RESUMO
AIMS: In previous studies, the present group showed that a factor, present in the cerebrospinal fluid of seven neuro-patients, was capable of inducing cell damages on cell cultures of epithelial cells (Vero), glial cells (DG54-MG) and human primary lymphocytes. The cytotoxicity, once induced, could be transmitted to fresh cell cultures using crude preparations obtained from the cytotoxic cell cultures. METHODS AND RESULTS: The present electron microscope study describes in detail the pathological changes occurring in the previously assayed cultured cell types, and for the first time in human fibroblasts, as a consequence of the treatment with crude cytotoxic preparation. It also demonstrates with reasonable certainty the absence of virus-like particles in both the treated cells and the partially purified cytotoxic fraction. Indeed high-resolution electron microscopy analysis shows that this fraction consists almost entirely of protein spheroids with a diameter of 8-12 nm. CONCLUSIONS: The possible significance of nanotubular aggregates, observed in particular in the glial and in the epithelial cells undergoing cytotoxic damage, is also discussed.
Assuntos
Citotoxinas/líquido cefalorraquidiano , Células Epiteliais/ultraestrutura , Fibroblastos/ultraestrutura , Linfócitos/ultraestrutura , Microscopia Eletrônica , Doenças do Sistema Nervoso/líquido cefalorraquidiano , Neuroglia/ultraestrutura , Animais , Chlorocebus aethiops , Citotoxinas/química , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Humanos , Linfócitos/metabolismo , Estrutura Molecular , Neuroglia/metabolismo , Tamanho da Partícula , Relação Estrutura-Atividade , Células VeroRESUMO
Human Tamm-Horsfall glycoprotein (THP) is synthesised in the thick ascending limb of Henle and convoluted distal tubules, inserted into luminal cell-surface by the glycosyl-phosphatidylinositol (GPI)-anchor and excreted in urine at a rate of 50-100 mg per day. Up to date there is no indication on the way in which THP is excreted into the urinary fluid. In this study, we examined by Western blotting THP from human kidney in comparison to urinary THP. As expected for a GPI-anchored protein, THP was recovered from the kidney lysate in a Triton X-100 insoluble form, which moved in a sucrose gradient to a zone of low density. The apparent molecular weight of kidney THP appeared greater than that of urinary THP, but no difference in the electrophoretic mobility was observed when the former was subjected to GPI-specific phospholipase-C treatment, strongly suggesting that a proteolytic cleavage at the juxtamembrane-ectodomain of kidney THP is responsible for the urinary excretion.
Assuntos
Glicosilfosfatidilinositóis/metabolismo , Rim/metabolismo , Mucoproteínas/metabolismo , Western Blotting , Humanos , Córtex Renal/metabolismo , Medula Renal/metabolismo , Mucoproteínas/isolamento & purificação , Mucoproteínas/urina , Fosfolipases Tipo C/metabolismo , UromodulinaRESUMO
Uroplakin III (UPIII) is one of the major transmembrane glycoproteins exposed at the luminal face of mammalian bladder. We investigated the terminal glycosylation of bovine UPIII in order to ascertain whether it contains the alpha 2,3-sialylated sequence thus potentially serving as a receptor for uropathogenic Escherichia coli expressing type S adhesins. We report the occurrence of sialic acid in alpha 2,3- and alpha 2,6-linkage to galactose in bovine UPIII glycans as evidenced by the sensitivity of UPIII to both Vibrio cholera and Newcastle disease virus neuraminidase and by the colocalization of UPIII antigen and material detected by lectins of Sambucus nigra and Maackia amurensis on the luminal face of the bladder. We also present evidence that UPIII glycans are capped by Gal-alpha 1,3-Gal epitope. Consistently, alpha 2,3- and alpha 2, 6-sialyltransferase, as well as alpha 1,3-galactosyltransferase were found to be present in the cells detached from the luminal side of bovine bladder, which are responsible for the UPIII biosynthesis. The putative role of UPIII sialylated glycans in enhancing the uropathogenicity of E. coli expressing type S adhesins is discussed.
Assuntos
Glicoproteínas de Membrana/metabolismo , Bexiga Urinária/metabolismo , Adesinas de Escherichia coli/metabolismo , Animais , Bovinos , Escherichia coli/metabolismo , Escherichia coli/patogenicidade , Galactose/análise , Galactosiltransferases/análise , Glicosilação , Imuno-Histoquímica , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/química , Ácido N-Acetilneuramínico/análise , Neuraminidase , Polissacarídeos/química , Sialiltransferases/análise , Bexiga Urinária/citologia , Bexiga Urinária/enzimologia , Uroplaquina IIIRESUMO
Hepatitis C virus glycoproteins E1 and E2 do not reach the plasma membrane of the cell but accumulate intracellularly, mostly in the endoplasmic reticulum. Previous studies based on transient expression assays have shown that the transmembrane domains of both glycoproteins are sufficient to localize reporter proteins in the endoplasmic reticulum and that other localization signals may be contained in the ectodomain of E1 protein. To identify such signals we generated chimeric proteins between E1 and two reporter proteins, the human CD8 glycoprotein and the human alkaline phosphatase, and analyzed their subcellular localization in stable as well as transient transfectants. Our results showed that (i) an independent localization determinant for the endoplasmic reticulum is present in the juxtamembrane region of the ectodomain of E1 protein and (ii) the localization dictated by this determinant is either due to direct retention or to a recycling mechanism from the intermediate compartment/cis-Golgi complex region, which is clearly different from those previously described for other retrieval signals. These results show for the first time in mammalian cells that the localization in the endoplasmic reticulum of transmembrane protein can be determined by specific targeting signals acting in the lumen of the compartment.
Assuntos
Retículo Endoplasmático/metabolismo , Sinais Direcionadores de Proteínas , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Antígenos CD8/genética , Antígenos CD8/metabolismo , Compartimento Celular , Técnica Indireta de Fluorescência para Anticorpo , Genes Reporter , Glicosilação , Hexosaminidases/metabolismo , Humanos , Manose , Dados de Sequência Molecular , Neuraminidase/metabolismo , Oligossacarídeos , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Proteínas do Envelope Viral/genéticaRESUMO
gp190 is a glycoprotein expressed on the cell surface of several human colon carcinoma cells in culture, on epithelial cells of fetal colon, but not on the normal mucosa of adult colon; thus it is referred to as an oncofetal crypt cell antigen. We report the characterisation of O-linked glycans carried by gp190 synthesised by [3H]glucosamine-labelled Caco-2 cells at the confluence (undifferentiated cells) and at three weeks of postconfluence (differentiated cells). By using a specific monoclonal antibody, gp190 was isolated and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The mobility of gp190 from differentiated cells was found to be lower than that from undifferentiated cells, suggesting a more extensive glycosylation process in the former glycoprotein. The major results of the glycan characterisation have been as follows: (i) gp190 carries mainly, if not exclusively, O-linked glycans with the core-2 structure; (ii) the elongation with N-acetyllactosamine units of the Gal beta1,4GlcNAc beta1,6(Gal beta1,3)GalNAc tetrasaccharide predominates in gp190 synthesised by differentiated cells, whereas the direct alpha2,3sialylation of the tetrasaccharide is prevalent in gp190 synthesised by undifferentiated cells. The increment in the core-2 beta1,6GlcNAc-transferase activity under the Caco-2 differentiation process may be relevant in producing the larger occurrence of polylactosaminoglycans in gp190 from differentiated cells. Since no change in the activity of the alpha2,3sialyltransferases upon cell differentiation was observed, we suggest that the lower alpha2,3sialylation in gp190 synthesised by polarised cells might be due to a changed transit-rate through the distal Golgi apparatus.
Assuntos
Antígenos de Neoplasias/metabolismo , Amino Açúcares/análise , Amino Açúcares/química , Amino Açúcares/metabolismo , Antígenos de Neoplasias/isolamento & purificação , Células CACO-2 , Configuração de Carboidratos , Sequência de Carboidratos , Diferenciação Celular , Fracionamento Químico , Glicosilação , Glicosiltransferases/metabolismo , Humanos , Dados de Sequência Molecular , Polissacarídeos/análise , Polissacarídeos/química , Ácidos Siálicos/químicaRESUMO
BACKGROUND: Human Tamm-Horsfall glycoprotein (T-H) is a glycosylphosphatidylinositol-anchored protein exposed at the surface of distal nephron cells, and urinary T-H is the released soluble counterpart. The latter has been implicated in tubulointerstitial nephritis, and the proinflammatory potential has been related to its ability to bind in vitro human neutrophils (PMNs). We have examined the conditions required for the binding of neutrophils to cell-surface anchored T-H and the consequent effects. METHODS: A HeLa cell-line derivative permanently transformed with human T-H cDNA and expressing T-H at the cell surface was used throughout the study. The adhesion of PMNs to cells expressing T-H was analyzed by immunofluorescence microscopy before and after the opsonization of cells with anti-T-H antibodies. The oxidative burst induced by adhesion of PMNs to the cells was determined by the activation of myeloperoxidase. Quantitative and qualitative changes in the release of T-H under the adhesion of activated PMNs were determined by dot-blot and Western blot analysis. RESULTS: No binding of neutrophils to cell-surface-anchored T-H was observed. On the contrary, the opsonization of cells with anti-T-H antibodies resulted in a dramatic adhesion of neutrophils. Such an adhesion induced the oxidative burst of PMNs and a large increment in the release of T-H, as well as the release of the slightly faster migrating T-H form, which is normally retained intracellularly. CONCLUSIONS: These results support the notion that, after the autoimmune response, the adhesion of neutrophils to cell-surface T-H contributes to the pathogenesis of tubulointerstitial nephritis, favoring a further accumulation of T-H in the interstitium and inducing the loss of cell integrity via reactive oxygen metabolites generated by activated neutrophils.
Assuntos
Adjuvantes Imunológicos/metabolismo , Mucoproteínas/urina , Nefrite Intersticial/metabolismo , Neutrófilos/metabolismo , Autoanticorpos/imunologia , Ligação Competitiva/imunologia , Western Blotting , Adesão Celular/efeitos dos fármacos , Adesão Celular/imunologia , Imunofluorescência , Expressão Gênica/imunologia , Glicosilação , Células HeLa , Humanos , Manose/metabolismo , Manose/farmacologia , Mucoproteínas/genética , Mucoproteínas/imunologia , Ácido N-Acetilneuramínico/metabolismo , Nefrite Intersticial/imunologia , Ativação de Neutrófilo/imunologia , Neutrófilos/imunologia , Transfecção , Trítio , Uromodulina , Aglutininas do Germe de Trigo/metabolismo , Aglutininas do Germe de Trigo/farmacologiaRESUMO
IgM are glycoproteins secreted by plasma cells as (mu2L2)5+J or (mu2L2)6 polymers. In most species, mu- and J-chains bear five and one N -glycans, respectively. Here we compare the terminal glycosylation patterns of 4-hydroxy-3-nitrophenylacetyl (NP)-specific IgM secreted by transfectants of the J558L mouse myeloma deficient in the alpha2,6 sialyltransferase [alpha2,6ST(N)] or by a hybridoma expressing this enzyme (B1.8 cells). The absence of alpha2,6-sialylation results in an increased addition of alpha1, 3-galactosyl residues to mu- and J-chain N-glycans. Since alpha1, 3-galactosyltransferase (alpha1,3Gal-T) is similarly expressed in the two cell lines, these results indicate that a competition reaction occurs in vivo between alpha2,6ST(N) and alpha1,3Gal-T. In the alpha2,6ST(N) deficient transfectants, mu-chains lacking the C-subterminal Cys575 residue, which are secreted mainly in the form of mu2L2 monomers, are more efficiently capped by alpha1, 3-galactosyl residues, confirming that polymerization significantly reduces the accessibility of mu-chain glycans to the Golgi processing enzymes involved in the biogenesis of antennary sugars. Functional assays indicate that IgM sialylation affects antigen-binding and complement-dependent hemolysis of haptenated red blood cells.
Assuntos
Dissacarídeos/metabolismo , Epitopos/metabolismo , Mieloma Múltiplo/metabolismo , Animais , Biopolímeros , Proteínas do Sistema Complemento/metabolismo , Glicosiltransferases/metabolismo , Imunoglobulina M/metabolismo , Camundongos , Mieloma Múltiplo/patologia , Polissacarídeos/metabolismo , Células Tumorais CultivadasRESUMO
Human Tamm-Horsfall glycoprotein (T-H), first described as the major urinary glycoprotein, is a glycosylphosphatidyl-inositol (GPI)-anchored membrane protein which mainly resides at the luminal face of cells of the thick ascending limb of Henle's loop (TAL) and early distal convoluted tubules of nephron. Since no human renal cell-line producing T-H is available, T-H cDNA was transfected in HeLa cells and a cell line was selected in which 95% of the cells stably expressed T-H, in order to elucidate the biosynthesis, mechanisms regulating the transport of T-H along the exocytic pathway, exposure at the cell surface and release in soluble form. Treatment of cells with an exogenous reducing agent results in a drastic delay in the conversion from precursor to mature T-H. Since the accumulating T-H-precursor carries glycans not yet processed by Golgi-mannosidases, we propose that the formation of a correct set of intrachain disulphide bonds is required for T-H exit out the endoplasmic reticulum. Even the treatment of cells with an inhibitor of GPI-anchor biosynthesis results in an intracellular accumulation of T-H precursor, loss of T-H localization into Golgi apparatus and reduced surface exposure. These results indicate that the GPI-anchor addition is necessary for T-H delivery to the cell-surface. The release rate of new synthesized T-H shows an initial lag time very likely depending on the time required for T-H surface exposure. A portion of released T-H appears to contain ethanolamine, a component of GPI anchor, indicating that, at least in HeLa cells, a GPI-specific phospholipase contributes to the T-H release. Exposure of cells to monensin and brefeldin A results in a loss of accumulation of T-H in the Golgi perinuclear region and a reduced delivery to the cell surface. Under monensin treatment an intermediate T-H form non-exposed at the cell surface is released in the medium, indicating that a soluble T-H may be produced inside the cell under conditions that alter the Golgi apparatus. If such an event occurs in polarized kidney cells, a T-H release from the basolateral face may be postulated, inasmuch as the GPI-anchor is an apical sorting signal. Since T-H is a powerful autoantigen, the accumulation of soluble T-H in the interstitium of TAL may cause the formation of immunocomplexes.
Assuntos
Mucoproteínas/farmacocinética , Transporte Biológico , Brefeldina A , Ciclopentanos/farmacologia , Células HeLa , Hexosaminas/farmacologia , Humanos , Mercaptoetanol/farmacologia , Monensin/farmacologia , Mucoproteínas/química , Proteínas Recombinantes/farmacocinética , UromodulinaRESUMO
We have previously documented a dramatic elevation in the activity of alpha 2,6-sialytransferase towards Gal beta 1,4GlcNAc (EC 2.4.99.1) (alpha 2,6ST) in CaCo-2 cells maintained in culture for several days after confluence to elicit a high degree of enterocytic differentiation phenotype. Northern analysis performed with a probe complementary to a region of human alpha 2,6ST mRNA common to all known transcripts demonstrated that the expression of alpha 2,6ST mRNA in CaCo-2 cells increased with the degree with the degree of differentiation. When probes complementary to 5'-untranslated exons (Y + Z or X) previously identified in transcripts isolated from human placenta and from several human lymphoblastoid cell lines were used, no hybridization signal with mRNA of CaCo-2 cells was found, as reported for the mRNA of hepatoma cell line HepG2 (Wang XC, Vertino A, Eddy RL, Byers MG, Jani-Sait SN, Shows TB, Lau JTY (1993) J Biol Chem 268: 4355-61). These results support the notion that the major alpha 2,6ST transcript of CaCo-2 cells was the hepatoma isoform or a new one, so far unreported. Consistent with the differentiation-dependent increase in alpha 2,6ST-mRNA expression, an elevation of the reactivity with Sambucus nigra agglutinin of differentiated CaCo-2 cell-surface was observed, indicating an enhanced alpha 2,6-sialylation of membrane glycoconjugates.
Assuntos
Células CACO-2/enzimologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sialiltransferases/genética , Sequência de Bases , Sequência de Carboidratos , Diferenciação Celular , Primers do DNA/genética , Expressão Gênica , Glicoconjugados/química , Glicoconjugados/metabolismo , Humanos , Cinética , Dados de Sequência Molecular , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Sialiltransferases/metabolismo , Especificidade por Substrato , beta-D-Galactosídeo alfa 2-6-SialiltransferaseRESUMO
In mammalian cells, resident luminal and type I transmembrane proteins of the endoplasmic reticulum usually contain KDEL and KKXX at the carboxyl terminus. These sequences induce retrieval from compartments located downstream in the secretory pathway. It has been suggested that the retrieval may occur from multiple sites, ranging from the intermediate compartment to the trans-Golgi network. To compare the retrieval of luminal and type I membrane proteins, we have used different forms of a single reporter, the human CD8 glycoprotein, stably expressed in FRT cells. Metabolic labeling and oligosaccharide analysis show that the mechanism based on the KDEL signal is leaky. With time, the KDEL-containing CD8 form reaches the trans/trans-Golgi network compartments, where the protein is terminally glycosylated. At this stage, the retrieval mechanism stops being effective and the protein is consequently secreted. Conversely, the mechanism based on the KKXX signal guarantees that most of the KKXX-containing CD8 form resides in the endoplasmic reticulum, little in the Golgi complex and undetectable levels at the plasma membrane. The O-glycosylation of this protein comprises for the vast majority the sole addition of peptide-bound GalNAc that occurs in an early Golgi compartment.
Assuntos
Antígenos CD8/metabolismo , Glicoproteínas de Membrana/metabolismo , Oligopeptídeos , Sinais Direcionadores de Proteínas , Sequência de Aminoácidos , Animais , Antígenos CD/biossíntese , Antígenos CD/química , Antígenos CD/metabolismo , Antígenos CD8/biossíntese , Antígenos CD8/química , Sequência de Carboidratos , Linhagem Celular , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Glicosilação , Complexo de Golgi/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Mamíferos , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/química , Modelos Biológicos , Dados de Sequência Molecular , Oligossacarídeos/química , Oligossacarídeos/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
We have previously shown that human colon carcinoma CaCo-2 cells express the Sda-beta 1,4-N-acetylgalactosaminyltransferase (Sda-beta GalNAc-transferase) and that the enzyme activity correlates with the degree of enterocytic differentiation. Here we report that a large amount of this glycosyltransferase is released in soluble form, particularly when CaCo-2 cells are maintained in culture for more than 3 weeks in order to ensure an higher degree of enterocyte differentiation. The soluble enzyme was concentrated and partially purified by Blue-Sepharose and fetuin-Sepharose chromatography. The substrate specificity of the partially purified enzyme was similar to that of Sda-enzyme from epithelial cells of colon mucosa, and for its activity strictly required the presence in acceptors of NeuAc in alpha 2,3-linkage to subterminal galactose. Among the low molecular glycans tested, NeuAc alpha 2,3Gal beta 1,4GlcNAc appeared to be the best acceptor, whereas sialyl-Lewisx and sialyl-Lewisa did not serve as acceptors, indicating that the fucosylation of sub-terminal GlcNAc hindered the transferase activity. Contrary to this, the activity towards a disialylated acceptor such as di-sialyl-lacto-N-tetraose was reduced but not abolished. When CaCo-2 cells were cultured on porous membranes and the transferase activity assayed in medium collected from chambers corresponding to either the apical or basolateral face of highly differentiated CaCo-2 cells, a preferential release from the basolateral surface was found. Considering that Sda-beta GalNAc-transferase is mainly located in the large intestine, current results support the notion that colonic cells largely contribute to the presence of the enzyme in human plasma.
Assuntos
N-Acetilgalactosaminiltransferases/metabolismo , Células CACO-2 , Sequência de Carboidratos , Diferenciação Celular/fisiologia , Humanos , Dados de Sequência Molecular , Solubilidade , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
The aim of the study was to ascertain whether the exposure to ethanol of human colon carcinoma CaCo-2 and HT-29 cell lines affects the differentiation process. As an index of enterocytic differentiation, the expression of sucrase, alkaline phosphatase, alpha 2,6-sialyltransferase toward the N-acetyllactosaminic sequence, and beta 1,4-N-acetylgalactosaminyltransferase (beta 1,4GalNAc-transferase) was examined. The latter enzyme is responsible for the biosynthesis of Sda carbohydrate histo-blood antigen, which mainly occurs in human colonic cells; its expression in CaCo-2 cells depends strictly on the enterocytic differentiation. The addition of ethanol in the culture medium resulted in a significant increment of sucrase and alpha 2,6-sialyltransferase activities in both cell lines, as well as the beta 1,4GalNAc-transferase activity in CaCo-2 cells and alkaline phosphatase activity in HT-29 cells. The increment was dose-dependent in the range between 50 and 200 mM ethanol and evident after 2 days of exposure in both cell systems. These results support the notion that, as occurs for cell lines of different origin, the ethanol in vitro positively affects the differentiation of intestinal cells, namely along the enterocytic lineage. The putative mechanism by which ethanol interferes with the maturation process of colonic cells is discussed.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Etanol/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo , Relação Dose-Resposta a Droga , Enzimas/metabolismo , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/enzimologiaRESUMO
The high occurrence in large intestine epithelial cells from pig of a beta-N-acetylgalactosaminyltransferase with a substrate specificity very similar to that of the Sda beta 1,4-N-acetylgalactosaminyltransferase from other tissues is reported. The enzyme strictly recognized the NeuAc alpha 2,3Gal beta terminal sequence of N- and O-linked oligosaccharides bound to glycoproteins. The transferase activity required Mn2+ and an optimum pH of 7.4. In contrast to the kidney Sda-enzyme from humans and other mammals, the microsomal fraction of pig colonic cells expressed a very high activity even in the absence of Triton X-100. A rapid procedure is presented for the large scale preparation of GalNAc beta 1,4(NeuAc alpha 2,3)Gal beta 1,4Glc from NeuAc alpha 2,3Gal beta 1,4Glc. The biosynthesized tetrasaccharide was completely resistant to the action of neuraminidase from Vibrio cholerae, whereas about 60% of N-acetylneuramic acid was cleaved by neuraminidase from Newcastle disease virus. HPLC separation of different compounds is reported.
Assuntos
Antígenos de Grupos Sanguíneos/metabolismo , Ceco/enzimologia , Colo/enzimologia , Mucosa Intestinal/enzimologia , N-Acetilgalactosaminiltransferases/metabolismo , Oligossacarídeos/biossíntese , Animais , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão , Epitélio/enzimologia , Humanos , Técnicas In Vitro , Rim/enzimologia , Dados de Sequência Molecular , Oligossacarídeos/química , Oligossacarídeos/isolamento & purificação , Especificidade de Órgãos , Ratos , Especificidade da Espécie , Especificidade por Substrato , SuínosRESUMO
In previous works we established that the alpha 2,6-sialyltransferase acting on N-acetyllactosaminic sequences [alpha 2,6(N)ST, E.C. 2.4.99.1] behaves, in colonic cells, as an oncodevelopmentally regulated enzyme. Subpopulations of the human colon cancer cell line HT-29 adapted to grow in 10(-5) M methotrexate (MTX), permanently retain the ability to differentiate as mucus-secreting cells when kept confluent for extended periods of time [Lesuffleur et al. (1991) J. Cell Biol. 115, 1409-1418]. In this study we have compared the activities of five sialyltransferases acting on N- or O-linked chains of glycoproteins in parental HT-29 and in the 10(-5) M MTX-resistant variant. Both cell lines were studied during the exponential phase of growth as well as after a long period of postconfluent culture (28-30 days). Regardless the culture conditions, resistance to 10(-5) M MTX is associated with a virtual disappearance of alpha 2,6(N)ST activity. This change results in a dramatic reduction of the reactivity of cell membranes with the fluorescent lectin from Sambucus nigra, specific for alpha 2,6-sialylated structures. The activity of the alpha 2,3-sialyltransferase which acts on N-acetyllactosaminic sequences increases about two times in postconfluent cultures of 10(-5) M MTX-resistant cells, suggesting a close relationship with the differentiation degree. No significative changes were observed in the activity of other sialyltransferases.
Assuntos
Resistência a Medicamentos/fisiologia , Metotrexato/toxicidade , Lectinas de Plantas , Sialiltransferases/metabolismo , Configuração de Carboidratos , Sequência de Carboidratos , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Neoplasias do Colo , Citometria de Fluxo , Humanos , Cinética , Lectinas , Dados de Sequência Molecular , Oligossacarídeos/biossíntese , Proteínas Inativadoras de Ribossomos , Especificidade por Substrato , Células Tumorais Cultivadas , beta-D-Galactosídeo alfa 2-6-Sialiltransferase , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
Human Tamm-Horsfall glycoprotein (T-H) is produced by renal cells of ascending limb of loop of Henle and is largely excreted in urine. N-linked glycans account for close to 30% of the weight of T-H. We studied the biosynthesis of recombinant T-H permanently expressed in HeLa cells. The conversion from the precursor (84 kDa) to the mature form (97 kDa) mainly depends on the processing of glycans from the high-mannose to polyantennary type. The conversion from precursor to mature form is very slow and the glycan structure of precursor appears to be that of a glycoprotein not yet processed by Golgi alpha 1,2 mannosidase. Since T-H has a very high number of disulfide bridges (more than 50 cysteine residues/mol) one may infer that the rate limiting step for the precursor export out of ER is the formation of a correct set of disulfide bonds. Mature T-H isolated from HeLa cells retained one N-linked chain with the high-mannose structure similarly to urinary T-H. This result indicates that the occurrence of one unprocessed high-mannose chain in mature T-H is host-cell independent and very likely related to the T-H primary structure.
Assuntos
Mucoproteínas/biossíntese , Oligossacarídeos/biossíntese , Processamento de Proteína Pós-Traducional , Sequência de Carboidratos , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Clonagem Molecular , Glicopeptídeos/isolamento & purificação , Glicosilação , Células HeLa , Humanos , Manose/metabolismo , Metionina/metabolismo , Dados de Sequência Molecular , Mucoproteínas/urina , Oligossacarídeos/isolamento & purificação , Proteínas Recombinantes/biossíntese , Transfecção , UromodulinaRESUMO
The biosynthesis of human CD8 glycoprotein in transfected rat epithelial cells produces an unglycosylated precursor, an intermediate species only initially O-glycosylated, and a doublet mature form carrying neutral and sialylated O-linked oligosaccharides with the core-2 structure (Pascale, M. C., Malagolini, N., Serafini-Cessi, F., Migliaccio, G., Leone, A., and Bonatti, S. (1992) J. Biol. Chem. 267, 9940-9947). In this study the most relevant post-translational events: dimerization, addition of the first O-linked GalNAc, fulfillment of O-linked chains, as well as expression of involved glycosyltransferases, have been examined and correlated with the localization and transit rate of CD8 through the exocytic pathway. The glycosyltransferase activities measured in rat epithelial cells transfected with human CD8 DNA are entirely consistent with the primary structure assigned to CD8 oligosaccharides. The half-time of appearance of the initially O-glycosylated precursor and mature form was estimated to be 4 and 14 min, respectively, and the half-time for delivery of mature CD8 to the cell surface was found to be about 30 min, indicating a very fast routing. Pulse experiments with [35S]cysteine at 37 degrees C followed by chase-periods at low temperatures showed that folding/dimerization occurs before routing to the Golgi apparatus, whereas the addiction of O-linked GalNAc appears to take place later, very likely in cis-Golgi cisternae. Treatment of cells with monensin accumulated the intermediate CD8 form carrying non-elongated O-linked GalNAc, whereas brefeldin A treatment produced a sialylated glycoprotein species with a mobility faster than the mature CD8. These results indicate that the two drugs affect assembly of O-linked chains at different time of their processing.
Assuntos
Antígenos CD8/biossíntese , Glicosiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Brefeldina A , Antígenos CD8/genética , Antígenos CD8/metabolismo , Sequência de Carboidratos , Linhagem Celular , Membrana Celular/imunologia , Ciclopentanos/farmacologia , Cisteína/metabolismo , Glicosilação , Humanos , Cinética , Dados de Sequência Molecular , Monensin/farmacologia , Neuraminidase/farmacologia , Ratos , Especificidade por Substrato , Temperatura , TransfecçãoRESUMO
The biosynthesis, post-translational modifications, and oligosaccharide structure of human CD8 glycoprotein have been studied in transfected rat epithelial cells. These cells synthesized and expressed on the plasma membrane high amounts of CD8 in a homodimeric form stabilized by a disulfide bridge. Three different CD8 forms were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis after metabolic labeling and immunoprecipitation: a newly synthesized, unglycosylated 27-kDa (CD8u), a palmitylated and initially O-glycosylated 29-kDa (CD8i), and the mature, terminally glycosylated 32-34-kDa doublet (CD8m). CD8i is a transient intermediate form between CD8u and CD8m: characterization of carbohydrate moiety of [3H]glucosamine-labeled CD8i showed that it comprises for the vast majority non-elongated O-linked GalNAc closely spaced on the peptide backbone. Structural analysis of oligosaccharides released by mild alkaline borohydride treatment from the [3H]glucosamine-labeled CD8 34-kDa form showed that the neutral tetrasaccharide Gal beta 1,4GlcNAc beta 1,6(Gal beta 1,3)GalNAcOH, and an homologous monosialylated pentasaccharide, predominate; the disialylated NeuAc2,3Gal beta 1,3(NeuAc alpha 2,6) GalNAcOH tetrasaccharide appeared to be poorly present. In the CD8 32-kDa form the neutral tetrasaccharide was by far the prominent O-linked chain, and no disialyloligosaccharides were identified. These results indicate that the maturation of CD8 glycoprotein in transfected rat epithelial cells results in the formation of branched O-linked oligosaccharides and that a higher degree of sialylation is responsible for the production of the heavier 34-kDa form.
Assuntos
Antígenos CD8/biossíntese , Glicoproteínas de Membrana/biossíntese , Oligossacarídeos/metabolismo , Animais , Antígenos CD8/química , Antígenos CD8/genética , Configuração de Carboidratos , Sequência de Carboidratos , Linhagem Celular , Cromatografia em Camada Fina , Epitélio/imunologia , Glucosamina/metabolismo , Glicopeptídeos/isolamento & purificação , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Oligossacarídeos/química , Oligossacarídeos/isolamento & purificação , Processamento de Proteína Pós-Traducional , Ratos , Transfecção , TrítioRESUMO
alpha 2,6 sialyltransferase towards the N-acetyllactosaminyl sequence (alpha 2,6 ST, E.C. 2.4.99.1) is one of the major sialyltransferases in human colonic cells; it strongly increases in human colorectal tumors and is largely expressed in fetal and neonatal rat colon. In this study we demonstrate that human colon carcinoma CaCo-2 cells, which differentiate spontaneously into enterocytes when maintained confluent for several days, exhibit a very high expression of alpha 2,6 ST both in the cell-bound and soluble form. When the CaCo-2 cells were cultured on porous membranes the soluble alpha 2,6 ST was mainly detected in the medium collected from the chamber corresponding to the basolateral face of the monolayer. The soluble alpha 2,6 ST could be concentrated and purified from the alpha 2,3 sialyltransferase by affinity chromatography on Blue Sepharose.
Assuntos
Diferenciação Celular , Sialiltransferases/metabolismo , Sequência de Carboidratos , Linhagem Celular , Membrana Celular/enzimologia , Cromatografia de Afinidade , Neoplasias do Colo , Meios de Cultura , Humanos , Cinética , Dados de Sequência Molecular , Sialiltransferases/isolamento & purificação , Fatores de Tempo , beta-D-Galactosídeo alfa 2-6-SialiltransferaseRESUMO
The activity of an alpha 2,6 sialyltransferase acting on N-acetyllactosaminic sequences (alpha 2,6 ST E.C. 2.4.99.1) has previously been found to be increased in 90% of human colon cancer specimens. In the present study, the alpha 2,6 ST activity of 6 human colon cancer cell lines grown in culture was compared with that expressed by the corresponding nude mice xenografts and by the cell lines derived from the xenografts. We found that xenografts of COLO 205, HT-29, SW 620, SW 948 and SW 948 FL (a non-adherent sub-line of SW 948) cells express an alpha 2,6 ST activity much higher than that of the in vitro-grown cells. SW 48 cells grown either in culture or as xenografts lack the enzyme activity. All the xenograft-derived cell lines except HT-29 retained the increased alpha 2,6 ST activity at least for the first 6 passages. Those derived from SW 948 xenografts showed an enrichment of round, non-adherent cells, strongly reactive with the NeuAc alpha 2,6 Gal/GalNAc-specific lectin from Sambucus nigra (SNA), thus indicating that a selection of these cells has occurred.