RESUMO
AIM: Abnormal glycosylation often occurs in tumor cells. T-synthase (core 1 beta 1,3- galactosyltransferase, C1GALT1, or T-synthase) is a key enzyme involved in O-glycosylation. Although T-synthase is known to be important in human tumors, the effects of T-synthase and T-antigen on human tumor responses remain poorly defined. METHODS: In this study, a T-synthase-specific short hairpin RNA (shRNA) or T-synthase-specific eukaryotic expression vector(pcDNA3.1(+)) was transfected into murine Osteosarcoma LM8 cells to assess the effects of T-synthase on T cells and cytokines. RESULTS: The up-regulation of T-synthase promoted the proliferation of osteosarcoma cells in vitro, but it promoted the proliferation of tumor initially up to 2-3 weeks but showed significant growth inhibitory effect after 3 weeks post-implantation in vivo. Osteosarcoma cells with high T-synthase expression in vitro promoted the proliferation and inhibited the apoptosis of CD8+ T cells. Further, T-synthase upregulation promoted CD8+ T-cell proliferation and the increased production of CD4+ T cell-derived IFN-γ cytokines to induce the increased tumor lethality of CTLs. CONCLUSION: Our data suggest that high T-synthase expression inhibits tumor growth by improving the body's anti-tumor immunity. Therefore, using this characteristic to prepare tumor cell vaccines with high immunogenicity provides a new idea for clinical immunotherapy of osteosarcoma.
Assuntos
Linfócitos T CD8-Positivos , Osteossarcoma , Humanos , Animais , Camundongos , Regulação para Cima , Interferon gama/metabolismo , Citocinas , RNA Interferente Pequeno , Osteossarcoma/genética , Osteossarcoma/metabolismo , Proliferação de Células , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Galactosiltransferases/farmacologiaRESUMO
Ischemic stroke is one of the major causes of morbidity and mortality. The ß-1, 3-galactosyltransferase 2 (B3galt2), a member of ß-1, 3-galactosyltransferase family, is playing a vital role in the pathological process of cerebral ischemic injury, but its underlying mechanisms remain unclear. In the present study, we examined the involvement of oxidative stress and NLRP3 inflammasome activation in the neuroprotective effect of B3galt2. Cerebral ischemia/reperfusion (I/R) injury was simulated in a mouse middle cerebral artery occlusion (MCAO) model. Recombinant human B3galt2 (rh-B3galt2) was administered intranasally 1 h post MCAO, and TGF-ß1-siRNA was administered intracerebroventricularly 24 h before MCAO. Outcome measures included brain infarct volume, neurological function, blood-brain barrier (BBB) permeability, neuronal apoptosis, oxidative stress, and the inflammatory response. First, we found that rh-B3galt2 significantly alleviated brain infarct volume and BBB permeability, improved neurological function, and attenuated I/R-induced neuron apoptosis and oxidative stress. Furthermore, rh-B3galt2 attenuated pro-inflammatory cytokines, NF-κB, IL-6, TNF-α, and IL-1ß, and inhibited NLRP3 inflammasome activation. Finally, inhibition of TGF-ß1 by TGF-ß1-siRNA abolished the anti-oxidative and anti-inflammatory effects of rh-B3galt2 in mice after I/R. Collectively, our study demonstrated that rh-B3galt2 exerts neuroprotective effects by regulating cerebral ischemia-induced oxidative stress and NLRP3 inflammasome, which is mainly dependent on the heightening of the TGF-ß1 pathway. Thus, B3galt2 might be considered a new therapeutic target for ischemic stroke treatment.
Assuntos
Isquemia Encefálica , AVC Isquêmico , Fármacos Neuroprotetores , Traumatismo por Reperfusão , Administração Intranasal , Animais , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/metabolismo , Modelos Animais de Doenças , Galactosiltransferases/metabolismo , Galactosiltransferases/farmacologia , Galactosiltransferases/uso terapêutico , Humanos , Infarto da Artéria Cerebral Média/metabolismo , Inflamassomos/metabolismo , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Neuroproteção , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Estresse Oxidativo , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Analysis of non-Gal antibody induced after pig-to-baboon cardiac xenotransplantation identified the glycan produced by porcine beta-1,4-N-acetyl-galactosaminyltransferase 2 (B4GALNT2) as an immunogenic xenotransplantation antigen. The porcine B4GALNT2 enzyme is homologous to the human enzyme, which synthesizes the human SDa blood group antigen. Most humans produce low levels of anti-SDa IgM which polyagglutinates red blood cells from rare individuals with high levels of SDa expression. The SDa glycan is also present on GM2 gangliosides. Clinical GM2 vaccination studies for melanoma patients suggest that a human antibody response to SDa can be induced. Expression of porcine B4GALNT2 in human HEK293 cells results in increased binding of anti-SDa antibody and increased binding of Dolichos biflorus agglutinin (DBA), a lectin commonly used to detect SDa. In pigs, B4GALNT2 is expressed by vascular endothelial cells and endothelial cells from a wide variety of pig backgrounds stain with DBA, suggesting that porcine vascular expression of B4GALNT2 is not polymorphic. Mutations in B4GALNT2 have been engineered in mice and pigs. In both species, the B4GALNT2-KO animals are apparently normal and no longer show evidence of SDa antigen expression. Pig tissues with a mutation in B4GALNT2, added to a background of alpha-1,3-galactosyltransferase deficient (GGTA1-KO) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase deficient (CMAH-KO), show reduced antibody binding, confirming the presence of B4GALNT2-dependent antibodies in both humans and non-human primates. Preclinical xenotransplantation using B4GALNT2-deficient donors has recently been reported. Elimination of this source of immunogenic pig antigen should minimize acute injury by preformed anti-pig antibody and eliminate an induced clinical immune response to this newly appreciated xenotransplantation antigen.
Assuntos
Animais Geneticamente Modificados/imunologia , Rejeição de Enxerto/imunologia , Xenoenxertos/imunologia , Transplante Heterólogo , Animais , Galactosiltransferases/farmacologia , Técnicas de Inativação de Genes/métodos , Humanos , Transplante Heterólogo/métodosRESUMO
Proteoglycans (PGs), including heparan sulfate forms, are important regulators of tumor progression. In the PGs biosynthetic process, the core protein is synthesized on a ribosomal template and the sugar chains are assembled post-translationally, one sugar at a time, starting with the linkage of xylose to a serine residue of the core protein and followed by galactosidation of the xylosylprotein. Hydrophobic xylopyranosides have been previously shown to prime heparan sulfate synthesis, a property that was required to cause growth inhibition of tumor cells. To know if the antiproliferative activity of synthetic xylopyranosides is related to their ability to act as "decoy acceptors" of xylosylprotein 4-ß-galactosyltransferase, we have heterologously expressed the catalytic domain of the human ß-1,4-GalT 7 and studied the ability of a variety of synthetic xylopyranoside derivatives to act as substrates or inhibitors of the recombinant enzyme.
Assuntos
Galactosiltransferases , Glicopeptídeos , Glicosídeos , N-Acetil-Lactosamina Sintase/metabolismo , Naftóis , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Galactosiltransferases/síntese química , Galactosiltransferases/metabolismo , Galactosiltransferases/farmacologia , Regulação Bacteriana da Expressão Gênica , Glicopeptídeos/síntese química , Glicopeptídeos/metabolismo , Glicopeptídeos/farmacologia , Glicosídeos/síntese química , Glicosídeos/metabolismo , Glicosídeos/farmacologia , Humanos , Dados de Sequência Molecular , N-Acetil-Lactosamina Sintase/genética , Naftóis/síntese química , Naftóis/metabolismo , Naftóis/farmacologia , Proteínas Recombinantes/genética , SolubilidadeRESUMO
BACKGROUND AND OBJECTIVES: The residual tumor cells remaining after completion of standard chemotherapy and radiation treatment in B lymphoma patients, may be eradicated by active immunotherapy that stimulates tumor-specific T lymphocytes. Irradiated autologous lymphoma cells expressing tumor-associated antigens (TAA) may serve as a potential tumor vaccine, provided that they are effectively targeted to the antigen-presenting cells (APC). We propose exploiting the natural anti-Gal antibody in order to target vaccinating tumor cells to APC. Anti-Gal constitutes 1% of IgG in human serum and interacts specifically with the alpha-gal epitope (Galalpha1-3Galphalbeta1-4GlcNAc-R). DESIGN AND METHODS: Alpha-gal epitopes were synthesized in vitro on the membrane of primary lymphoma cells by using the recombinant glycosylation enzyme alpha1,3galactosyltransferase (alpha1,3GT). Processed tumor cells were opsonized by purified anti-Gal antibodies and studied for uptake (phagocytosis) by APC including monocyte-derived macrophages and dendritic cells. Cross-presentation of tumor antigens after phagocytosis of processed MHC-I negative lymphoma cells was measured by activation of a tumor-specific CD8+ T-cell line. RESULTS: We demonstrate synthesis of alpha-gal epitopes on freshly isolated B lymphoma cells of various types following the use of the recombinant enzyme alpha1,3GT. The subsequent binding of anti-Gal to the de novo synthesized alphagal epitopes opsonizes these tumor cells for effective uptake by macrophages and dendritic cells, through phagocytosis mediated by FcgammaR1 (CD64). Moreover, anti-Gal-mediated phagocytosis resulted in cross-presentation of TAA by dendritic cells. INTERPRETATION AND CONCLUSIONS: This study suggests that immunization with irradiated autologous lymphoma cells processed to express alpha-gal epitopes will result in anti-Gal-mediated, in vivo targeting of the autologous tumor vaccine to APC.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Vacinas Anticâncer/uso terapêutico , Imunoglobulina G/imunologia , Imunoterapia/métodos , Linfoma de Células B/patologia , Células-Tronco Neoplásicas/imunologia , Trissacarídeos/imunologia , Amino Açúcares/imunologia , Animais , Apresentação de Antígeno , Antígenos de Neoplasias/imunologia , Callithrix , Células Cultivadas/imunologia , Galactosiltransferases/genética , Galactosiltransferases/farmacologia , Glicosilação , Humanos , Imunidade Inata , Imunização , Ativação Linfocitária , Linfoma de Células B/imunologia , Linfoma de Células B/terapia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos da radiação , Neuraminidase/farmacologia , Fagocitose , Receptores de IgG/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Subpopulações de Linfócitos T/imunologiaRESUMO
c-H-ras is located in lipid/rafts and undergoes cholesterol dependent regulation. To analyze the regulatory effects of ganglioside GM1 on the proliferation of fibroblasts transformed with mutated ras gene, GM1 synthase cDNA was transfected into NIH3T3/H-ras cells containing mutation. In the transient expression system with EGFP-fused GM1 synthase, the ratio of BrdU-positive cells among EGFP-positive cells was compared between GM1(+) transfectant cells and control cells, indicating that the transient GM1 expression suppresses cell proliferation. Then, established transfectant cells C21 and C34 expressed definite levels of GM1, and analyzed for the cell growth with the control cells D2 and D4 expressing no GM1. GM1(+) cells showed reduced proliferation compared with controls. Phosphorylation levels of ERK1/2 after FCS treatment were examined, showing that those on the GM1(+) transfectant cells increased slowly, while those in the controls rapidly reached the plateau. Fractionation of Triton X-100 extracts with sucrose density gradient ultra-centrifugation revealed that activated H-ras proteins from controls as well as NIH3T3/H-ras were completely localized in non-GEM/raft fraction. On the other hand, some portions of activated H-ras were transferred to GEM/raft fraction, i.e., 32% in C21, and 8% in C34. Since the Ras effector Raf-1 was localized in non-GEM/raft, the growth suppression might be caused, at least partly, by the movement of activated H-ras to GEM/raft fraction.
Assuntos
Proliferação de Células , Galactosiltransferases/biossíntese , Galactosiltransferases/farmacologia , Perfilação da Expressão Gênica , Genes ras , Microdomínios da Membrana/metabolismo , Animais , DNA Complementar , Fibroblastos/fisiologia , Citometria de Fluxo , Gangliosídeo Galactosiltransferase , Camundongos , Células NIH 3T3 , Fosforilação , TransfecçãoRESUMO
OBJECTIVE: Poor presentation of tumor-associated antigens (TAA) to the immune system remains a major obstacle to effective anti-tumor vaccine therapy. The aim of this study is to demonstrate the feasibility of producing a novel autologous tumor vaccine from ovarian carcinoma that is expected to have increased immunogenicity. The strategy is based on the ability of the anti-Gal IgG antibody (a natural antibody comprising 1% of IgG in humans) to target tumor membranes expressing alpha-gal epitopes (Galalpha1-3Galbeta1-4GlcNAc-R) to antigen-presenting cells (APC). STUDY DESIGN: Freshly obtained ovarian carcinoma tumors are homogenized, washed, and incubated with a mixture of neuraminidase, recombinant alpha1,3 galactosyltransferase (ralpha1,3GT) and uridine diphosphate galactose (UDP-Gal) to synthesize alpha-gal epitopes on carbohydrate chains of glycoproteins of these membranes. Subsequently, the processed membranes are analyzed for expression of alpha-gal epitopes and for the binding of anti-Gal. RESULTS: Incubation of 3 g of ovarian carcinoma membranes, from five different patients, at 100 mg/ml, mixed together with ralpha1,3GT (50 microg/ml), neuraminidase (1 mU/ml), and UDP-Gal (2 mM), resulted in the effective synthesis of alpha-gal epitopes to the extent of approximately 2 x 10(11) epitopes/mg of tumor membranes. As a result of this de novo expression of alpha-gal epitopes, the tumor membranes readily bound purified anti-Gal antibody, as well as anti-Gal in autologous serum. CONCLUSIONS: The method described in this study is very effective in the synthesis of many alpha-gal epitopes on tumor membranes obtained from ovarian carcinoma. These novel epitopes readily bind the naturally occurring anti-Gal antibody. This technique of opsonization of alpha-gal-modified autologous tumor membranes carrying TAA is expected to increase effective uptake of the vaccine by APC, which is key to successful anti-tumor vaccination.
Assuntos
Vacinas Anticâncer/imunologia , Neoplasias Ovarianas/imunologia , Trissacarídeos/biossíntese , Trissacarídeos/imunologia , Células Apresentadoras de Antígenos/imunologia , Sequência de Carboidratos , Membrana Celular/imunologia , Membrana Celular/metabolismo , Ensaio de Imunoadsorção Enzimática , Epitopos/biossíntese , Epitopos/imunologia , Feminino , Galactosiltransferases/metabolismo , Galactosiltransferases/farmacologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Glicoproteínas de Membrana/imunologia , Dados de Sequência Molecular , Neuraminidase/metabolismo , Neuraminidase/farmacologia , Neoplasias Ovarianas/metabolismo , Uridina Difosfato Galactose/metabolismo , Uridina Difosfato Galactose/farmacologiaRESUMO
It is currently under debate whether the mechanism of Golgi retention of different glycosyltransferases is determined by sequences in the transmembrane, luminal, or cytoplasmic domains or a combination of these domains. We have shown that the cytoplasmic domains of alpha1,3-galactosyltransferase (GT) and alpha1,2-fucosyltransferase (FT) are involved in Golgi localization. Here we show that the cytoplasmic tails of GT and FT are sufficient to confer specific Golgi localization. Further, we show that the expression of only the cytoplasmic tail of GT can lead to displacement or inhibition of binding of the whole transferase and that cells expressing the cytoplasmic tail of GT were not able to express full-length GT or its product, Galalpha1,3Gal. Thus, the presence of the cytoplasmic tail prevented the localization and function of full-length GT, suggesting a possible specific Golgi binding site for GT. The effect was not altered by the inclusion of the transmembrane domain. Although the transmembrane domain may act as an anchor, these data show that, for GT, only the cytoplasmic tail is involved in specific localization to the Golgi.
Assuntos
Galactosiltransferases/química , Complexo de Golgi/metabolismo , Animais , Células COS , Citoplasma/metabolismo , DNA/metabolismo , Relação Dose-Resposta a Droga , Galactosiltransferases/farmacologia , Microscopia Confocal , Microscopia de Fluorescência , Peptídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , TransfecçãoRESUMO
Many cytosolic and nuclear proteins are modified by monomeric O-linked N-acetyl-d-glucosamine (O-GlcNAc). The biological functions of this form of glycosylation are unclear but evidence suggests that it heightens regulation of protein function. To assess the biological function of O-GlcNAc addition, we examined the biological effects of galactosyltransferase (GalT) microinjected into the cytoplasm of Xenopus ovarian oocytes. GalT, which catalyzes beta1-4-galactose addition to O-GlcNAc, should inhibit deglycosylation and lectin-like interactions requiring unmodified O-GlcNAc residues. Although GalT injection into diplotene-arrested oocytes has no detectable effects on cell viability, it is toxic to oocytes entering meiosis. Cell-cycle-specific toxicity is recapitulated in vitro as GalT inhibits formation of nuclei and microtubule asters from cell-free extracts of ovulated frog eggs. These observations suggest that regulation of O-GlcNAc is important for cell cycle progression and may be important in diseases in which O-GlcNAc metabolism is abnormal. The methods described here outline a viable experimental scheme for ascribing a biological function to this form of glycosylation.
Assuntos
Acetilglucosamina/metabolismo , Ciclo Celular , Galactosiltransferases/farmacologia , Animais , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sistema Livre de Células , Galactosiltransferases/metabolismo , Microinjeções , Microscopia de Fluorescência , Membrana Nuclear/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Progesterona/farmacologia , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/metabolismo , Xenopus laevisRESUMO
Injection of rats with a single dose of epidermal growth factor (EGF) or isoproterenol increased parotid gland acinar cell levels of cyclic AMP (cAMP) significantly above control basal concentrations (34, 177 and 11.5 pmol/g tissue/100 g body weight, respectively). Following a chronic regimen of isoproterenol (3 days), EGF, bovine galactosyltransferase (Gal Tase, EC 2.4.1.22) and isoproterenol increased cAMP levels, albeit to a lower level than observed for the single dose (21, 17 and 51 pmol, respectively). Using isolated parotid gland membranes, EGF and bovine galactosyltransferase also stimulated adenylate cyclase (EC 2.7.4.3) activity in a concentration-dependent manner. Introduction of the beta-adrenergic receptor antagonist propranolol blocked isoproterenol-stimulated adenylate cyclase activity and cAMP accumulation, but not that observed with EGF or the transferase treatment. Growth factor-stimulated adenylate cyclase activity required the presence of the guanosine triphosphate (GTP) analogue, guanyl-5'-imidodiphosphate (p[NH]ppG), while cAMP accumulation could additionally be blocked by introducing the GDP analog, guanosine 5'[beta-thio]diphosphate (GDP[S]). The ability of EGF to activate adenylate cyclase was not affected by pretreatment of acinar cell membranes with pertussis toxin, whereas pretreatment with cholera toxin eliminated EGF-stimulated cyclase activity. The experimental results presented here expand to the parotid gland our knowledge of the ability of EGF to stimulate the cAMP second messenger signalling pathway via a G-binding regulatory protein, by a mechanism independent of beta-adrenergic receptor activation.
Assuntos
Adenilil Ciclases/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Glândula Parótida/efeitos dos fármacos , Animais , AMP Cíclico/análise , Ativação Enzimática/efeitos dos fármacos , Feminino , Galactosiltransferases/farmacologia , Guanosina Trifosfato/farmacologia , Isoproterenol/farmacologia , Glândula Parótida/enzimologia , Ratos , Ratos Endogâmicos , Transdução de Sinais/efeitos dos fármacosRESUMO
Neurite outgrowth from PC12 pheochromocytoma cells, as well as from peripheral and central nervous system neurons in vitro, is mediated by the extracellular matrix molecule, laminin. We have recently shown that mesenchymal cell spreading and migration on laminin is mediated, in part, by the cell surface enzyme, beta 1,4 galactosyltransferase (GalTase). GalTase is localized on lamellipodia of migrating cells where it functions as a laminin receptor by binding to specific N-linked oligosaccharides in laminin (Runyan et al., 1988; Eckstein and Shur, 1989). In the present study, we examined whether GalTase functions similarly during neutrite outgrowth on laminin using biochemical and immunological analyses. PC12 neurite outgrowth was inhibited by reagents that perturb cell surface GalTase activity, including anti-GalTase IgG and Fab fragments, as well as the GalTase modifier protein alpha-lactalbumin. Control reagents had no effect on neurite outgrowth. Furthermore, blocking GalTase substrates on laminin matrices by earlier galactosyltion or enzymatic removal of GalTase substrates also inhibited neurite outgrowth. Conversely, neurite outgrowth was enhanced by the addition of UDP-galactose, which completes the GalTase enzymatic reaction, while inappropriate sugar nucleotides had no effect. The effects of all these treatments were dose and/or time dependent. Surface GalTase was shown to function during both neurite initiation and elongation, although the effects of GalTase perturbation were most striking during the initiation stages of neurite formation. Consistent with this, surface GalTase was localized by indirect immunofluorescence to the growth cone and developing neurite. Collectively, these results demonstrate that GalTase mediates the initiation of neurite outgrowth on laminin, and to a lesser extent, neurite elongation. Furthermore, this study demonstrates that process extension from both mesenchymal cells and neuronal cells is partly dependent upon specific oligosaccharide residues in laminin.
Assuntos
Axônios/fisiologia , Galactosiltransferases/fisiologia , Células Tumorais Cultivadas/patologia , Animais , Axônios/efeitos dos fármacos , Axônios/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Membrana Celular/ultraestrutura , Transformação Celular Neoplásica/efeitos dos fármacos , Galactosiltransferases/metabolismo , Galactosiltransferases/farmacologia , Imuno-Histoquímica , Laminina/metabolismo , Fatores de Crescimento Neural/farmacologia , Feocromocitoma/metabolismo , Feocromocitoma/patologia , Feocromocitoma/ultraestrutura , Ratos , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/ultraestruturaRESUMO
An inhibitor of N-acetylgalactosamine:GM3, N-acetylgalactosaminyltransferase (EC 2.4.1.92) from chicken blood serum, was tested for its activity on embryonic chicken neural retina in culture. The inhibitor did not change the cellular protein content of the cultures but produced a significant reduction of the labeling of gangliosides. The ratio of labeling of GD3 to GD1a increased from about 0.1 to about 0.8 in the cells cultured without or with the inhibitor, respectively. A striking effect of the inhibitor was seen on the morphology of the neurons, those cultured in its presence being practically devoid of neurites. Glial flat cells were apparently not affected.
Assuntos
Inibidores Enzimáticos/farmacologia , Galactosiltransferases/farmacologia , Galactosiltransferases/fisiologia , Gangliosídeos/metabolismo , N-Acetilgalactosaminiltransferases , Retina/citologia , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Dendritos/efeitos dos fármacos , Galactosiltransferases/metabolismo , Gangliosídeos/fisiologia , Retina/efeitos dos fármacos , Retina/enzimologia , Polipeptídeo N-AcetilgalactosaminiltransferaseRESUMO
Past studies from this laboratory have suggested that mouse sperm binding to the egg zona pellucida is mediated by a sperm galactosyltransferase (GalTase), which recognizes and binds to terminal N-acetylglucosamine (GlcNAc) residues in the zona pellucida (Shur, B. D., and N. G. Hall, 1982, J. Cell Biol. 95:567-573; 95:574-579). We now present evidence that directly supports this mechanism for gamete binding. GalTase was purified to homogeneity by sequential affinity-chromatography on GlcNAc-agarose and alpha-lactalbumin-agarose columns. The purified enzyme produced a dose-dependent inhibition of sperm binding to the zona pellucida, relative to controls. To inhibit sperm/zona binding, GalTase had to retain its native conformation, since neither heat-inactivated nor Mn++-deficient GalTase inhibited sperm binding. GalTase inhibition of sperm/zona binding was not due to steric blocking of an adjacent sperm receptor on the zona, since GalTase could be released from the zona pellucida by forced galactosylation with UDPGal, and the resulting galactosylated zona was still incapable of binding sperm. In control experiments, when UDPGal was replaced with the inappropriate sugar nucleotide, UDPglucose, sperm binding to the zona pellucida remained normal after the adsorbed GalTase was washed away. The addition of UDPGal produced a dose-dependent inhibition of sperm/zona binding, and also dissociated preformed sperm/zona adhesions by catalyzing the release of the sperm GalTase from its GlcNAc substrate in the zona pellucida. Under identical conditions, UDP-glucose had no effect on sperm binding to the zona pellucida. The ability of UDPGal to dissociate sperm/zona adhesions was both time- and temperature-dependent. UDPGal produced nearly total inhibition of sperm/zona binding when the zonae pellucidae were first galactosylated to reduce the number of GalTase binding sites. Finally, monospecific anti-GalTase IgG and its Fab fragments produced a dose-dependent inhibition of sperm/zona binding and concomitantly blocked sperm GalTase catalytic activity. Preimmune IgG or anti-mouse brain IgG, which also binds to the sperm surface, had no effect. The sperm GalTase was localized by indirect immunofluorescence to a discrete plasma membrane domain on the dorsal surface of the anterior head overlying the intact acrosome. These results, along with earlier studies, show clearly that sperm GalTase serves as a principal gamete receptor during fertilization.
Assuntos
Galactosiltransferases/fisiologia , Óvulo/análise , Receptores de Superfície Celular/metabolismo , Interações Espermatozoide-Óvulo , Espermatozoides/enzimologia , Zona Pelúcida/análise , Acetilglucosaminidase/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Feminino , Galactosiltransferases/imunologia , Galactosiltransferases/farmacologia , Imunoglobulina G/imunologia , Masculino , Camundongos , Interações Espermatozoide-Óvulo/efeitos dos fármacos , Espermatozoides/fisiologia , Especificidade por Substrato , Uridina Difosfato Galactose/farmacologiaRESUMO
The effect of cancer-associated galactosyltransferase acceptor (CAGA) on incorporation of a variety of macromolecular precursors has been studied in transformed and nontransformed cells. Incorporation of [3H]-mannose, [3H]-galactose, and [3H]-glucosamine into acid precipitable material after one-hour pulse was inhibited more than 70% within four hours after exposure to CAGA in polyoma-transformed BHK cells and within eight hours after exposure in chick embryo fibroblasts infected with a temperature-sensitive RSV mutant (Ts68) grown at the permissive temperature (CEF-RSV 37 degrees C). Initial short-term rate of uptake (less than one minute) and total long-term uptake (one hour) of the labelled carbohydrates (acid-soluble and acid-insoluble material) was inhibited less than 15% over this period. Incorporation of 14C-leucine, 3H-serine, 3H-uridine, and 3H-thymidine into acid-precipitable material was also inhibited greater than 85% in transformed cells, but more than 12-hour exposure to CAGA was required before maximal inhibition was detected. Uptake of these labelled precursors was inhibited less than 20% up to eight hours after exposure to CAGA. In nontransformed cells (BHK and CEF) incorporation of labelled monosaccharides as well as protein and nucleic acid precursors into acid-precipitable material was reduced less than 25% up to 12 hours following exposure to CAGA. Infected CEF grown at the nonpermissive temperature (CEF-RSV 41 degrees C) were affected to an extent similar to other nontransformed cells. These data suggest that the specific action of CAGA on transformed cells may be due to inhibition of glycoconjugate synthesis.
Assuntos
Metabolismo dos Carboidratos , Transformação Celular Neoplásica/efeitos dos fármacos , Galactosiltransferases/farmacologia , Glicopeptídeos/farmacologia , Animais , Linhagem Celular , Cricetinae , Galactose/metabolismo , Glucosamina/metabolismo , Rim/efeitos dos fármacos , Leucina/metabolismo , Timidina/metabolismo , Uridina/metabolismoRESUMO
Cancer-associated galactosyltransferase acceptor (CAGA glycoprotein), a small glycoprotein purified from human malignant effusion that selectively kills transformed cells, was tritiated by reductive methylation in the presence of NaB(3)H(4). CAGA-glycoprotein-sensitive cells (baby-hamster kidney cells transformed by polyoma virus and chick-embryo fibroblasts infected with Ts68 temperature-sensitive mutant of Rous sarcoma virus grown at 37 degrees C, the permissive temperature) bound 3-5-fold more (3)H-labelled CAGA glycoprotein than did their CAGA-glycoprotein-resistant non-transformed counterparts. The Rous-sarcoma-virus-infected chick-embryo fibroblasts grown at non-permissive temperature (41 degrees C) bound an intermediate amount of (3)H-labelled CAGA glycoprotein; however, this intermediate amount appeared to be sufficient to induce inhibition of cell growth when the infected chick-embryo fibroblasts treated at 41 degrees C were switched to 37 degrees C. Binding of (3)H-labelled CAGA glycoprotein was time- and temperature-dependent and was not inhibited by monosaccharide. Binding was completely inhibited by the oligosaccharide liberated by endoglucosaminidase H treatment or by exhaustive Pronase digestion of intact CAGA glycoprotein. However, the isolated oligosaccharide failed to demonstrate the growth-inhibition characteristics of the intact glycopeptide. Binding of (3)H-labelled CAGA glycoprotein was unaffected by co-incubation with the peptide core released by endoglucosaminidase H treatment. (3)H-labelled CAGA glycoprotein bound to intact cells could be removed by trypsin treatment up to 4h after addition of the glycoprotein but not thereafter. This time course paralleled the decreasing reversibility of growth inhibition. However, all (3)H-labelled CAGA glycoprotein was found in the supernatant when cells were first disrupted by sonication followed by trypsin treatment for up to 12h. (3)H-labelled CAGA glycoprotein linked to Sepharose 4B failed to cause growth inhibition in CAGA-glycoprotein-sensitive cells. These findings suggest that binding of CAGA glycoprotein occurs via its oligosaccharide moiety. Binding appears to be a necessary but not sufficient condition to induce cell killing. Growth inhibition appears to depend on internalization of the glycoprotein and the presence of a transformation-specific cell process.
Assuntos
Transformação Celular Neoplásica/efeitos dos fármacos , Galactosiltransferases/farmacologia , Glicopeptídeos/farmacologia , Aminoácidos/análise , Animais , Carboidratos/análise , Carboidratos/farmacologia , Gatos , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Transformação Celular Viral/efeitos dos fármacos , Células Cultivadas , Cricetinae , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Galactosiltransferases/metabolismo , Glicopeptídeos/metabolismo , Oligossacarídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Ligação ProteicaRESUMO
Possible genetic models for the inheritance of the ABO blood groups are discussed in terms of the glycosyltransferase enzymes which complete the synthesis of the A and B determinants. Recent immunologic evidence in support of the allelic status of the ABO genes is reviewed. Results are presented of experiments which demonstrate that the B gene associated alpha-3-D-galactosyltransferase can be used to synthesis blood group A determinants.
Assuntos
Sistema ABO de Grupos Sanguíneos/genética , Epitopos , N-Acetilgalactosaminiltransferases , Acetilgalactosamina/farmacologia , Alelos , Animais , Galactosiltransferases/imunologia , Galactosiltransferases/farmacologia , Código Genético , Glucosiltransferases/biossíntese , Glucosiltransferases/genética , Humanos , Modelos Biológicos , CoelhosRESUMO
When the blood group H sites of cord erythrocytes obtained from newborn infants of groups O, A, B and AB were labelled specifically by incubation of the whole cells with the A1 gene dependent alpha-N-acetylgalactosaminyl transferase in the presence of UDP-N-acetyl [14C]-galactosamine, the incorporation of radioactivity was considerably lower than that found for cells from adults. Based on the amount of label recovered in the membranes, average values of 326,000 H sites per single O cell and 68,000 H sites per single A, B and AB cell were calculated. Following fractionation of the stromal blood group substances thus labelled, it was found that, on the average, 66% of the radioactivity was bound to glycoprotein material, 2.7% to glycosphingolipids with short carbohydrate chains, and about 24% to polyglycosylceramides. As these values are similar to those previously determined for O cells from adults, this result shows that there are probably no substantial differences between erythrocytes from adults and newborn infants concerning the overall membrane disposition, but rather provides evidence for variations in the carbohydrate chains of the stromal glycoconjugates.
Assuntos
Sistema ABO de Grupos Sanguíneos/imunologia , Sangue Fetal/imunologia , Acetilgalactosamina/farmacologia , Membrana Celular/imunologia , Fucosil Galactose alfa-N-Acetilgalactosaminiltransferase/farmacologia , Galactosiltransferases/farmacologia , Glicolipídeos , Glicoproteínas , Humanos , Recém-Nascido , Peptídeo Hidrolases/farmacologia , Pronase/farmacologiaRESUMO
A distinctive glycopeptide, which acts as an acceptor for a cancer-associated galactosyltransferase, has been detected in sera and effusions of patients with extensive carcinoma. Cancer-associated galactosyltransferase acceptor (CAGA) purified from human malignant effusions was tested for its effects on cell growth in vitro and in vivo. Addition of the glycopeptide to the media of cells growing in tissue culture significantly inhibited the attachment and growth of transformed cells but had minimal effect on nontransformed cells. Transformed hamster cells (BHKpy, BHKpygiv, Nilpy) and human malignant cells (BT-20 human breast, pancreatic and colonic carcinoma cells) were killed by the addition of as little as 0.5 microgram of acceptor (per ml of medium); whereas nontransformed counterparts did not show a significant change in growth or morphology. In vivo studies showed that the acceptor inhibited development and progression of tumors in hamsters inoculated with tumorigenic BHKpy cells and in nude mice inoculated with human carcinoma cells. Growth of tumors was inhibited 69--94% in animals given 20 micrograms of acceptor subcutaneously and 39--67% when acceptor was given intraperitoneally at the time of tumor cell inoculation. Administration of the acceptor after the development of palpable tumor (congruent to 0.5 cm) caused a 60--85% reduction in growth rate and, in some cases, actual reduction in size and disappearance of palpable tumor. These studies demonstrate that a galactosyltransferase glycopeptide acceptor purified from human malignant effusions produces selective inhibition of transformed cell growth in animal and tissue culture systems.
Assuntos
Transformação Celular Neoplásica , Galactosiltransferases/farmacologia , Glicopeptídeos/farmacologia , Neoplasias Experimentais/tratamento farmacológico , Neoplasias/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Neoplasias do Colo/tratamento farmacológico , Cricetinae , Galactosiltransferases/metabolismo , Glicopeptídeos/metabolismo , Humanos , Camundongos , Neoplasias Pancreáticas/tratamento farmacológicoRESUMO
The authors determined the agglutination percentages with anti-B, eel and Ulex europeus anti-H reagents for the kinetic study of the in vitro conversion O red blood cells in B. The agglutination with the anti-B increases in proportion as the agglutination with eel anti-H decreases; the agglutination with Ulex anti-H remains constant. They converted (time=18 h) O red blood cells with a more or less high substance H content (adults in good health, africans and europeans, patients, newborn and one "Bombay" phenotype). They showed that there is a good correlation between the agglutination percentages with eel anti-H or Ulex anti-H before conversion and the agglutination percentages with anti-B after conversion. These "O converted" look like the B phenotype defined by a check sample of B subjects.
Assuntos
Sistema ABO de Grupos Sanguíneos , Galactosiltransferases/farmacologia , Adulto , Eritrócitos/metabolismo , Testes de Hemaglutinação , Humanos , Recém-Nascido , Cinética , Fatores de TempoRESUMO
The effects of dibutyryl cyclic AMP (B2cAMP) and theophylline on glyoprotein synthesis in rabbit colon were studied in mucosal organ cultures using [3H]glucosamine as a precursor. Addition of B2cAMP (1 mm) to culture medium caused a significant increase in glycoprotein synthesis after 12 and 24 hr compared with biopsies cultured in control medium. The increase in glycoprotein synthesis was observed only if the cyclic nucleotide was present continuously in the incubation medium for at least 12 hr. The stimulatory effect of B2cAMP on [3H]glucosamine incorporation was blocked by cycloheximide. B2cAMP also stimulated mucosal uptake of glucosamine into the intracellular pool and markedly enhanced specific activity of mucosa galactosyltransferase, an enzyme involved in glycoprotein synthesis. Addition of 5mM theophylline caused a greater than 2-fold increase in cAMP levels, which was also accompanied by an increase in glucosamine uptake and incorporation into mucosal glycoproteins. This study demonstrates that elevation of intracellular cAMP concentration in colon epithelium in vitro is associated with an increase in glycoprotein synthesis. These effects may be mediated in part by (1) increased uptake of glycoprotein precursors such as glucosamine, and (2) increased activity of glycoprotein synthetic enzymes.