RESUMO
Leukocyte adhesion deficiency II (LAD II) is characterized by the lack of fucosylated glycoconjugates, including selectin ligands, causing immunodeficiency and severe mental and growth retardation. No deficiency in fucosyltransferase activities or in the activities of enzymes involved in GDP-fucose biosynthesis has been found. Instead, the transport of GDP-fucose into isolated Golgi vesicles of LAD II cells appeared to be reduced. To identify the gene mutated in LAD II, we cloned 12 cDNAs from Caenorhabditis elegans, encoding multi-spanning transmembrane proteins with homology to known nucleotide sugar transporters, and transfected them into fibroblasts from an LAD II patient. One of these clones re-established expression of fucosylated glycoconjugates with high efficiency and allowed us to identify a human homolog with 55% identity, which also directed re-expression of fucosylated glycoconjugates. Both proteins were localized to the Golgi. The corresponding endogenous protein in LAD II cells had an R147C amino acid change in the conserved fourth transmembrane region. Overexpression of this mutant protein in cells from a patient with LAD II did not rescue fucosylation, demonstrating that the point mutation affected the activity of the protein. Thus, we have identified the first putative GDP-fucose transporter, which has been highly conserved throughout evolution. A point mutation in its gene is responsible for the disease in this patient with LAD II.
Assuntos
Proteínas de Transporte/genética , Guanosina Difosfato Fucose/metabolismo , Síndrome da Aderência Leucocítica Deficitária/genética , Proteínas de Transporte de Monossacarídeos , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Proteínas de Transporte/metabolismo , Compartimento Celular , Teste de Complementação Genética , Glicosilação , Complexo de Golgi/química , Humanos , Síndrome da Aderência Leucocítica Deficitária/classificação , Síndrome da Aderência Leucocítica Deficitária/etiologia , Modelos Moleculares , Dados de Sequência Molecular , Mutação Puntual , Homologia de Sequência de AminoácidosRESUMO
The neural cell adhesion molecule NCAM and its dynamically regulated posttranslational modification polysialic acid (PSA) are major determinants of cellular interactions during ontogeny. While NCAM in the absence of PSA stabilizes cell-cell interactions, the attachment of the large and polyanionic PSA negatively influences cell adhesion and promotes plasticity. Disease-associated changes in the polysialylation state of NCAM raise the question whether the PSA-NCAM system can affect CNS pharmacology. Here we investigated the pharmacological effects of the competitive AMPA antagonist NBQX in genetic mouse models either lacking NCAM and PSA (female NCAM knockout mice) or being drastically reduced in the level of PSA expression (female ST8SiaIV knockout mice). Studies were carried out with the respective wildtype littermate controls. In mice lacking NCAM and PSA, NBQX-induced ataxia proved to be more intense as compared with wild-type mice. On both mutant backgrounds, NBQX significantly elevated seizure thresholds during i.v. infusion of the chemoconvulsant pentylenetetrazole. In summary, the data demonstrate that the PSA-NCAM system impacts AMPA receptor pharmacology under in vivo conditions. The fact that comparable effects were observed in NCAM- and ST8SiaIV-knockout mice indicates that this impact is not due to a stabilizing effect of NCAM in the absence of PSA. Thus, disease-related changes in the polysialylation of NCAM are likely to be associated with effects on the efficacy and tolerability of AMPA receptor antagonists.
Assuntos
Antagonistas de Aminoácidos Excitatórios/farmacologia , Moléculas de Adesão de Célula Nervosa/deficiência , Quinoxalinas/farmacologia , Convulsões/fisiopatologia , Sialiltransferases/deficiência , Análise de Variância , Animais , Ataxia/induzido quimicamente , Ataxia/genética , Comportamento Animal/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pentilenotetrazol , Convulsões/induzido quimicamente , Convulsões/genéticaRESUMO
The addition of poly-alpha2,8-N-acetylneuraminic acid (polysialic acid; PSA) to the neural cell adhesion molecule NCAM plays a crucial role in neural development [1-3], neural regeneration [4], and plastic processes in the vertebrate brain associated with neurite outgrowth [5], axonal pathfinding [6], and learning and memory [7,-9]. PSA levels are decreased in people affected by schizophrenia [10], and PSA has been identified as a specific marker for some neuroendocrine and lymphoblastoid tumours [11-13]; expression of PSA on the surface of these tumour cells modulates their metastatic potential [11-13]. Studies aimed at understanding PSA biosynthesis and the dynamics of its production have largely been promoted by the cloning of polysialyltransferases (PST-1 in hamster; PST in human and mouse) [14-16]. However, the number of enzymes involved in the biosynthesis of PSA has not been identified. Using incompletely glycosylated NCAM variants and soluble recombinant glycosyltransferases, we reconstituted the site at which PST-1 acts to polysialylate NCAM in vitro. The data presented here clearly demonstrate that polysialylation of NCAM is catalyzed by a single enzyme, PST-1, and that terminal sialylation of the N-glycan core is sufficient to generate the PSA acceptor site. Our results also show that PST-1 can act on core structures with the terminal sialic acid connected to galactose via an alpha2,3 or alpha2,6 linkage.
Assuntos
Moléculas de Adesão de Célula Nervosa/metabolismo , Ácidos Siálicos/metabolismo , Sialiltransferases/metabolismo , Animais , Linhagem Celular , Cricetinae , Humanos , Camundongos , Especificidade por SubstratoRESUMO
The neural cell adhesion molecule (NCAM) has been implicated in regulating synaptic plasticity mechanisms as well as memory consolidation processes. Attachment of polysialic acid to NCAM (PSA-NCAM) has been reported to down-regulate its adhesive forces, a process hypothesized to be implicated in synapse selection after learning experiences. PSA-NCAM has been critically implicated in hippocampus-related synaptic plasticity and memory storage, but information about its functional role in other brain areas remains scarce. Here, we studied mice deficient for polysialyltransferase-1 (ST8SialV/PST-1), an enzyme which attaches PSA to NCAM during postnatal development and adulthood, and whose deficiency results in a drastic reduction of PSA-NCAM expression throughout the brain in adulthood. Mice were tested for their performance in the water maze and auditory fear conditioning (AFC). We report that ST8SiaIV knockout mice were impaired in spatial as well as reversal learning in the water maze. On the other hand, AFC was intact and ST8SiaIV mice exhibited no impairments in the acquisition or retention of cued fear memories. Spatial orientation learning and reversal learning require complex integration of spatial information and response selection involving the hippocampus and prefrontal cortex, whereas cued fear conditioning is an associative type of emotional memory that highly depends on amygdala function. Therefore, our results indicate that PSA-NCAM contributes differentially to learning processes that differ in the nature of the neural computations involved, which probably reflects a differential role of this molecule in different brain regions.
Assuntos
Encéfalo/metabolismo , Deficiências da Aprendizagem/metabolismo , Transtornos da Memória/metabolismo , Molécula L1 de Adesão de Célula Nervosa/deficiência , Ácidos Siálicos/deficiência , Tonsila do Cerebelo/metabolismo , Tonsila do Cerebelo/fisiopatologia , Animais , Aprendizagem da Esquiva/fisiologia , Encéfalo/fisiopatologia , Condicionamento Psicológico/fisiologia , Medo/fisiologia , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Deficiências da Aprendizagem/genética , Deficiências da Aprendizagem/fisiopatologia , Aprendizagem em Labirinto/fisiologia , Transtornos da Memória/genética , Transtornos da Memória/fisiopatologia , Camundongos , Camundongos Knockout , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/fisiopatologia , Sialiltransferases/genéticaRESUMO
Although peripheral nerves exhibit regeneration capacities after transection injuries, the success of nerve repair depends crucially on the length of the gap. In addition to autologous nerve grafting as the conventional neurosurgical treatment to overcome long gaps, alternative strategies are needed. Numerous experimental studies have been undertaken to find the optimal material for production of artificial prostheses, which can be introduced as conduits between the nerve stumps. The current study follows the aim to establish polysialic acid (polySia), a homopolymer of alpha2,8-linked sialic acid residues, as a novel, biocompatible, and bioresorbable material for nerve tissue engineering. As a first step towards this goal, protocols for efficient coating of cell culture dishes with soluble polySia were established. In addition, primary nerve cells which are candidates for reconstructive therapies, including neonatal and adult Schwann cells, neural progenitor cells, spinal ganglionic neurons and motoneurons were cultured on polySia substrates. Cultures were evaluated with regard to cell survival and cell proliferation capacities. polySia turned out to be stable under cell culture conditions, and induced degradable and degradation products had no negative effects on cell cultures. Furthermore, polySia revealed its compatibility for several cell types derived from rat embryonic, postnatal and adult nervous tissue when used as a substrate.
Assuntos
Técnicas de Cultura de Células/métodos , Neurônios/citologia , Neurônios/fisiologia , Células de Schwann/citologia , Células de Schwann/fisiologia , Ácidos Siálicos/química , Engenharia Tecidual/métodos , Animais , Animais Recém-Nascidos , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Materiais Revestidos Biocompatíveis/química , Teste de Materiais , Ratos , Ratos Sprague-DawleyRESUMO
Polysialic acid is a unique cell surface polysaccharide found in the capsule of neuroinvasive bacteria and as a highly regulated post-translational modification of the neural cell adhesion molecule. Recent progress has been achieved in research on both the physicochemical properties of polysialic acid and the biosynthetic pathways leading to polysialic acid expression in bacteria and mammals.
Assuntos
Bactérias/metabolismo , Ácidos Siálicos/química , Ácidos Siálicos/metabolismo , Animais , Regulação da Expressão Gênica , Ácidos Siálicos/genéticaRESUMO
In human serum, at least two molecular species of the neural cell adhesion molecule (NCAM) with molecular weights of 110,000-130,000 and 150,000-180,000, respectively, can be identified by Western blotting. Both are characterized by the absence of epitopes for monoclonal antibodies KD11 and MG5, which specifically recognize intracellular domains of the human NCAM transmembrane isoforms, NCAM-140 and NCAM-180. In contrast to the M(r) 110,000-130,000 molecule also detectable in serum samples from healthy blood donors, the M(r) 150,000-180,000 molecule appears to be tumor associated. The only difference between these two species is shown to be the presence of long chains of alpha-(2,8)-linked N-acetylneuraminic acids, which are characteristic for the so-called embryonic NCAM form. After treatment with endoneuraminidase N, the M(r) 150,000-180,000 molecule can no longer be discriminated from the M(r) 110,000-130,000 molecule in Western blotting as well as gel and anion exchange chromatography experiments. The experimental data clearly show that only the embryonic NCAM molecule carrying the poly-alpha-(2,8)-linked N-acetylneuraminic acid moiety can be regarded as a specific serum marker for small cell lung cancer.
Assuntos
Carcinoma de Células Pequenas/sangue , Moléculas de Adesão Celular Neuronais/sangue , Neoplasias Pulmonares/sangue , Proteínas de Neoplasias/sangue , Western Blotting , Moléculas de Adesão Celular Neuronais/imunologia , Cromatografia/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos/análise , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Proteínas de Neoplasias/imunologiaRESUMO
Neuroblastomas and cell lines derived from these tumors bear the oncodevelopmental antigen polysialic acid (PSA) bound to the neural cell adhesion molecule. Polysialyation of neural cell adhesion molecule can be achieved by two different polysialyltransferases, ST8SiaII and ST8SiaIV. This study was undertaken to investigate the pattern of polysialyltransferases expressed in the human neuroblastoma cell line SH-SY5Y. Reverse transcription-PCR showed simultaneous expression of the two enzymes, and in situ hybridization demonstrated that the polysialyltransferase mRNA expression parallels immunoreactivity with the PSA-specific monoclonal antibody 735. After retinoic acid-induced differentiation, only the PSA-positive, neuron-like cell type gave clear signals for ST8SiaII and ST8SiaIV in in situ hybridization, whereas both signals were drastically reduced in the weakly PSA-positive substrate adherent phenotype. Like the SH-SY5Y cells, a primary, PSA-positive neuroblastoma specimen revealed expression of the two polysialyltransferases. To investigate the role of PSA for cell growth and differentiation, SH-SY5Y cells were treated with the PSA-specific endo-N-acetylneuraminidase E. Although loss of PSA was accompanied with a marked reduction of cell growth, it did not interfere with retinoic acid-induced differentiation. Together, our results suggest that PSA surface expression is regulated on the level of polysialyltransferase transcription. Moreover, the similarity to the primary neuroblastoma tissue makes SH-SY5Y cells a suitable model system to examine further the role of polysialylation in tumor cell growth and the orchestration of PSA synthesis in neuroblastoma.
Assuntos
Neuroblastoma/metabolismo , Ácidos Siálicos/fisiologia , Sialiltransferases/metabolismo , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Humanos , Células Tumorais CultivadasRESUMO
The neural cell adhesion molecule (NCAM) is a glycoprotein implicated in cell-cell adhesion, neurite outgrowth and synaptic plasticity. Polysialic acid (polySia) is mainly attached to NCAM (polySia-NCAM) and has an essential role in regulating NCAM-dependent developmental processes that require plasticity, that is, cell migration, axon guidance and synapse formation. Post-mortem and genetic evidence suggests that dysregulation of polySia-NCAM is involved in schizophrenia (SZ). We enrolled 45 patients diagnosed with SZ and 45 healthy individuals who were submitted to polySia-NCAM peripheral quantification, cognitive and psychopathological assessment and structural neuroimaging (brain volumes and diffusion tensor imaging). PolySia-NCAM serum levels were increased in SZ patients, independently of antipsychotic treatment, and were associated with negative symptoms, blunted affect and declarative memory impairment. The increased polySia-NCAM levels were associated with decreased volume in the left prefrontal cortex, namely Brodmann area 46, in patients and increased volume in the same brain area of healthy individuals. As this brain region is involved in the pathophysiology of SZ and its associated phenomenology, the data indicate that polySia-NCAM deserves further scrutiny because of its possible role in early neurodevelopmental mechanisms of the disorder.
Assuntos
Encéfalo/patologia , Transtornos Cognitivos/complicações , Moléculas de Adesão de Célula Nervosa/sangue , Esquizofrenia/complicações , Ácidos Siálicos/sangue , Adulto , Mapeamento Encefálico , Movimento Celular/genética , Transtornos Cognitivos/sangue , Transtornos Cognitivos/genética , Imagem de Tensor de Difusão , Feminino , Humanos , Masculino , Moléculas de Adesão de Célula Nervosa/genética , Plasticidade Neuronal/genética , Tamanho do Órgão , Esquizofrenia/sangue , Esquizofrenia/genética , Ácidos Siálicos/genéticaRESUMO
Polysialic acid (PSA) is a developmentally regulated carbohydrate consisting of alpha-2,8-linked sialic acid residues attached to the neural cell adhesion molecule NCAM. PSA promotes plasticity of cell-cell interactions in the nervous system and appears linked to the malignant potential of several tumors. Two enzymes, the polysialyltransferases ST8SiaII (STX) and ST8SiaIV (PST) have been identified and shown to be independently able to synthesize PSA. However, in vivo studies have demonstrated that in the majority of PSA-positive tissues the two polysialyltransferases are expressed simultaneously. Therefore, this study was undertaken to elucidate in which way the individual enzymes contribute to PSA expression under in vivo conditions. Using a semiquantitative RT-PCR strategy PSA-positive human tumor cell lines were screened for expression of ST8SiaII and ST8SiaIV at the mRNA level. Divergent patterns observed in some cell lines suggest that polysialyltransferases are independently regulated at the transcriptional level. In subsequent analyses the different mRNA levels of ST8SiaII and ST8SiaIV in these tumor cells were correlated with the degree of PSA expression and the cellular capacity to rapidly synthesize PSA. Our data indicate that ST8SiaIV is the major regulator of NCAM polysialylation in vivo.
Assuntos
Moléculas de Adesão de Célula Nervosa/metabolismo , Sialiltransferases/metabolismo , Northern Blotting , Western Blotting , Ensaio de Imunoadsorção Enzimática , Regulação Enzimológica da Expressão Gênica , Humanos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ácidos Siálicos/biossíntese , Sialiltransferases/biossíntese , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Antibodies specific to the neural cell adhesion molecule (NCAM-total), the 180 x 10(3) M(r) component of NCAM (NCAM-180) and polysialic acid (PSA) were used in immunohistochemistry and Western blots to detect the spatiotemporal dynamics of these molecules in development and regeneration of the retinotectal system of Pleurodeles waltl. NCAM-total and NCAM-180 are continuously expressed in the retina, optic nerve, and tectum of the developing and adult salamander. This is also found for the 140 x 10(3) M(r) component of NCAM in Western blots of the retina. In the larval retina, PSA is present in the inner plexiform layer (IPL) and a few cells in all nuclear layers. At metamorphosis, PSA expression in the retina strongly increases in the layer of cone photoreceptor somata. Several cells in the inner nuclear layer and Müller cell processes also begin to express PSA. This pattern persists into adulthood. The optic nerve and the tectum are strongly PSA-immunoreactive throughout development. In the adult optic nerve and optic fiber pathway in the brain, PSA expression is selectively downregulated. In the crush-lesioned adult optic nerve, regenerating fibers are NCAM-180-positive but PSA-negative. This demonstrates a molecular difference between growing nerve fibers of Pleurodeles in development and in regeneration. PSA regulation is closely correlated with metamorphosis, thus suggesting that PSA expression may be under hormonal control. Some aspects of PSA and NCAM isoform expression patterns in the retinotectal system of salamanders differ considerably from that of other vertebrates. The sustained expression of NCAM isoforms in adult salamanders might be due to secondary simplification (paedomorphosis).
Assuntos
Moléculas de Adesão Celular Neuronais/biossíntese , Regeneração Nervosa/fisiologia , Pleurodeles/fisiologia , Retina/fisiologia , Ácidos Siálicos/biossíntese , Colículos Superiores/fisiologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Western Blotting , Imuno-Histoquímica , Larva , Metamorfose Biológica/fisiologia , Compressão Nervosa , Nervo Óptico/crescimento & desenvolvimento , Nervo Óptico/metabolismo , Nervo Óptico/fisiologia , Retina/crescimento & desenvolvimento , Retina/metabolismo , Células Ganglionares da Retina/metabolismo , Colículos Superiores/crescimento & desenvolvimento , Colículos Superiores/metabolismoRESUMO
The Golgi apparatus serves as the major site of glycosylation reactions. Nucleotide sugars which are substrates of the Golgi localized glycosyltransferases are synthesized in the cytoplasm (cell nucleus in case of CMP-sialic acid) and must be transported into the compartment lumen. This transport function is carried out by nucleotide sugar transporters. The first genes were cloned in the year 1996 and revealed a family of structurally conserved multi-transmembrane-spanning proteins. Due to the high structural and functional conservation, the identification of many putative nucleotide sugar transporter sequences has become possible in the existing gene data bases and accelerates the increase in knowledge on structure-function-relationships. Recent developments in the nucleotide sugar transporter field are discussed in this article.
Assuntos
Proteínas de Transporte/fisiologia , Proteínas de Membrana/fisiologia , Ribose/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/classificação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Clonagem Molecular , Humanos , Síndrome da Aderência Leucocítica Deficitária/fisiopatologia , Proteínas de Membrana/classificação , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Relação Estrutura-AtividadeRESUMO
The biological activity of oligopeptide analogues of C3a is markedly increased by N-terminal attachment of a hydrophobic group as, for instance, 9-fluorenylmethoxycarbonyl (Fmoc), either direct or via a flexible 6-aminohexanoyl (Ahx) spacer. This study presents evidence from fluorescence anisotropy decay measurements that the hydrophobic appendix mediates non-specific binding of the synthetic peptide analogues to phospholipid vesicles. According to quantitative considerations no alternative or additional rate-enhancing mechanisms other than surface diffusion are required to account for the gain in biopotency.
Assuntos
Complemento C3a/análogos & derivados , Complemento C3a/metabolismo , Sequência de Aminoácidos , Polarização de Fluorescência/métodos , Cinética , Matemática , Membranas/metabolismo , Modelos Teóricos , Dados de Sequência Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/metabolismo , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
BACKGROUND/PURPOSE: Some malignant pediatric tumors express the polysialylated form of the neural cell adhesion molecule (PSA-NCAM), which normally becomes restricted to a few regions of neural plasticity and regenerating nerve tissue after embryogenesis. Recently, serum concentrations of polysialylated NCAM have been shown to be tumor associated in children with rhabdomyosarcoma and neuroblastoma. This study was undertaken to evaluate polysialylated NCAM as a marker helpful in distinguishing between various embryonal tumors and other lesions suspicious of a malignancy. METHODS: Fresh frozen specimens from exemplary tumors of the thorax, adrenal glands, and kidneys of 17 children were investigated immunohistochemically by the Alkaline Phosphatase anti-Alkaline Phosphatase (APAAP) technique. Simultaneously, the patients' serum was investigated by a chemiluminescent immunoassay, which likewise used the polysialic acid-specific monoclonal antibody 735. RESULTS: Serum concentrations correlated with the expression of PSA-NCAM in immunohistochemistry. They were elevated in children with PSA-NCAM-positive tumors as alveolar rhabdomyosarcoma, undifferentiated neuroblastoma, and anaplastic Wilms tumor, but negative in all other patients. CONCLUSION: PSA-NCAM serves as a useful marker for differential diagnosis during workup of tumors suspicious of a malignant neoplasm.
Assuntos
Neoplasias das Glândulas Suprarrenais/diagnóstico , Biomarcadores Tumorais/metabolismo , Neoplasias Renais/diagnóstico , Molécula L1 de Adesão de Célula Nervosa , Moléculas de Adesão de Célula Nervosa/metabolismo , Rabdomiossarcoma/diagnóstico , Ácidos Siálicos/metabolismo , Neoplasias Torácicas/diagnóstico , Adolescente , Neoplasias das Glândulas Suprarrenais/metabolismo , Biomarcadores Tumorais/sangue , Criança , Pré-Escolar , Diagnóstico Diferencial , Feminino , Humanos , Imuno-Histoquímica , Lactente , Neoplasias Renais/metabolismo , Medições Luminescentes , Masculino , Moléculas de Adesão de Célula Nervosa/sangue , Neuroblastoma/diagnóstico , Neuroblastoma/metabolismo , Derrame Pleural Maligno/diagnóstico , Derrame Pleural Maligno/metabolismo , Rabdomiossarcoma/metabolismo , Ácidos Siálicos/sangue , Neoplasias Torácicas/metabolismoRESUMO
Chinese hamster ovary (CHO) glycosylation mutants of the Lec2 complementation group are unable to express sialylated glycoproteins and glycolipids due to a defect in the Golgi CMP-sialic acid transporter (CMP-Sia-Tr). Using an expression cloning strategy, we isolated a cDNA encoding the hamster CMP-Sia-Tr which complements the Lec2 phenotype. The deduced amino acid sequence of the cloned cDNA shows 95% identity to the recently cloned murine CMP-Sia-Tr. The expression of a hamster CMP-Sia-Tr fusion protein with an N-terminal MDYKDDDDK (FLAG) sequence revealed Golgi localisation of the transporter. Amino acid sequence comparison revealed strong similarity (44.6% identity and 19.3% similarity) of CMP-Sia-Tr to the recently cloned human UDP-galactose transporter (UDP-Gal-Tr). In contrast, sequence similarities to the yeast UDP-N-acetylglucosamine transporter (UDP-GlcNAc-Tr) and the GDP-mannose transporter (GDP-Man-Tr) of Leishmania donovani are restricted to a region encoding the two most C-terminally located transmembrane helices. A computer-based structural analysis of CMP-Sia-Tr proposes an eight transmembrane helix model with the N- and C-termini located on the cytosolic side of the Golgi membrane.
Assuntos
Proteínas de Transporte/genética , Proteínas de Membrana/genética , Proteínas de Transporte de Nucleotídeos , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Clonagem Molecular , Cricetinae , Monofosfato de Citidina/metabolismo , Primers do DNA/genética , Epitopos/genética , Glicosilação , Complexo de Golgi/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Estrutura Molecular , Mutação , Ácido N-Acetilneuramínico/metabolismo , Reação em Cadeia da Polimerase , Estrutura Secundária de Proteína , Homologia de Sequência de AminoácidosRESUMO
Monoclonal antibodies (MAbs) ranked together as small-cell-lung-cancer (SCLC) Cluster I MAbs are directed against the neural cell adhesion molecule NCAM (CD 56) and have been shown to be useful reagents in SCLC diagnosis and therapy. We analyzed the epitopes recognized by 5 SCLC cluster-I MAbs (123C3, 123A8, ERIC-I, MB2, and Leu 19) and a closely related anti CD 56 MAb (T199). Our results show that within the NCAM molecule Ig-like domain 3 and the segment of about 200 amino acids comprised by exons 11-13 are immunodominant regions. The MAbs investigated in this study can be combined into 2 groups. Group 1 consists of MAbs MB2, Leu 19 and T199, which are directed against epitopes located in the 3rd Ig-like domain. These MAbs recognize closely related but distinctive conformational epitopes. MAbs ERIC-1, 123C3 and 123A8 form Group 2 and are directed against a membrane-proximal region of the NCAM molecule. The data presented suggest that the 3 Group-2 MAbs bind to closely related or identical epitopes.
Assuntos
Anticorpos Monoclonais , Carcinoma de Células Pequenas/terapia , Moléculas de Adesão Celular Neuronais/imunologia , Epitopos/análise , Neoplasias Pulmonares/terapia , Adulto , Anticorpos Monoclonais/uso terapêutico , Sequência de Bases , Western Blotting , Carcinoma de Células Pequenas/diagnóstico , Moléculas de Adesão Celular Neuronais/análise , Moléculas de Adesão Celular Neuronais/biossíntese , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Feto , Humanos , Neoplasias Pulmonares/diagnóstico , Dados de Sequência Molecular , Peso Molecular , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/imunologiaRESUMO
Polysialic acid (PSA) is an important regulator of cellular interactions. Two enzymes (ST8SiaII and ST8SiaIV) are capable of synthesizing PSA. In the present study, the gene encoding the murine ST8SiaIV (PST-1) has been isolated and characterized. In contrast to the ST8SiaII (STX) gene which contains six exons and spans about 80 kb, the ST8SiaIV gene comprises only five exons spanning over at least 55 kb. However, alignment of the two genes revealed that exon-intron boundaries of exons 2-5 of ST8SiaIV and exons 3-6 of ST8SiaII are located at identical sites. Differences are restricted to the 5'-region encoded by one exon in the case of ST8SiaIV, whereas the corresponding region of ST8SiaII is interrupted by a very long intron. 5'-RACE analysis of the ST8SiaIV transcript using mRNA from AtT20 cells identified two transcription start sites at positions -324 and -204 relative to the translation start codon. The promoter region of ST8SiaIV lacks TATA- and CAAT-like sequences and is enriched in G+C (60%). The promoter contains putative Sp1, AP-1, AP-2, and PEA3 binding sites, as well as a purine- and a pyrimidine-rich region. Luciferase reporter gene assays demonstrated that the region between nucleotides -443 and -162 is sufficient to direct gene expression. The induction of luciferase activity was 30- and 10-fold in the PSA-positive AtT20 and CHO cells, but only 5- and 7-fold in the PSA-negative NIH-3T3 cells and in a PSA-negative subline of AtT20. Thus, although decreased in activity in PSA-negative cell lines, the basal promoter is not sufficient for the strong cell-type and tissue specific regulation of the ST8SiaIV gene, suggesting regulatory elements in the more upstream 5'-region.
Assuntos
Sialiltransferases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Éxons/genética , Genes Reporter/genética , Íntrons/genética , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , Sequências Reguladoras de Ácido Nucleico/genética , Análise de Sequência de DNA , Transcrição Gênica/genéticaRESUMO
Nucleotide-sugar transporters (NSTs) are critical components of glycosylation pathways in eukaryotes. The identification of structural elements that are involved in NST functions provides an important task. Chinese hamster ovary glycosylation mutants defective in nucleotide-sugar transport provide access to inactive transporters that can define such structure/function relationships. In this study, we have cloned the hamster UDP-galactose transporter (UGT) and identified defects in UGT gene transcripts from nine independent Chinese hamster ovary mutants that belong to the Lec8 complementation group. Reverse transcription polymerase chain reaction with primers that span the UGT open reading frame showed that three Lec8 mutants express a full-length open reading frame, while six Lec8 mutants predominantly express truncated UGT gene transcripts. Sequencing identified different single or triplet nucleotide changes in full-length UGT transcripts from three of the mutants. These mutations translate into three different amino acid changes at positions that are highly conserved in all the known mammalian NSTs. Transfection of a cDNA encoding either of the mutations Delta serine 213 or G281D failed to correct the UDP-galactose transport defect in Lec8 transfectants. Most importantly, introducing these same mutations into the homologous region of the murine CMP-sialic acid transporter caused inactivation of this transporter. Thus, identifying point mutations that inactivate UGT in Lec8 mutants resulted in the discovery of amino acids that are critical to the activity of both UGT and CST, the two most divergent mammalian NSTs.
Assuntos
Proteínas de Transporte/genética , Proteínas de Membrana/genética , Proteínas de Transporte de Monossacarídeos/genética , Sequência de Aminoácidos , Animais , Células CHO , Proteínas de Transporte/metabolismo , Cricetinae , Regulação da Expressão Gênica , Glicosilação , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Proteínas de Transporte de Monossacarídeos/metabolismo , Mutação Puntual , Alinhamento de SequênciaRESUMO
Translocation of nucleotide sugars across the membrane of the Golgi apparatus is a prerequisite for the synthesis of complex carbohydrate structures. While specific transport systems for different nucleotide sugars have been identified biochemically in isolated microsomes and Golgi vesicles, none of these transport proteins has been characterized at the molecular level. Chinese hamster ovary (CHO) mutants of the complementation group Lec2 exhibit a strong reduction in sialylation of glycoproteins and glycolipids due to a defect in the CMP-sialic acid transport system. By complementation cloning in the mutant 6B2, belonging to the Lec2 complementation group, we were able to isolate a cDNA encoding the putative murine Golgi CMP-sialic acid transporter. The cloned cDNA encodes a highly hydrophobic, multiple membrane spanning protein of 36.4 kDa, with structural similarity to the recently cloned ammonium transporters. Transfection of a hemagglutinin-tagged fusion protein into the mutant 6B2 led to Golgi localization of the hemagglutinin epitope. Our results, together with the observation that the cloned gene shares structural similarities to other recently cloned transporter proteins, strongly suggest that the isolated cDNA encodes the CMP-sialic acid transporter.
Assuntos
Proteínas de Transporte/biossíntese , Ácido N-Acetilneuramínico do Monofosfato de Citidina/metabolismo , Glicolipídeos/biossíntese , Glicoproteínas/biossíntese , Proteínas de Membrana/biossíntese , Proteínas de Transporte de Nucleotídeos , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Clonagem Molecular , Cricetinae , Teste de Complementação Genética , Complexo de Golgi/metabolismo , Hemaglutininas/biossíntese , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Peso Molecular , Proteínas Recombinantes de Fusão/biossínteseRESUMO
Chinese hamster ovary (CHO) mutants belonging to the Lec2 complementation group are unable to translocate CMP-sialic acid to the lumen of the Golgi apparatus. Complementation cloning in these cells has recently been used to isolate cDNAs encoding the CMP-sialic acid transporter from mouse and hamster. The present study was carried out to determine the molecular defects leading to the inactivation of CMP-sialic acid transport. To this end, CMP-sialic acid transporter cDNAs derived from five independent clones of the Lec2 complementation group, were analyzed. Deletions in the coding region were observed for three clones, and single mutants were found to contain an insertion and a point mutation. Epitope-tagged variants of the wild-type transporter protein and of the mutants were used to investigate the effect of the structural changes on the expression and subcellular targeting of the transporter proteins. Mutants derived from deletions showed reduced protein expression and in immunofluorescence showed a diffuse staining throughout the cytoplasm in transiently transfected cells, while the translation product derived from the point-mutated cDNA (G189E) was expressed at the level of the wild-type transporter and co-localized with the Golgi marker alpha-mannosidase II. This mutation therefore seems to directly affect the transport activity. Site-directed mutagenesis was used to change glycine 189 into alanine, glutamine, and isoleucine, respectively. While the G189A mutant was able to complement CMP-sialic acid transport-deficient Chinese hamster ovary mutants, the exchange of glycine 189 into glutamine or isoleucine dramatically affected the transport activity of the CMP-sialic acid transporter.