RESUMEN
Integrin α1ß1 is an adhesion receptor that binds to collagen and laminin. It regulates cell adhesion, cytoskeletal organization, and migration. The cytoplasmic tail of the α1 subunit consists of 15 amino acids and contains six positively charged lysine residues. In this study, we present evidence that the α1 integrin cytoplasmic tail (α1CT) directly associates with phosphoinositides, preferentially with phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). Since the association was disrupted by calcium, magnesium and phosphate ions, this interaction appears to be in ionic nature. Here, the peptide-lipid interaction was driven by the conserved KIGFFKR motif. The exchange of both two potential phospholipid-binding lysines for glycines in the KIGFFKR motif increased α1ß1 integrin-specific adhesion and F-actin cytoskeleton formation compared to cells expressing the unmodified α1 subunit, whereas only mutation of the second lysine at position 1171 increased levels of constitutively active α1ß1 integrins on the cell surface. In addition, enhanced focal adhesion formation and increased phosphorylation of focal adhesion kinase, but decreased phosphorylation of AKT was observed in these cells. We conclude that the KIGFFKR motif, and in particular lysine1171 is involved in the dynamic regulation of α1ß1 integrin activity and that the interaction of α1CT with phosphoinositides may contribute to this process.
Asunto(s)
Integrina alfa1 , Proteínas Proto-Oncogénicas c-akt , Integrina alfa1/química , Fosfatidilinositoles , Lisina , Adhesión Celular/genéticaRESUMEN
Enzymatic sequencing of oligosaccharides provides structural information on sequence of monosaccharides and type of linkage within the oligosaccharide chain. This data can be obtained by stepwise enzymatic digestion of a single, isolated oligosaccharide using individual or mixtures of specific exoglycosidases. N-glycans have to be fractionated from mixtures prior to sequence analysis to assign this type of structural information to a specific glycan. Enzymatic sequencing can as well be applied to oligosaccharide mixtures to evaluate the occurrence of distinct oligosaccharide motives of functional and/or structural interest.Here we describe the application of enzymatic sequence analysis to a mixture of N-glycans released from α1-acid glycoprotein. The experimental conditions are optimized for detection of possible Lewis X structures after stepwise exoglycosidase digestion by MALDI-TOF mass spectrometry. However, the described method is generally applicable to analyze other structural properties of N-glycans using (respective) specific exoglycosidases.
Asunto(s)
Glicósido Hidrolasas/química , Polisacáridos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Hidrólisis , Antígeno Lewis X/química , Estructura Molecular , Orosomucoide/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
PURPOSE: Previous studies have identified alkyl-phospholipids as promising compounds for cancer therapy by targeting constituents of the cell membrane and different signaling pathways. We previously showed that the alkylphospholipid Inositol-C2-PAF inhibits the proliferation and migration of immortalized keratinocytes and the squamous carcinoma-derived cell line SCC-25. Here, we investigated the effect of this compound on growth and motility as well as its mode of action in mammary carcinoma-derived cell lines. METHODS: Using BrdU incorporation and haptotactic cell migration assays, we assessed the effects of Inositol-C2-PAF on MCF-7 and MBA-MB-231 cell proliferation and migration. The phosphorylation status of signaling molecules was investigated by Western blotting as well as indirect immunofluorescence analysis and capillary isoelectric focusing. RESULTS: We found that Inositol-C2-PAF inhibited the growth as well as the migration in MCF-7 and MBA-MB-231 cells. Furthermore, we found that this compound inhibited phosphorylation of the protein kinase Akt at serine residue 473, but had no impact on phosphorylation at threonine 308. Phosphorylation of other kinases, such as Erk1/2, FAK and Src, which are targeted by Inositol-C2-PAF in other cells, remained unaffected by the compound in the mammary carcinoma-derived cell lines tested. In MCF-7 cells, we found that IGF-1-induced growth, as well as phosphorylation of AktS473, mTOR and the tumor suppressor pRB, was inhibited in the presence of Inositol-C2-PAF. Moreover, we found that in these cells IGF-1 had no impact on migration and did not seem to be linked to full Akt activity. Therefore, MCF-7 cell migration appears to be inhibited by Ino-C2-PAF in an Akt-independent manner. CONCLUSION: The antagonistic effects of Inositol-C2-PAF on cell migration and proliferation are indicative for its potential for breast cancer therapy, alone or in combination with other cytostatic drugs.
Asunto(s)
Neoplasias de la Mama/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Inositol/análogos & derivados , Factor de Activación Plaquetaria/análogos & derivados , Antineoplásicos/farmacología , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Femenino , Humanos , Inositol/farmacología , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Células MCF-7 , Factor de Activación Plaquetaria/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
In cutaneous inflammatory diseases, such as psoriasis, atopic dermatitis and allergic contact dermatitis, skin-infiltrating T lymphocytes and dendritic cells modulate keratinocyte function via the secretion of pro-inflammatory cytokines. Keratinocytes then produce mediators that recruit and activate immune cells and amplify the inflammatory response. These pathophysiological tissue changes are caused by altered gene expression and the proliferation and maturation of dermal and epidermal cells. We recently demonstrated that the glycosidated phospholipid Ino-C2-PAF down-regulates a plethora of gene products associated with innate and acquired immune responses and inflammation in the HaCaT keratinocyte cell line. To further evaluate the influence of Ino-C2-PAF we established an in vitro 2D-model of epidermal inflammation. The induction of inflammation and the impact of Ino-C2-PAF were assessed in this system using a genome-wide microarray analysis. In addition, the expression of selected genes was validated using qRT-PCR and flow cytometry. Treatment of the keratinocytes with a mix of proinflammatory cytokines resulted in transcriptional effects on a variety of genes involved in cutaneous inflammation and immunity, while additional treatment with Ino-C2-PAF counteracted the induction of many of these genes. Remarkably, Ino-C2-PAF suppressed the expression of a group of targets that are implicated in antigen processing and presentation, including MHC molecules. Thus, it is conceivable that Ino-C2-PAF possess therapeutic potential for inflammatory skin disorders, such as psoriasis and allergic contact dermatitis.
Asunto(s)
Antiinflamatorios/farmacología , Inflamación/tratamiento farmacológico , Inositol/análogos & derivados , Queratinocitos/efectos de los fármacos , Factor de Activación Plaquetaria/análogos & derivados , Inmunidad Adaptativa/efectos de los fármacos , Antígenos CD/genética , Antígenos CD/metabolismo , Técnicas de Cultivo de Célula , Citocinas/genética , Citocinas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inmunidad Innata/efectos de los fármacos , Inflamación/metabolismo , Inositol/farmacología , Factor de Activación Plaquetaria/farmacología , Análisis por Matrices de ProteínasRESUMEN
AIMS: A20 (TNFAIP3) is a nuclear factor-κB (NF-κB)-inducible component of tumour necrosis factor and Toll-like receptor intracellular signal transduction. It negatively regulates NF-κB, and has been identified as a tumour suppressor. Several studies have described A20 inactivation by deletion of the A20 locus at 6q23, inactivating mutations, and/or methylation of the A20 promoter in various lymphoma entities. METHODS AND RESULTS: We generated a monoclonal antibody against the C-terminus of A20 (Ber-A20) and investigated full-length A20 expression of normal lymphoid tissue and lymphomas for the first time. We identified loss of A20 expression in tumour cells of 24% of classical Hodgkin lymphoma, 27% of diffuse large B-cell lymphoma, 20% of chronic lymphocytic leukaemia, 19% of follicular lymphoma, 13% of mantle cell lymphoma and 8% of primary mediastinal B-cell lymphoma cases by immunohistology. Loss of A20 expression rarely occurred in T-cell non-Hodgkin lymphoma. CONCLUSIONS: Our data are in agreement with cytogenetic and molecular analyses. Among 21 cases of ocular adnexal marginal zone lymphomas with known A20 mutation status, we detected complete absence of A20 expression, whereas cases with wild-type A20 were weakly A20-positive. We demonstrate that A20 loss can be detected by immunohistology with a sensitivity similar to that of complex molecular and genetic methods.
Asunto(s)
Anticuerpos Monoclonales/química , Proteínas de Unión al ADN/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Anticuerpos Monoclonales/metabolismo , Proteínas de Unión al ADN/inmunología , Neoplasias del Ojo/metabolismo , Enfermedad de Hodgkin/metabolismo , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular/inmunología , Linfoma no Hodgkin/metabolismo , Mutación , Proteínas Nucleares/inmunología , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfaRESUMEN
Improving the accessibility and functions of therapeutic and diagnostic glycoproteins is one of the major goals of glycobiotechnology. Here we present that stable knock-down of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), the key enzyme in the sialic acid biosynthetic pathway, dramatically increases incorporation of N-acetylmannosamine analogues into glycoproteins of HEK293 cells. By means of these GNE-deficient cells highly sialylated glycoproteins can efficiently be decorated with reactive functional groups, which can be employed in bioorthogonal functionalization strategies for fluorescence labelling or biotinylation.
Asunto(s)
Técnicas de Silenciamiento del Gen , Glicoproteínas/metabolismo , Oligosacáridos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Ingeniería de Proteínas/métodos , Antígenos CD/metabolismo , Conformación de Carbohidratos , Carbohidrato Epimerasas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Glicoproteínas/química , Células HEK293 , Humanos , Ácido N-Acetilneuramínico/metabolismo , Oligosacáridos/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/deficiencia , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/metabolismo , Coloración y EtiquetadoRESUMEN
Members of the family of carcinoembryonic antigen (CEA)-related cell adhesion molecules (CEACAMs) belonging to the immunoglobulin (Ig) superfamily are expressed in a variety of normal and malignant human tissues. As components of the cell membrane, these glycoproteins can make contact with adjacent cells. CEACAM1 and CEACAM5 (CEA) express Lewis(x) (Le(x)) structures. As shown by mass spectrometry in conjunction with enzymatic digestion, CEACAM1 contains at least seven Le(x) residues. Fucosyltransferase IX is the main fucosyltransferase responsible for attachment of terminal fucose, the key feature of the Le(x) structure, to CEA and CEACAM1. The Le(x) residues of both, CEACAM1 and CEA, interact with the human Le(x)-binding glycan receptors DC-SIGN and SRCL. Since subpopulations of human macrophages express DC-SIGN or SRCL, Le(x)-carrying CEACAMs may modulate the immune response in normal tissues such as the human placenta or in malignant tumours, for example in colorectal, pancreatic or lung carcinomas.
Asunto(s)
Antígenos CD/metabolismo , Antígeno Carcinoembrionario/metabolismo , Moléculas de Adhesión Celular/metabolismo , Colectinas/metabolismo , Lectinas Tipo C/metabolismo , Polisacáridos/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Depuradores/metabolismo , Proteínas Recombinantes/metabolismo , Línea Celular , Neoplasias Colorrectales/metabolismo , Femenino , Fucosa/metabolismo , Fucosiltransferasas/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Antígeno Lewis X/metabolismo , Melanoma/metabolismo , Placenta/metabolismo , Embarazo , Unión Proteica , Neoplasias Cutáneas/metabolismo , Extractos de TejidosRESUMEN
Although Moraxella catarrhalis and Neisseria meningitidis are important human pathogens, they often colonize the human respiratory tract without causing overt clinical symptoms. Both pathogens express structurally unrelated proteins that share the ability to stimulate the adhesion molecule CEACAM1 expressed on human cells. Here we demonstrate that the interaction of CEACAM1 with ubiquitous surface protein A1 expressed on M. catarrhalis or with opacity-associated proteins on N. meningitidis resulted in reduced Toll-like receptor 2-initiated transcription factor NF-kappaB-dependent inflammatory responses of primary pulmonary epithelial cells. These inhibitory effects were mediated by tyrosine phosphorylation of the immunoreceptor tyrosine-based inhibitory motif of CEACAM1 and by recruitment of the phosphatase SHP-1, which negatively regulated Toll-like receptor 2-dependent activation of the phosphatidylinositol 3-OH kinase-Akt kinase pathway. Our results identify a CEACAM1-dependent immune-evasion strategy.
Asunto(s)
Antígenos CD/inmunología , Bronquios/inmunología , Moléculas de Adhesión Celular/inmunología , Moraxella catarrhalis/inmunología , Neisseria meningitidis/inmunología , Mucosa Respiratoria/inmunología , Receptor Toll-Like 2/inmunología , Secuencias de Aminoácidos/fisiología , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Antígenos CD/química , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Bronquios/metabolismo , Bronquios/microbiología , Moléculas de Adhesión Celular/química , Células Cultivadas , Citocinas/metabolismo , Regulación hacia Abajo , Humanos , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/microbiología , Transducción de Señal/inmunología , Receptor Toll-Like 2/metabolismoRESUMEN
Enzymatic sequencing of oligosaccharides gives structural information on sequence of monosaccharides and type of linkage within the oligosaccharide chain. This data can be obtained by stepwise enzymatic digestion of a single, isolated oligosaccharide using individual or mixtures of specific exoglycosidases. N-glycans have to be fractionated from mixtures prior to sequence analysis to assign this type of structural information to a specific glycan. Enzymatic sequencing can be applied to oligosaccharide mixtures as well to evaluate the occurrence of distinct oligosac-charide motives of functional and/or structural interest. Here we describe the application of enzymatic sequence analysis to a mixture of N-glycans released from alpha1-acid glycoprotein. The experimental conditions are optimized for detection of possible Lewis X structures after stepwise exoglycosi-dase digestion by MALDI-TOF mass spectrometry. However, the described method is generally applicable to analyze other structural properties of N-glycans by use of the respective specific exoglycosidases.
Asunto(s)
Glicósido Hidrolasas/metabolismo , Antígeno Lewis X/química , Polisacáridos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Conformación de Carbohidratos , Polisacáridos/metabolismoRESUMEN
Negative ion tandem mass spectrometry (MS/MS) spectra of three isomeric triantennary N-linked glycans provided clear differentiation between the isomers and confirmed the occurrence of an isomer that was substituted with galactose on a bisecting GlcNAc (1 --> 4-substituted on the core mannose) residue recently reported by Takegawa et al. from N-glycans released from human immunoglobulin G (IgG). We extend this analysis of human serum IgG to reveal an analogue of the fucosylated triantennary glycan reported by Takegawa et al. together with a third compound that lacked both the sialic acid and the fucose residues. In addition, we demonstrate the biosynthesis of bisected hybrid-type glycans with the galactose modification, with and without core fucose, on the stem cell marker glycoprotein, 19A, expressed in a partially ricin-resistant human embryonic kidney cell line. It would appear, therefore, that this modification of N-linked glycans containing a galactosylated bisecting GlcNAc residue may be more common than originally thought. Negative ion MS/MS analysis of glycans is likely to prove an invaluable tool in the analysis and monitoring of therapeutic glycoproteins.
Asunto(s)
Biomarcadores de Tumor/química , Galactosa/química , Inmunoglobulina G/química , Oligopéptidos/química , Polisacáridos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Aniones , Isomerismo , N-Acetilgalactosaminiltransferasas , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
The homophilic cell-cell adhesion receptor CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1, CD66a) acts as a regulator of contact-dependent cell survival, differentiation, and growth. It is involved in the control of proliferation in hematopoietic and epithelial cells and can act as a tumor suppressor. In this study, we identify DNA polymerase delta-interacting protein 38 (PDIP38) as a novel binding partner for CEACAM1-L and CEACAM1-S. We show that PDIP38 can occur in the nucleus, in the cytoplasm and at the plasma membrane in NBT-II, IEC18, RBE, and HeLa cells and that the distribution in NBT-II cells is influenced by the confluency of the cells. We also demonstrate that the interaction of CEACAM1 and PDIP38 is of functional importance in NBT-II cells, which co-express the long and the short CEACAM1 isoform. In subconfluent, proliferating NBT-II cells, perturbation of CEACAM1 by antibody clustering induces increased binding to PDIP38 and results in rapid recruitment of PDIP38 to the plasma membrane. The same treatment of confluent, quiescent NBT-II cells leads to a different response, i.e. translocation of PDIP38 to the nucleus. Together, our data show that PDIP38 can shuttle between the cytoplasmic and the nuclear compartments and that its subcellular localization is regulated by CEACAM1, implicating that PDIP38 may constitute a novel downstream target of CEACAM1 signaling.
Asunto(s)
Antígenos CD/metabolismo , Moléculas de Adhesión Celular/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Transducción de Señal/fisiología , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/fisiología , Animales , Anticuerpos Monoclonales/farmacología , Antígenos CD/genética , Células CHO , Moléculas de Adhesión Celular/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Núcleo Celular/genética , Cricetinae , Cricetulus , Citoplasma/genética , Células HeLa , Humanos , Recubrimiento Inmunológico/efectos de los fármacos , Proteínas de la Membrana/genética , Proteínas Nucleares/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratas , Transducción de Señal/efectos de los fármacosRESUMEN
The HIV envelope has evolved a dense array of immunologically "self" carbohydrates that efficiently protect the virus from antibody recognition. Nonetheless, one broadly neutralising antibody, IgG1 2G12, has been shown to recognise a cluster of oligomannose glycans on the HIV-1 surface antigen gp120. Thus the self carbohydrates of HIV are now regarded as potential targets for viral neutralisation and vaccine design. Here, we show that chemical inhibition of mammalian glycoprotein synthesis, with the plant alkaloid kifunensine, creates multiple HIV (2G12) epitopes on the surface of previously non-antigenic self proteins and cells, including HIV gp120. This formally demonstrates the structural basis for self/non-self discrimination between viral and host glycans, by a neutralising antibody. Moreover, this study provides an alternative protein engineering approach to the design of a carbohydrate vaccine for HIV-1 by chemical synthesis.
Asunto(s)
Metabolismo de los Hidratos de Carbono/efectos de los fármacos , Anticuerpos Anti-VIH/metabolismo , VIH-1/inmunología , Manosidasas/antagonistas & inhibidores , Polisacáridos/biosíntesis , Polisacáridos/inmunología , Alcaloides/farmacología , Animales , Fármacos Anti-VIH/metabolismo , Metabolismo de los Hidratos de Carbono/inmunología , Epítopos/inmunología , Antígenos VIH/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Humanos , Modelos Biológicos , Modelos Moleculares , Pruebas de NeutralizaciónRESUMEN
The bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is a key enzyme for the biosynthesis of sialic acids, the terminal sugars of glycoconjugates associated with a variety of physiological and pathological processes such as cell adhesion, development, inflammation and cancer. In this study, we characterized rat GNE by different biophysical methods, analytical ultracentrifugation, dynamic light-scattering and size-exclusion chromatography, all revealing the native hydrodynamic behavior and molar mass of the protein. We show that GNE is able to reversibly self-associate into different oligomeric states including monomers, dimers and tetramers. Additionally, it forms non-specific aggregates of high molecular mass, which cannot be unequivocally assigned a distinct size. Our results also indicate that ligands of the epimerase domain of the bifunctional enzyme, namely UDP-N-acetylglucosamine and CMP-N-acetylneuraminic acid, stabilize the protein against aggregation and are capable of modulating the quaternary structure of the protein. The presence of UDP-N-acetylglucosamine strongly favors the tetrameric state, which therefore likely represents the active state of the enzyme in cells.
Asunto(s)
Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Animales , Biofisica/métodos , Carbohidrato Epimerasas/química , Cinética , Ligandos , Luz , Conformación Molecular , Ácido N-Acetilneuramínico/química , Unión Proteica , Ratas , Ratas Wistar , Proteínas Recombinantes/química , Dispersión de Radiación , Ultracentrifugación/métodosRESUMEN
Lymphomatoid papulosis (LyP), primary cutaneous anaplastic large T-cell lymphoma (cALCL), and cutaneous infiltrates of systemic anaplastic large cell lymphoma (sALCL) are CD30-positive lymphoproliferative disorders of the skin that overlap clinically, histopathologically, immunophenotypically, and genetically but differ considerably in their prognosis. In particular, lesions of LyP regress spontaneously, whereas those of cALCL and sALCL persist and may progress and spread to extracutaneous sites. In contrast to patients with cALCL, LyP patients do not benefit from an aggressive radio- and/or chemotherapeutic approach. We generated a novel tumor necrosis factor receptor (TNFR)-associated factor 1 (TRAF1) antibody that recognizes a formalin-resistant epitope (Ber-TRAF1A) and investigated the expression of TRAF1, an intracellular component of TNFR signaling, in LyP and ALCL. We could show a strong TRAF1 expression in the tumor cells of most LyP cases (42/49, 84%). In contrast, tumor cells of primary and secondary cALCL revealed TRAF1 expression in only a few cases (3/41, 7%) as shown for sALCL without skin manifestation. The data indicate that TRAF1 expression reliably distinguishes LyP from primary or secondary cALCL. This might be of crucial diagnostic importance and has a strong impact on the treatment decision for patients with cALCL and LyP.
Asunto(s)
Antígeno Ki-1/análisis , Linfoma de Células B Grandes Difuso/química , Linfoma Cutáneo de Células T/química , Linfoma de Células T/química , Papulosis Linfomatoide/metabolismo , Factor 1 Asociado a Receptor de TNF/análisis , Animales , Humanos , Ratones , Ratones Endogámicos BALB C , Factor 1 Asociado a Receptor de TNF/inmunología , Factor 1 Asociado a Receptor de TNF/fisiologíaRESUMEN
Intercellular adhesion molecule-3 (ICAM-3) binds to the alpha(L)beta(2) integrin and mediates the contact between T cells and antigen-presenting cells. It has been suggested that dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN), a C-type lectin of macrophages and DCs, is an additional ligand of ICAM-3. So far, the glycan structure mediating the interaction of native ICAM-3 with DC-SIGN is undefined. Here, we demonstrate that native ICAM-3 from human peripheral leukocytes binds recombinant DC-SIGN, is recognized by monoclonal Lewis x antibodies, and specifically interacts with DC-SIGN on immature DCs. The presence of Lewis x residues on ICAM-3 was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy. Investigations on different peripheral blood cell populations revealed that only ICAM-3 from granulocytes bound DC-SIGN. Cotransfection studies demonstrated that fucosyltransferase (FUT) IX and, to a significantly lesser extent, FUT IV, but not FUTs III and VII, mediate the synthesis of Lewis x residues on ICAM-3. These findings indicate that FUT IX is the main FUT mediating the synthesis of Lewis x residues of ICAM-3 in cells of the myeloid lineage, and that these residues bind DC-SIGN. The results suggest that ICAM-3 assists in the interaction of granulocytes with DC-SIGN of DCs.
Asunto(s)
Antígenos CD/química , Moléculas de Adhesión Celular/química , Granulocitos/inmunología , Lectinas Tipo C/química , Antígeno Lewis X/química , Receptores de Superficie Celular/química , Antígenos CD/aislamiento & purificación , Moléculas de Adhesión Celular/aislamiento & purificación , Fucosiltransferasas/química , Humanos , Leucocitos/inmunología , Antígeno Lewis X/análisis , Antígeno Lewis X/biosíntesis , Proteínas Recombinantes/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Hereditary inclusion body myopathy (HIBM), a neuromuscular disorder, is caused by mutations in UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), the key enzyme of sialic acid biosynthesis. To date, more than 40 different mutations in the GNE gene have been reported to cause the disease. Ten of them, representing mutations in both functional domains of GNE, were recombinantly expressed in insect cells (Sf9). Each of the mutants that was analyzed displayed a reduction in the two known GNE activities, thus revealing that mutations may also influence the function of the domain not harboring them. The extent of reduction strongly differs among the point mutants, ranging from only 20% reduction found for A631T and A631V to almost 80% reduction of at least one activity in D378Y and N519S mutants and more than 80% reduction of both activities of G576E, underlined by structural changes of N519S and G576E, as observed in CD spectroscopy and gel filtration analysis, respectively. We therefore generated models of the three-dimensional structures of the epimerase and the kinase domains of GNE, based on Escherichia coli UDP-N-acetylglucosamine 2-epimerase and glucokinase, respectively, and determined the localization of the HIBM mutations within these proteins. Whereas in the kinase domain most of the mutations are localized inside the enzyme, mutations in the epimerase domain are mostly located at the protein surface. Otherwise, the different mutations result in different enzymatic activities but not in different disease phenotypes and, therefore, do not suggest a direct role of the enzymatic function of GNE in the disease mechanism.
Asunto(s)
Miositis por Cuerpos de Inclusión/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/farmacología , Baculoviridae/metabolismo , Carbohidrato Epimerasas/genética , Carbohidrato Epimerasas/metabolismo , Carbohidrato Epimerasas/farmacología , Humanos , Modelos Moleculares , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Mutación , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Estructura Terciaria de Proteína/genética , Transporte de Proteínas , Relación Estructura-ActividadRESUMEN
The CEA-related cell adhesion molecule 1, CEACAM1, is a glycoprotein expressed on the surface of human granulocytes and lymphocytes, endothelia, and many epithelia. CEACAM1 is involved in the regulation of important biological processes, such as tumor growth, angiogenesis, and modulation of the immune response. CEACAM1, a member of the immunoglobulin superfamily carries several Lewis x (Lex) structures as we recently demonstrated by mass spectrometry of native CEACAM1 from human granulocytes. Since Lex residues of pathogens bind to the C-type lectin dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) expressed on human DCs, we hypothesized that Lex glycans of CEACAM1 are recognized by DC-SIGN. Here, we demonstrate that CEACAM1, the major carrier of Lex residues in human granulocytes, is specifically recognized by DC-SIGN via Lex residues mediating the internalization of CEACAM1 into immature DCs. Expression studies with CEACAM1 in combination with different fucosyltransferases (FUTs) revealed that FUTIX plays a key role in the synthesis of Lex groups of CEACAM1. As Lex groups on CEACAM1 are selectively attached and specifically interact with DC-SIGN, our findings suggest that CEACAM1 participates in immune regulation in physiological conditions and in pathological conditions, such as inflammation, autoimmune disease, and cancer.
Asunto(s)
Antígenos CD/metabolismo , Moléculas de Adhesión Celular/metabolismo , Células Dendríticas/metabolismo , Fucosa/metabolismo , Fucosiltransferasas/metabolismo , Granulocitos/metabolismo , Lectinas Tipo C/metabolismo , Antígeno Lewis X/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Antígenos CD/química , Moléculas de Adhesión Celular/química , Diferenciación Celular , Línea Celular , Cricetinae , Células Dendríticas/citología , Fucosa/química , Expresión Génica , Humanos , Ligandos , Unión ProteicaRESUMEN
Binding of integrins to proteins of the extracellular matrix (ECM) provides structural and signaling information for biological processes such as cell proliferation, migration, neurite outgrowth, and differentiation. Integrins represent a family of heterodimeric transmembrane cell surface receptors. Besides connecting the ECM with the cytoskeleton, integrins also induce various signaling pathways in response to ligand binding. Integrin ligation leads to cytoplasmic protein-protein interactions requiring both integrin cytoplasmic tails. These sequences are initiation points for focal adhesion formation and subsequent signal transduction cascades. In this study, we addressed the question of whether the short cytoplasmic tail of the alpha(3) integrin subunit of alpha(3)beta(1) integrin is required for alpha(3)beta(1) integrin-dependent processes. For this purpose, cDNA representing the extracellular and transmembrane domain of the interleukin 2 receptor (IL2R) alpha subunit and the cytoplasmic sequence of the alpha(3) integrin subunit was transfected into PC12 cells. Autonomous expression of the cytoplasmic alpha(3) tail does not affect attachment but leads to inhibition of neuronal differentiation on laminin 5. This indicates that the cytoplasmic alpha(3) sequence is not required for cell attachment but is necessary for long-term adhesion and for the reorganization of the cytoskeleton that precedes neuronal differentiation. Inhibition of neurite outgrowth by chimeric IL2R-alpha(3) can be rescued by treatment of transfected cells with the pharmacological inhibitor Y27632, which inhibits the RhoA downstream effector Rho kinase alpha.
Asunto(s)
Citoplasma/metabolismo , Integrina alfa3beta1/fisiología , Neuritas/fisiología , Animales , Anticuerpos/farmacología , Western Blotting/métodos , Carcinoma/patología , Moléculas de Adhesión Celular/inmunología , Moléculas de Adhesión Celular/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Línea Celular Tumoral , Citoplasma/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Citometría de Flujo/métodos , Técnica del Anticuerpo Fluorescente/métodos , Regulación de la Expresión Génica/fisiología , Humanos , Células PC12 , Estructura Terciaria de Proteína , ARN Mensajero/biosíntesis , Ratas , Receptores de Interleucina-2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Transfección/métodos , KalininaRESUMEN
The carcinoembryonic antigen-related cell adhesion molecule CEACAM1 (CD66a) and the scaffolding protein filamin A have both been implicated in tumor cell migration. In the present study we identified filamin A as a novel binding partner for the CEACAM1-L cytoplasmic domain in a yeast two-hybrid screen. Direct binding was shown by surface plasmon resonance analysis and by affinity precipitation assays. The association was shown for human and rodent CEACAM1-L in endogenous CEACAM1-L expressing cells. To address functional aspects of the interaction, we used a well-established melanoma cell system. We found in different migration studies that the interaction of CEACAM1-L and filamin A drastically reduced migration and cell scattering, whereas each of these proteins when expressed alone, acted promigratory. CEACAM1-L binding to filamin A reduced the interaction of the latter with RalA, a member of the Ras-family of GTPases. Furthermore, co-expression of CEACAM1-L and filamin A led to a reduced focal adhesion turnover. Independent of the presence of filamin A, the expression of CEACAM1-L led to an increased phosphorylation of focal adhesions and to altered cytoskeletal rearrangements during monolayer wound healing assays. Together, our data demonstrate a novel mechanism for how CEACAM1-L regulates cell migration via its interaction with filamin A.
Asunto(s)
Antígenos CD/fisiología , Moléculas de Adhesión Celular/fisiología , Movimiento Celular/fisiología , Proteínas Contráctiles/fisiología , Proteínas de Microfilamentos/fisiología , Animales , Antígenos CD/genética , Antígenos CD/farmacología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/farmacología , Línea Celular Tumoral , Proteínas Contráctiles/genética , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Filaminas , Proteína-Tirosina Quinasas de Adhesión Focal/efectos de los fármacos , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Regulación de la Expresión Génica , Humanos , Proteínas de Microfilamentos/genética , Paxillin/efectos de los fármacos , Paxillin/metabolismo , Fosforilación , Unión Proteica , Ratas , Técnicas del Sistema de Dos Híbridos , Tirosina/efectos de los fármacos , Tirosina/metabolismoRESUMEN
Growth factor receptors, extracellular matrix receptors, and cell-cell adhesion molecules co-operate in regulating the activities of intracellular signaling pathways. Here, we demonstrate that the cell adhesion molecule CEACAM1 co-regulates growth-factor-induced DNA synthesis in NBT-II epithelial cells in a cell-density-dependent manner. CEACAM1 exerted its effects by regulating the activity of the Erk 1/2 MAP kinase pathway and the expression levels of the cyclin-dependent kinase inhibitor p27(Kip1). Interestingly, both inhibitory and stimulatory effects were observed. Confluent cells continuously exposed to fetal calf serum showed little Erk activity and DNA synthesis compared with sparse cells. Under these conditions, anti-CEACAM1 antibodies strongly stimulated Erk activation, decreased p27 expression, and induced DNA synthesis. In serum-starved confluent cells, re-addition of 10% fetal calf serum activated the Erk pathway, decreased p27 expression, and stimulated DNA synthesis to the same levels as in sparse cells. Under these conditions anti-CEACAM1 antibodies de-activated Erk, restored the level of p27, and inhibited DNA synthesis. These data indicate that CEACAM1 mediates contact inhibition of proliferation in cells that are constantly exposed to growth factors, but co-activates growth-factor-induced proliferation in cells that have been starved for growth factors; exposure to extracellular CEACAM1 ligands reverts these responses.