-1 isoforms associate with CD9 leading to additional level of complexity of this primary complex in the tetraspanin web.     

Tetraspanin CD9 modulates human lymphoma cellular proliferation via histone deacetylase activity

Non-Hodgkin Lymphoma (NHL) is a type of hematological malignancy that affects two percent of the overall population in the United States. Tetraspanin CD9 is a cell surface protein that has been thoroughly demonstrated to be a molecular facilitator of cellular phenotype. CD9 expression varies in two human lymphoma cell lines, Raji and BJAB. In this report, we investigated the functional relationship between CD9 and cell proliferation regulated by histone deacetylase (HDAC) activity in these two cell lines. Introduction of CD9 expression in Raji cells resulted in significantly increased cell proliferation and HDAC activity compared to Mock transfected Raji cells. The increase in CD9–Raji cell proliferation was significantly inhibited by HDAC inhibitor (HDACi) treatment. Pretreatment of BJAB cells with HDAC inhibitors resulted in a significant decrease in endogenous CD9 mRNA and cell surface expression. BJAB cells also displayed decreased cell proliferation after HDACi treatment. These results suggest a significant relationship between CD9 expression and cell proliferation in human lymphoma cells that may be modulated by HDAC activity.     

The tetraspanin CD151 is required for Met-dependent signaling and tumor cell growth

CD151, a transmembrane protein of the tetraspanin family, is implicated in the regulation of cell-substrate adhesion and cell migration through physical and functional interactions with integrin receptors. In contrast, little is known about the potential role of CD151 in controlling cell proliferation and survival. We have previously shown that β4 integrin, a major CD151 partner, not only acts as an adhesive receptor for laminins but also as an intracellular signaling platform promoting cell proliferation and invasive growth upon interaction with Met, the tyrosine kinase receptor for hepatocyte growth factor (HGF). Here we show that RNAi-mediated silencing of CD151 expression in cancer cells impairs HGF-driven proliferation, anchorage-independent growth, protection from anoikis, and tumor progression in xenograft models in vivo. Mechanistically, we found that CD151 is crucially implicated in the formation of signaling complexes between Met and β4 integrin, a known amplifier of HGF-induced tumor cell growth and survival. CD151 depletion hampered HGF-induced phosphorylation of β4 integrin and the ensuing Grb2-Gab1 association, a signaling pathway leading to MAPK stimulation and cell growth. Accordingly, CD151 knockdown reduced HGF-triggered activation of MAPK but not AKT signaling cascade. These results indicate that CD151 controls Met-dependent neoplastic growth by enhancing receptor signaling through β4 integrin-mediated pathways, independent of cell-substrate adhesion.     

Mutation of juxtamembrane cysteines in the tetraspanin CD81 affects palmitoylation and alters interaction with other proteins at the cell surface

Palmitoylation of tetraspanins affects protein-protein interactions, suggesting a key role in the assembly of the tetraspanin web. Since palmitoylation occurs on intracellular cysteine residues, we examined whether mutating these residues in the human tetraspanin CD81 would affect the association of CD81 with other surface membrane proteins. Mutation of at least six of the eight juxtamembrane cysteines was required to completely eliminate detectable CD81 palmitoylation, indicating that several sites can be palmitoylated. Interestingly, these mutated proteins exhibited reduced cell surface detection by antibody compared to wild-type CD81, but this was not due to differences in the level of protein expression, trafficking to the cell surface, protein stability, or anti-CD81 antibody binding affinity. Instead, the mutant CD81 proteins appeared to be partially hidden from detection by anti-CD81 antibody, presumably due to altered interactions with other proteins at the cell surface. Associations with the known CD81-interacting proteins CD9 and EWI-2 were also impaired with the mutant CD81 proteins. Taken together, these findings indicate that mutation of juxtamembrane cysteines alters the interaction of CD81 with other proteins, either because of reduced palmitoylation, structural alterations in the mutant proteins, or a combination of both factors, and this affects the CD81 microenvironment on the cell surface.     

Infertility of CD9-deficient mouse eggs is reversed by mouse CD9, human CD9, or mouse CD81; polyadenylated mRNA injection developed for molecular analysis of sperm-egg fusion

CD9 is a membrane protein belonging to the tetraspanin family. Despite CD9's broad tissue distribution, the only abnormality observed in CD9-deficient mice was infertility of females, which was responsible for a defect in the sperm-egg fusion process. However, the function of CD9 in sperm-egg fusion is not clear at all because the technique to analyze the activity of molecules in sperm-egg fusion has not been established. We demonstrated that the exogenous mouse CD9, expressed by polyadenylated mRNA injection at the germinal-vesicle stage oocytes, was precisely localized to the egg plasma membrane, and the expression reversed the infertility of CD9(-/-) eggs. Then, two other tetraspanins, human CD9 and mouse CD81, overexpressed with this technique on CD9(-/-) eggs restored the fertilization rate up to approximately 90 and approximately 50% against that of wild type eggs, respectively. Moreover, in the presence of an anti-mouse CD9 mAb, which blocks sperm-egg fusion, expression of human CD9 or mouse CD81 on eggs also rescued the fusibility. These results suggested that human CD9 plays a crucial role in human fertilization, and mouse CD81 has the potential to compensate for CD9 function in sperm-egg fusion. In addition, the polyadenylated mRNA injection is effective for molecular analysis of sperm-egg fusion.     

In situ chemical cross-linking on living cells reveals CD9P-1 cis-oligomer at cell surface

Tetraspanins are integral membrane proteins involved in a variety of physiological and pathological processes. They associate with each other in multimolecular complexes containing numerous membrane proteins. As a first step towards the study of the supramolecular organization of tetraspanin complexes, we have implemented a proteomic approach based on in situ protein cross-linking on living cells followed by affinity purification of tetraspanin complexes. This allowed observing the presence of high molecular weight protein complexes that were characterized as containing CD9P-1/CD315 using LC-MS/MS. Western blot analyses and the use of different tags demonstrated the presence of CD9P-1 oligomer in cis-association at cell surface. A significant amount of CD9P-1 oligomer was observed on various cell types. We have shown that CD9P-1 self-associates independently from its association with tetraspanins. However, the expression level of CD9 or CD81 that associate directly and specifically with CD9P-1, positively modulates the cross-linking efficiency of CD9P-1. Thus, tetraspanins can play a role on CD9P-1 oligomerization status.     

CD95 tyrosine phosphorylation is required for CD95 oligomerization

Proapoptotic stimuli, such as CD95 ligand and hydrophobic bile acids induce an epidermal growth factor receptor (EGFR)-catalyzed tyrosine phosphorylation of CD95-death receptor in hepatocytes, as a prerequisite for CD95-translocation to the plasma membrane, formation of the death-inducing signalling complex and execution of apoptotic cell death. However, the molecular role played by CD95 tyrosine phosphorylation remained unclear. The present study shows that CD95-tyrosine phosphorylation is required for CD95-oligomerization. Fluorescence resonance energy transfer (FRET)-analysis in Huh7 hepatoma cells, which were cotransfected with CD95-YFP/CD95-CFP revealed that stimulation of these cells with CD95 ligand, proapoptotic bile acids or hyperosmolarity resulted within 30 min in an intracellular FRET-signal, suggestive for CD95/CD95-oligomerization. After 120 min the FRET-signal was detected in the plasma membrane, indicating translocation of the CD95/CD95-oligomer to the plasma membrane. CD95/CD95-oligomerization was abolished in presence of AG1478 or a JNK-inhibitory peptide, i.e. maneuvers known to prevent EGFR-catalyzed CD95-tyrosine phosphorylation. Transfection studies with YFP/CFP-coupled CD95-mutants, which contain tyrosine/phenylalanine-exchanges in positions 232 and 291 (CD95^Y232,291F), revealed that at least one tyrosine (Y^232,291)-phosphorylated CD95 is required for CD95/CD95-oligomerization. FRET-studies in mouse embryonic fibroblasts, which in contrast to Huh7 express endogenous CD95, revealed that EGF, but not CD95L induced EGFR-homomerization, whereas CD95 ligand, but not EGF resulted in EGFR/CD95-heteromerization. These findings suggest that EGFR-catalyzed CD95-tyrosine phosphorylation is involved in the CD95/CD95-oligomerization process, which is induced by proapoptotic stimuli and is required for apoptosis induction.     

Murine CD146 is widely expressed on endothelial cells and is recognized by the monoclonal antibody ME-9F1

The endothelium plays an important role in the exchange of molecules, but also of immune cells between blood and the underlying tissue. The endothelial molecule S-Endo 1 antigen (CD146) is preferentially located at endothelial junctions and has been claimed to support endothelial integrity. In this study we show that the monoclonal antibody ME-9F1 recognizes the extracellular portion of murine CD146. Making use of ME-9F1 we found CD146 highly expressed and widely spread on endothelial cells in the analyzed murine tissues. In contrast to humans that express CD146 also on T cells or follicular dendritic cells, murine CD146 albeit at low levels was only found on a subset of NK1.1⁺ cells. The antibody against murine CD146 is useful for immunomagnetic sorting of primary endothelial cells not only from the liver but from various other organs. In vitro, no evidence was seen that the formation and integrity of endothelial monolayers or the transendothelial migration of T cells was affected by antibody binding to CD146 or by crosslinking of the antigen. This makes the antibody ME-9F1 an excellent tool especially for the ex vivo isolation of murine endothelial cells intended to be used in functional studies.     

Vacuole membrane protein 1 marks endoplasmic reticulum subdomains enriched in phospholipid synthesizing enzymes and is required for phosphoinositide distribution

The multispanning membrane protein vacuole membrane protein 1 (VMP1) marks and regulates endoplasmic reticulum (ER)‐domains associated with diverse ER‐organelle membrane contact sites. A proportion of these domains associate with endosomes during their maturation and remodeling. We found that these VMP1 domains are enriched in choline/ethanolamine phosphotransferase and phosphatidylinositol synthase (PIS1), 2 ER enzymes required for the synthesis of various phospholipids. Interestingly, the lack of VMP1 impairs the formation of PIS1‐enriched ER domains, suggesting a role in the distribution of phosphoinositides. In fact, depletion of VMP1 alters the distribution of PtdIns4P and proteins involved in the trafficking of PtdIns4P. Consistently, in these conditions, defects were observed in endosome trafficking and maturation as well as in Golgi morphology. We propose that VMP1 regulates the formation of ER domains enriched in lipid synthesizing enzymes. These domains might be necessary for efficient distribution of PtdIns4P and perhaps other lipid species. These findings, along with previous reports that involved VMP1 in regulating PtdIns3P during autophagy, expand the role of VMP1 in lipid trafficking and explain the pleiotropic effects observed in VMP1‐deficient mammalian cells and other model systems.      

Mouse CD24 is required for homeostatic cell renewal

Under physiological conditions, some adult tissues retain a capacity for self-renewal. This property is attributable to the proliferation and differentiation of stem, transit-amplifying, and differentiating cells, which are regulated by cell-cell or cell-matrix interactions or by secreted factors. By gain and loss of function experiments, we demonstrate the involvement of mouse CD24 (mouse cluster of differentiation 24), which is a glycosyl phosphatidylinositol (GPI)-anchored cell-surface glycoprotein, in the regulation of homeostatic cell renewal. BrdU incorporation observations, at optical and electron-microscopic levels, have revealed increased cell proliferation in the developing brain and in the epithelia of mCD24-deleted mice. We have observed ectopic proliferative cells in the suprabasal layers of the mutant skin leading to a general disruption of basal and suprabasal layers. By contrast, ectopic mCD24 expression mediated by retroviral infection of the embryonic brain leads to a decreased number of clusters of cells generated in the progeny. Together, these results and our previous published data indicate that mCD24 contributes to the regulation of the production of differentiated cells by controlling the proliferation/differentiation balance between transit-amplifying and committed differentiated cells.     

Trafficking and function of the tetraspanin CD63

Tetraspanins comprise a large superfamily of cell surface-associated membrane proteins characterized by four transmembrane domains. They participate in a variety of cellular processes, like cell activation, adhesion, differentiation and tumour invasion. At the cell surface, tetraspanins form networks with a wide diversity of proteins called tetraspanin-enriched microdomains (TEMs). CD63 was the first characterized tetraspanin. In addition to its presence in TEMs, CD63 is also abundantly present in late endosomes and lysosomes. CD63 at the cell surface is endocytosed via a clathrin-dependent pathway, although recent studies suggest the involvement of other pathways as well and we here present evidence for a role of caveolae in CD63 endocytosis. In late endosomes, CD63 is enriched on the intraluminal vesicles, which by specialized cells are secreted as exosomes through fusion of endosomes with the plasma membrane. The complex localization pattern of CD63 suggests that its intracellular trafficking and distribution must be tightly regulated. In this review we discuss the latest insights in CD63 trafficking and its emerging function as a transport regulator of its interaction partners. Finally, the involvement of CD63 in cancer will be discussed.     

Palisade is required in the Drosophila ovary for assembly and function of the protective vitelline membrane

The innermost layer of the Drosophila eggshell, the vitelline membrane, provides structural support and positional information to the embryo. It is assembled in an incompletely understood manner from four major proteins to form a homogeneous, transparent extracellular matrix. Here we show that RNAi knockdown or genetic deletion of a minor constituent of this matrix, Palisade, results in structural disruptions during the initial synthesis of the vitelline membrane by somatic follicle cells surrounding the oocyte, including wide size variation among the precursor vitelline bodies and disorganization of follicle cell microvilli. Loss of Palisade or the microvillar protein Cad99C results in abnormal uptake into the oocyte of sV17, a major vitelline membrane protein, and defects in non-disulfide cross-linking of sV17 and sV23, while loss of Palisade has additional effects on processing and disulfide cross-linking of these proteins. Embryos surrounded by the abnormal vitelline membranes synthesized when Palisade is reduced are fertilized but undergo developmental arrest, usually during the first 13 nuclear divisions, with a nuclear phenotype of chromatin margination similar to that described for wild-type embryos subjected to anoxia. Our results demonstrate that Palisade is involved in coordinating assembly of the vitelline membrane and is required for functional properties of the eggshell.     

Preparation of fibroblast-free cytotrophoblast cultures utilizing differential expression of the CD9 antigen

Summary The leukemia-associated antigen CD9 is present on a variety of normal cells, with apparent variable expression on normal human fibroblasts. In this study, we demonstrate by immunoperoxidase staining and direct binding studies that the CD9 antigen is uniformly expressed on normal human fibroblasts grown from first trimester and term placenta, embryonic fetal fibroblasts, and from human adult and fetal skin fibroblasts. Higher CD9 expression was present on fetal cells. CD9 antigen was not present on trophoblast. Over 99% of fibroblasts could be absorbed onto antibody to the CD9 antigen conjugated to magnetic beads. By applying this selective immunoadsorption of fibroblasts to term placental cytotrophoblast preparations, we demonstrated that fibroblast contamination could be nearly completely eliminated. This is a novel technique for purifying primary trophoblast cultures and may have wider applicability in cell culture of other cell types.     

Interaction between membrane proteins PBP3 and rodA is required for normal cell shape and division in Escherichia coli.

In Escherichia coli, the products of several genes are required for septation, and the products of several others are required for the maintenance of the rod shape of the cells. We show here that the combination of certain mutations in a division gene (ftsI) with a specific mutation in one of the shape genes (rodA) could produce cells with normal shape and division, although separately these mutations led to a loss of the capacity to divide (ftsI) or to form normal rod-shaped cells (rodA). In contrast, combinations between other mutant alleles of these genes produced double mutants which had lost the capacity both to divide and to form rod-shaped cells. The mutual phenotypic correction observed within particular pairs of mutant genes suggests that the normal morphogenetic cycle of growth and division may require direct interaction between the two membrane proteins which are the products of these genes.     

PKCtheta is required for the activation of human T lymphocytes induced by CD43 engagement

The turnover of phosphoinositides leading to PKC activation constitutes one of the principal axes of intracellular signaling. In T lymphocytes, the enhanced and prolonged PKC activation resulting from the engagement of the TcR and co-receptor molecules ensures a productive T cell response. The CD43 co-receptor promotes activation and proliferation, by inducing IL-2 secretion and CD69 expression. CD43 engagement has been shown to promote phosphoinositide turnover and DAG production. Moreover, PKC activation was found to be required for the activation of the MAP kinase pathway in response to CD43 ligation. Here we show that CD43 engagement led to the membrane translocation and enzymatic activity of specific PKC isoenzymes: cPKC (alpha/beta), nPKC (epsilon and theta;), aPKC (zeta) and PKCmu. We also show that activation of PKCtheta; resulting from CD43 ligation induced CD69 expression through an ERK-dependent pathway leading to AP-1, NF-kappaB activation and an ERK independent pathway promoting NFAT activation. Together, these data suggest that PKCtheta; plays a critical role in the co-stimulatory functions of CD43 in human T cells.     

Structural requirements for the inhibitory action of the CD9 large extracellular domain in sperm/oocyte binding and fusion

CD9 has been shown to be essential for sperm/oocyte fusion in mice, the only non-redundant role found for a member of the tetraspanin family. CD9 can act in cis, reconstituting sperm/oocyte fusion when ectopically expressed in oocytes from CD9 null mice, or in trans, inhibiting sperm fusion when the large extracellular domain (LED) is added to CD9-positive oocytes as a soluble protein. In contrast to cis inhibition, the structural requirements of the trans inhibition by soluble CD9 LED are unknown. Here we show that human CD9 LED is as potent an inhibitor as mouse CD9 LED in mouse sperm/oocyte fusion assays and that CD9 LED can also inhibit sperm/oocyte binding. The two disulphide bridges that define membership of the tetraspanin family are critical for structure and function of human CD9 LED and mutation of a pentapeptide sequence in the hypervariable region further defines the critical region for trans inhibition.     

Galectin-9 induces osteoblast differentiation through the CD44/Smad signaling pathway

Galectin-9 is a β-galactoside-binding lectin expressed in various tissues. It binds various glycoconjugates and modulates a variety of biological functions in various cell types. Although galectin-9 is expressed in bone, its function in human osteoblasts remains unclear. We demonstrate that galectin-9 induces osteoblast differentiation through the CD44/Smad signaling pathway in the absence of bone morphogenetic proteins (BMPs). Galectin-9 increases alkaline phosphatase activities in human osteoblasts and induces the phosphorylation of Smad1/5/8 and translocation of Smad4 to the nucleus in the absence of BMPs. Galectin-9 also induces binding of Smad4 to the Id1 promoter and increases its activity. Anti-CD44 antibody inhibits Smad1/5/8 phosphorylation by galectin-9. Galectin-9 binds to CD44 and induces the formation of a CD44/BMP receptor complex. Because Smad1 is phosphorylated by BMP receptors, we propose that formation of the CD44/BMP receptor complex induced by galectin-9 may provide a trigger for the activation of Smads.