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1.
Biochem Biophys Res Commun ; 508(2): 380-386, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30502090

RESUMO

Little is known about an oncogenic signal transducer ß-1,4-galactosyltransferase-V (ß-1,4-GalT-V), in human colorectal cancer. Using quantitative RT-PCR, immunohistochemical staining and ELISA assays, we determined that ß-1,4-GalT-V gene/protein expression is specifically increased in human colorectal cancer (CRC) tumors, compared to visibly normal tissue. Furthermore, we observed a marked increase in its enzymatic activity, and its product lactosylceramide. Moreover, we found increased dihydrosphingolipid metabolites, in particular dihydrosphingomyelin in cancer tissue compared to normal. Further, inhibition of glycosphingolipid synthesis by the synthetic ceramide analog, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), concurrently inhibited colorectal cancer cell (HCT-116) proliferation, as well as ß-1,4-GalT-V mass and several glycosphingolipid levels. We conclude that ß-1,4-GalT-V may serve as a diagnostic and therapeutic biomarker for the progression of human colorectal cancer, and consequently, inhibition of GSL synthesis may be a novel approach for the treatment of this life-threatening disease.


Assuntos
Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/enzimologia , Galactosiltransferases/genética , Galactosiltransferases/metabolismo , Biomarcadores Tumorais/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/tratamento farmacológico , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Galactosiltransferases/antagonistas & inibidores , Células HCT116 , Humanos , Imuno-Histoquímica , Lactosilceramidas/biossíntese , Morfolinas/administração & dosagem , Morfolinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Esfingolipídeos/biossíntese , Regulação para Cima
2.
Methods Mol Biol ; 1804: 189-205, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29926409

RESUMO

Protocols for assay of 24 different Glycolipid-Glycosyltransferases (GSL-GLTs) of the eukaryotic systems are described. Problems of quantitating the activities in crude membranes are also described. Different separation methods (for separation of substrate, donors, and the product of the reaction) have been described based on the paper chromatography or high voltage paper electrophoresis in 1.0% Na2B4O7. Liquid Scintillation counting system was used for quantitation of the enzymatic product. In the assay of each GSL-GLT it is recommended to compare the selected method to be used with the exact conditions used by the authors published previously. As a test case for these assays the following kinetic parameters for Lactosylceramide Synthase, GalT-2 (UDP-Gal: Glc-Cer ß1-4-galactosyltransferase), (Km of glucosylceramide = 1.65 × 10-4 M; Km for UDP-Gal = 0.5 × 10-4 M; V max is determined in the presence of optimum detergent concentrations (2-15 mg/ml of Cutscum-Triton X-100, 2:1); Mn++ and Mg++, 10-20 mM) has been reported. The importance of use of GalT-2 assay method (as a model system) in the purified Golgi-rich membranes from 13-day-old embryonic chicken brains (13-ECB) is described.


Assuntos
Ensaios Enzimáticos/métodos , Células Eucarióticas/metabolismo , Gangliosídeos/metabolismo , Globosídeos/metabolismo , Glicosiltransferases/metabolismo , Lactosilceramidas/biossíntese , Antígenos CD15/metabolismo , Animais , Apoptose , Encéfalo/metabolismo , Galinhas
3.
Cell Physiol Biochem ; 41(3): 1208-1218, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28427052

RESUMO

BACKGROUND: Cystic fibrosis (CF) is the most common autosomal-recessive disorder in western countries. Previous studies have demonstrated an important role of sphingolipids in the pathophysiology of cystic fibrosis. It has been shown that ceramide has a central role in various pulmonary infections, including those with Pseudomonas aeruginosa (P. aeruginosa). Ceramide is accumulated in the airways of CF mice and patients. However, little is known about a potential role of glucosylceramide in cystic fibrosis. METHODS: We investigated the expression of glucosylceramide and lactosylceramide in the respiratory tract of murine and human CF samples by immunohistochemistry and analyzed effects of glucosylceramide on P. aeruginosa in vitro. We performed pulmonary infections with P. aeruginosa and tested inhalation with glucosylceramide. RESULTS: We demonstrate that glucosylceramide is down-regulated on the apical surface of bronchial and tracheal epithelial cells in cystic fibrosis mice. Although glucosylceramide did not have a direct bactericidal effect on Pseudomonas aeruginosa in vitro, inhalation of CF mice with glucosylceramide protected these mice from infection with P. aeruginosa, while non-inhaled CF mice developed severe pneumonia. CONCLUSION: Our data suggest that glucosylceramide acts in vivo in concert with ceramide and sphingosine to determine the pulmonary defense against P. aeruginosa.


Assuntos
Antibacterianos/farmacologia , Antígenos CD/farmacologia , Fibrose Cística/imunologia , Glucosilceramidas/farmacologia , Lactosilceramidas/farmacologia , Infecções por Pseudomonas/prevenção & controle , Administração por Inalação , Animais , Antibacterianos/biossíntese , Antígenos CD/biossíntese , Fibrose Cística/microbiologia , Fibrose Cística/patologia , Glucosilceramidas/biossíntese , Humanos , Lactosilceramidas/biossíntese , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Camundongos Transgênicos , Testes de Sensibilidade Microbiana , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento
4.
Glycobiology ; 25(6): 655-68, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25595946

RESUMO

Lactosylceramide (LacCer), which is essential for many cellular processes, is highly expressed on the plasma membranes of human neutrophils and mediates innate immune functions. Less is known, however, about the properties and biological functions of LacCer in mouse neutrophils. This study therefore analyzed the properties of mouse neutrophil LacCer. LacCer was observed on the surface of these cells, with flow cytometry indicating that mouse neutrophil LacCer could be detected by the anti-LacCer mAb T5A7, but not by the anti-LacCer antibodies Huly-m13 and MEM-74. The molecular species of LacCer were nearly identical in mouse and human neutrophils, including C24:0 and C24:1 fatty acid chain-containing species, although the LacCer content in plasma membranes was ∼ 20-fold lower in mouse than in human neutrophils. Surface plasmon resonance analysis revealed that T5A7 bound to a lipid monolayer composed of LacCer, DOPC, cholesterol and sphingomyelin (molar ratio 0.1 : 10 : 10 : 1), whereas Huly-m13 did not. T5A7 induced neutrophil migration, which was abolished by inhibitors of Src-family kinases, PI-3 kinases, and trimeric G (o/i) proteins. T5A7 also inhibited phagocytosis of non-opsonized zymosans by neutrophils. Taken together, these findings suggest that in mouse neutrophils, (i) LacCer is expressed as LacCer-enriched microdomains in cell surface plasma membranes, (ii) these microdomains are recognized by T5A7 but not by other known anti-LacCer antibodies and (iii) LacCer is involved in cell migration and phagocytosis.


Assuntos
Antígenos CD/imunologia , Antígenos CD/metabolismo , Lactosilceramidas/imunologia , Lactosilceramidas/metabolismo , Neutrófilos/química , Animais , Anticorpos Monoclonais/imunologia , Reações Antígeno-Anticorpo , Antígenos CD/biossíntese , Cálcio/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Voluntários Saudáveis , Humanos , Lactosilceramidas/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/citologia , Neutrófilos/imunologia , Neutrófilos/metabolismo
5.
Glycobiology ; 24(6): 532-41, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24653215

RESUMO

Our previous studies showed that mouse ß4-galactosyltransferase 5 (ß4GalT5) is a lactosylceramide (Lac-Cer) synthase, and that its gene expression increases by 2- to 3-fold upon malignant transformation of cells. In the present study, we examined whether or not the tumorigenic and metastatic potentials of B16-F10 mouse melanoma cells can be suppressed by reducing the expression of the ß4GalT5 gene. We isolated a stable clone named E5 whose ß4GalT5 gene expression level was reduced to 35% that of a control clone C1 by transfection of its antisense cDNA. Thin-layer chromatography analysis of glycosphingolipids showed that the amounts of Lac-Cer and ganglioside GM3 are significantly less in clone E5 than in clone C1. Clone C1 and E5 cells were each transplanted subcutaneously or injected intravenously into C57BL/6 mice, and the sizes of tumors and numbers of colonies formed in the lungs were determined. The average tumor size and average number of colonies formed with clone E5 were decreased to 44 and 49%, respectively, of those formed with clone C1. Furthermore, the numbers and sizes of colonies formed in the soft agarose gels, and the volumes of tumors formed in athymic mice with fibroblasts from wild type, heterozygous and homozygous ß4GalT5-knockout mouse embryos upon transformation with the polyoma virus oncogene correlated with the ß4GalT5 gene dosage. These results strongly indicate that the amounts of Lac-Cer synthesized by ß4GalT5 correlate with the tumorigenic potentials of malignantly transformed cells.


Assuntos
Antígenos CD/biossíntese , Carcinogênese/genética , Galactosiltransferases/biossíntese , Lactosilceramidas/biossíntese , Melanoma Experimental/genética , Animais , Linhagem Celular Tumoral , Galactosiltransferases/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma Experimental/patologia , Camundongos
6.
Glycobiology ; 23(10): 1175-83, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23882130

RESUMO

Glycosphingolipids are expressed on the cell membrane and act as important factors in various events that occur across the plasma membrane. Lactosylceramide (LacCer) is synthesized from glucosylceramide and is a common precursor of various glycosphingolipids existing in whole body. Based on the enzyme purification, ß1,4-galactosyltransferase 6 (B4galt6) cDNA was isolated as a LacCer synthase-coding gene in the rat brain. We generated B4galt6 gene knockout (KO) mice and analyzed their phenotypes to examine roles of ß4GalT6. B4galt6 KO mice were born and grew up apparently normal. LacCer synthase activity and the composition of acidic glycosphingolipids in the brain were almost equivalent or minimally different between wild-type and KO mice. Studies by mouse embryonic fibroblasts (MEFs) revealed that the silencing of B4galt5 gene resulted in the marked reduction in LacCer synthase activity and this reduction was more severe in MEFs derived from B4galt6 KO mice than those from wild-type mice. These results suggested that ß4GalT6 plays a role as a LacCer synthase, whereas ß4GalT5 acts as a main enzyme for LacCer biosynthesis in these tissues and cells.


Assuntos
Galactosiltransferases/metabolismo , Lactosilceramidas/biossíntese , Animais , Encéfalo/metabolismo , Linhagem Celular , Galactosiltransferases/genética , Lactosilceramidas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo
7.
Yakugaku Zasshi ; 132(6): 691-7, 2012.
Artigo em Japonês | MEDLINE | ID: mdl-22687727

RESUMO

ß-1,4-Galactosyltransferase (ß-1,4-GalT) V - whose human and mouse genes were cloned by us - has been suggested to be involved in the biosyntheses of N-glycans, O-glycans, and lactosylceramide by in vitro studies. Our recent study showed that ß-1,4-GalT V-knockout mice are embryonic lethal, suggesting the importance of the glycans synthesized by ß-1,4-GalT V for embryonic development. A subsequent study showed that murine ß-1,4-GalT V is involved in the biosynthesis of lactosylceramide. It is well known that the glycosylation of cell surface glycoproteins and glycolipids changes dramatically upon the malignant transformation of cells. We found that among six ß-1,4-GalTs the gene expression of only ß-1,4-GalT V increases upon malignant transformation. The expression of the ß-1,4-GalT V gene has been shown to be regulated by transcription factors Sp1 and Ets-1 in cancer cells. Both transcription factors regulate the gene expression levels of not only glycosyltransferases, but also key molecules involved in tumor growth, invasion and metastasis. Therefore, the abnormal glycosylation and malignant phenotypes of cancer cells are considered to be suppressed by regulating the expression levels of the transcription factor genes. This review gives a summary account of the gene discovery, in vivo function, and transcriptional mechanism of ß-1,4-GalT V. Also, a perspective on applications of the manipulation of transcription factor genes to cancer therapy will be discussed.


Assuntos
Transformação Celular Neoplásica/genética , Galactosiltransferases/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Animais , Antígenos CD/biossíntese , Transformação Celular Neoplásica/metabolismo , Desenvolvimento Embrionário/genética , Galactosiltransferases/metabolismo , Galactosiltransferases/fisiologia , Terapia Genética , Glicolipídeos/metabolismo , Glicosilação , Humanos , Lactosilceramidas/biossíntese , Glicoproteínas de Membrana/metabolismo , Camundongos , Neoplasias/enzimologia , Neoplasias/patologia , Neoplasias/terapia , Polissacarídeos/biossíntese , Proteína Proto-Oncogênica c-ets-1/fisiologia , Fator de Transcrição Sp1/fisiologia , Transcrição Gênica
8.
J Dent Res ; 91(1): 78-83, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21979133

RESUMO

Neurotrophin 4 (NT-4) and its receptors regulate the differentiation of ameloblasts in tooth development. Gangliosides, sialic acids that contain glycosphingolipids (GSLs), are involved in a variety of membrane-associated cell physiological functions such as ligand-receptor signal transmission. However, the expression patterns and functions of GSLs during tooth development remain unclear. In this study, we identified strong expressions of GM3 and LacCer in dental epithelium, which give rise to differentiation into enamel-secreting ameloblasts. Exogenous GM3 and LacCer in dental epithelial cells induced the expression of ameloblastin (Ambn), while it was also interesting that GM3 synergistically exerted enhancement of NT-4-mediated Ambn expression. In addition, consistently exogenous GM3 and LacCer in dental epithelial cells induced distinct activation of extracellular signal-regulated kinase 1/2 (ERK1/2), an event upstream of the expression of Ambn. Furthermore, depletion of GSLs from dental epithelial cells by D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) inhibited Ambn expression as well as phosphorylation of ERK1/2. In contrast, exogenous addition of GM3 or LacCer rescued the phosphorylation of ERK1/2 repressed by pre-treatment with D-PDMP. Taken together, these results suggest that GM3 and LacCer are essential for NT-4-mediated Ambn expression, and contribute to dental epithelial cell differentiation into ameloblasts.


Assuntos
Ameloblastos/citologia , Amelogênese/genética , Antígenos CD/fisiologia , Proteínas do Esmalte Dentário/biossíntese , Gangliosídeo G(M3)/fisiologia , Glicoesfingolipídeos/fisiologia , Lactosilceramidas/fisiologia , Ameloblastos/efeitos dos fármacos , Ameloblastos/metabolismo , Amelogênese/efeitos dos fármacos , Animais , Antígenos CD/biossíntese , Antígenos CD/farmacologia , Diferenciação Celular , Linhagem Celular , Proteínas do Esmalte Dentário/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Gangliosídeo G(M3)/biossíntese , Gangliosídeo G(M3)/farmacologia , Glicoesfingolipídeos/biossíntese , Lactosilceramidas/biossíntese , Lactosilceramidas/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Fatores de Crescimento Neural/fisiologia , Fosforilação , Ratos , Transdução de Sinais
9.
PLoS One ; 5(11): e14010, 2010 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-21103358

RESUMO

The sialidase activity of neuraminidase-1 (Neu-1) is responsible for ERK 1/2 pathway activation following binding of elastin peptide on the elastin receptor complex. In this work, we demonstrate that the receptor and lipid rafts colocalize at the plasma membrane. We also show that the disruption of these microdomains as well as their depletion in glycolipids blocks the receptor signaling. Following elastin peptide treatment, the cellular GM(3) level decreases while lactosylceramide (LacCer) content increases consistently with a GM(3)/LacCer conversion. The use of lactose or Neu-1 siRNA blocks this process suggesting that the elastin receptor complex is responsible for this lipid conversion. Flow cytometry analysis confirms this elastin peptide-driven LacCer generation. Further, the use of a monoclonal anti-GM(3) blocking antibody shows that GM(3) is required for signaling. In conclusion, our data strongly suggest that Neu-1-dependent GM(3)/LacCer conversion is the key event leading to signaling by the elastin receptor complex. As a consequence, we propose that LacCer is an early messenger for this receptor.


Assuntos
Antígenos CD/biossíntese , Elastina/farmacologia , Fibroblastos/efeitos dos fármacos , Lactosilceramidas/biossíntese , Neuraminidase/metabolismo , Peptídeos/farmacologia , Adulto , Antígenos CD/metabolismo , Western Blotting , Membrana Celular/metabolismo , Células Cultivadas , Elastina/química , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/metabolismo , Citometria de Fluxo , Gangliosídeo G(M3)/metabolismo , Humanos , Lactose/farmacologia , Lactosilceramidas/metabolismo , Microdomínios da Membrana/metabolismo , Pessoa de Meia-Idade , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neuraminidase/genética , Interferência de RNA , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Adulto Jovem
10.
Glycoconj J ; 27(7-9): 685-95, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21057870

RESUMO

Human ß-1,4-galactosyltransferase (ß-1,4-GalT) V was shown to be involved in the biosynthesis of N-glycans, O-glycans and lactosylceramide (Lac-Cer) by in vitro studies. To determine its substrate specificity, enzymatic activity and its products were analyzed using mouse embryonic fibroblast (MEF) cells from ß-1,4-GalT V (B4galt5)-mutant mice. Analysis of expression levels of the ß-1,4-GalT I-VI genes revealed that the expression of the ß-1,4-GalT V gene in B4galt5 ( +/- ) - and B4galt5 ( -/- ) -derived MEF cells are a half and null when compared to that of B4galt5 ( +/+ )-derived MEF cells without altering the expression levels of other ß-1,4-GalT genes. These MEF cells showed no apparent difference in their growth. When ß-1,4-GalT activities were determined towards GlcNAcß-S-pNP, no significant difference in its specific activity was obtained among B4galt5 ( +/+ )-, B4galt5 ( +/- ) - and B4galt5 ( -/- ) -derived MEF cells. No significant differences were obtained in structures and amounts of N-glycans and lectin bindings to membrane glycoproteins among B4galt5 ( +/+ )-, B4galt5 ( +/- ) - and B4galt5 ( -/- ) -derived MEF cells. However, when cell homogenates were incubated with glucosylceramide in the presence of UDP-[(3)H]Gal, Lac-Cer synthase activity in B4galt5 ( +/- ) - and B4galt5 ( -/- ) -derived MEF cells decreased to 41% and 11% of that of B4galt5 ( +/+ )-derived MEF cells. Consistent with this, amounts of Lac-Cer and its derivative GM3 in B4galt5 ( -/- ) -derived MEF cells decreased remarkably when compared with those of B4galt5 ( +/+ )-derived MEF cells. These results indicate that murine ß-1,4-GalT V is involved in Lac-Cer biosynthesis.


Assuntos
Antígenos CD/biossíntese , Galactosiltransferases/fisiologia , Lactosilceramidas/biossíntese , Animais , Linhagem Celular , Proliferação de Células , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Galactosiltransferases/biossíntese , Galactosiltransferases/química , Glicolipídeos/metabolismo , Glicosilação , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Mutantes , Especificidade por Substrato
11.
Glycobiology ; 20(12): 1631-42, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20693232

RESUMO

We have established hybridoma cell lines producing monoclonal antibodies (mAbs) directed to N-acetylglucosaminylß1-3galactose (GlcNAcß1-3Gal) residue by immunizing BALB/c mice with lactotriaosylceramide (Lc(3)Cer). These obtained hybridoma cells, specific to Lc(3)Cer, were dual immunoglobulin (Ig)-producing cells which secreted both IgM and IgG molecules as antibodies. The established mAbs are able to react with not only Lc(3)Cer but also GlcNAcß1-3-terminal glycosphingolipids (GSLs) despite branching or lactosamine chain lengths and human transferrin with terminal GlcNAc residues. Comparison of the variable regions of the cloned IgM and IgG by reversed transcription-polymerase chain reaction analysis confirmed that the variable regions determine the specificity, the other amino acids are conserved, and these mAbs are encoded by J558 and Vκ-21family genes. Furthermore, we have analyzed the expression of GSLs with GlcNAcß1-3 epitope in acute leukemia cell lines and mouse fetal tissues using these mAbs, in which antigens were distributed comparatively. These mAbs are useful for studying the precise distribution of GlcNAcß1-3Gal-terminating GSL expression in tissues as well as for detecting GSLs carrying terminal GlcNAcß1-3Gal carbohydrate structure.


Assuntos
Anticorpos Monoclonais Murinos/imunologia , Especificidade de Anticorpos/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Região Variável de Imunoglobulina/imunologia , Lactosilceramidas/imunologia , Animais , Anticorpos Monoclonais Murinos/biossíntese , Anticorpos Monoclonais Murinos/genética , Especificidade de Anticorpos/genética , Feminino , Células HL-60 , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/genética , Imunoglobulina M/biossíntese , Imunoglobulina M/genética , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/genética , Células K562 , Lactosilceramidas/biossíntese , Lactosilceramidas/genética , Camundongos , Camundongos Endogâmicos BALB C , Células U937
12.
Glycobiology ; 19(4): 418-27, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19136585

RESUMO

In general, an elevated expression of beta 3-galactosyltransferase (beta 3GalT) activity contributed by beta 3GalT5 correlates well with increased biosynthesis and expression of type 1 chain (Gal beta 1-3GlcNAc beta 1-) derivatives such as Lewis A and sialyl Lewis A, which are mostly recognized as terminal epitopes and not further extended. Most known beta 3-N-acetylglucosaminyltransferases show a higher activity toward extending type 2 chain (Gal beta 1-4GlcNAc beta 1-), and an over-expression of beta 3GalT5 could suppress the formation of the type 2 chain poly-N-acetyllactosaminoglycans. The potential of extending instead the predominant type 1 chain termini synthesized under such circumstances was, however, not investigated, partly due to technical difficulty in unambiguous identification of extended type 1 chains. Using an advanced mass spectrometry-based glycomic mapping and glycan sequencing approach, we show here that type 1 chains carried on the lacto-series glycosphingolipids of colonic carcinoma cells can be extended when the endogenous beta 3GalT activity relative to competing beta 4GalT activity, as defined against a common GlcNAc beta 1-3Gal beta 1-4Glc acceptor, is sufficiently high, as found in Colo205 and SW1116, but not in DLD-1 cells. In support of this positive correlation, the lacto-series glycosphingolipids isolated from stably transfected DLD-1 clones over-expressing beta 3GalT5 were shown to comprise fucosylated dimeric type 1 chains, whereas a mock transfectant and the DLD-1 parent carried only fucosylated dimeric type 2 chains on their lactosylceramides. It suggests that while the natural expression of extended type 1 chain is likely to be determined by many contributing factors including the relative amounts of competing glycosyltransferases and the UDP-Gal level, the enhanced expression of beta 3GalT5 is sufficient to promote in vivo extension of type 1 chains by furnishing a significantly higher amount of type 1 chain precursors relative to competing type 2 chains.


Assuntos
Antígenos CD/biossíntese , Galactosiltransferases/biossíntese , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Lactosilceramidas/biossíntese , Antígenos CD/genética , Configuração de Carboidratos , Linhagem Celular Tumoral , Neoplasias do Colo , Galactosiltransferases/genética , Humanos , Lactosilceramidas/genética , Antígenos do Grupo Sanguíneo de Lewis , Espectrometria de Massas , Oligossacarídeos/biossíntese , Oligossacarídeos/genética
13.
Curr Drug Targets ; 9(4): 272-81, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18393821

RESUMO

Lactosylceramide is a ubiquitously present glycosphingolipid in mammalian tissues and has been implicated in cell proliferation, adhesion, migration and angiogenesis. This glycosphingolipid is synthesized by Golgi-localized enzyme LacCer synthase. According to recent nomenclature and gene mapping studies, two LacCer synthases beta1,4GalT-V and beta1,4GalT-VI have been identified and characterized. In addition, beta1,4GalT-V has been implicated in the synthesis of N-glycans of cell surface glycoproteins. During the past two decades data have accumulated suggesting that the cellular level of LacCer can be regulated by various growth factors, cytokines, lipids, lipoproteins and hemodynamic factors, such as fluid shear stress, by altering the activity of LacCer synthase. An interesting feature is that a nuclear regulating factor (SP1) plays a critical role in transcriptional regulation of this enzyme in cancer cells. Moreover, in human umbilical vein endothelial cells, NF-kappaB has been also shown to regulate this enzyme which, in turn, regulates the gene/protein expression of platelet endothelial cell adhesion molecule, intercellular cell adhesion molecule and angiogenesis. Since new blood supply via formation of capillaries is critical in tumor growth, metastasis, and atherogenesis, these findings expand the role of enzyme in these pathologies. Additional studies are warranted to understand the molecular and biochemical basis of how LacCer synthases are regulated. These studies will facilitate advances in discovery of drugs which mitigate diseases, such as atherosclerosis and cancer due to an aberrant regulation of these LacCer synthases.


Assuntos
Sistemas de Liberação de Medicamentos , Galactosiltransferases/metabolismo , Lactosilceramidas/metabolismo , Animais , Galactosiltransferases/efeitos dos fármacos , Galactosiltransferases/genética , Humanos , Lactosilceramidas/biossíntese , Lipoproteínas LDL/metabolismo , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Transcrição Gênica
14.
J Biochem ; 141(3): 309-17, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17190787

RESUMO

By comparing ovarian carcinoma-derived KF28 cells with the corresponding anticancer drug-resistant cells, the taxol- and cisplatin-resistant properties were found to be closely related with MDR1 and BSEP, and MRP2 transporters, respectively. In addition to the transporters expression, the amounts of glycolipids, particularly their longer carbohydrate structures, in the resistant cells increased to 3-4-fold of those in the sensitive cells due to enhanced transcription of the respective glycosyltransferases. The major glycolipids in the sensitive and resistant cells were GlcCer and Gb(3)Cer, respectively, and extension of the carbohydrate structure into Lewis antigen characteristically occurred in the resistant cells. Le(b), which was not detected in the cisplatin-resistant cells, was present in the taxol-resistant cells, while Le(x) was present in the cisplatin-resistant cells at a higher concentration than in the taxol-resistant cells. 2-Hydroxy fatty acids were significantly abundant in glycolipids of the resistant cells, but they were not detected in free ceramides or sphingomyelin, indicating that the enhanced synthesis of glycolipids in the resistant cells was not linked with the removal pathway for virulent ceramides derived from sphingomyelin. The resistant cells with abundant glycolipids exhibited lower membrane fluidity than the KF28 cells, and this property might be involved in the anticancer drug-resistance.


Assuntos
Resistencia a Medicamentos Antineoplásicos/fisiologia , Glicolipídeos/biossíntese , Antígenos do Grupo Sanguíneo de Lewis/biossíntese , Neoplasias Ovarianas/metabolismo , Cisplatino/farmacologia , Ácidos Graxos/análise , Feminino , Regulação Neoplásica da Expressão Gênica , Glucosilceramidas/biossíntese , Glicosiltransferases/genética , Humanos , Lactosilceramidas/biossíntese , Antígenos CD15/biossíntese , Oligossacarídeos/biossíntese , Paclitaxel/farmacologia , Esfingolipídeos/química , Triexosilceramidas/biossíntese , Células Tumorais Cultivadas
15.
J Biol Chem ; 279(9): 7867-76, 2004 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-14662772

RESUMO

Transfection studies have implicated the multiple drug resistance pump, MDR1, as a glucosyl ceramide translocase within the Golgi complex (Lala, P., Ito, S., and Lingwood, C. A. (2000) J. Biol. Chem. 275, 6246-6251). We now show that MDR1 inhibitors, cyclosporin A or ketoconazole, inhibit neutral glycosphingolipid biosynthesis in 11 of 12 cell lines tested. The exception, HeLa cells, do not express MDR1. Microsomal lactosyl ceramide and globotriaosyl ceramide synthesis from endogenous or exogenously added liposomal glucosyl ceramide was inhibited by cyclosporin A, consistent with a direct role for MDR1/glucosyl ceramide translocase activity in their synthesis. In contrast, cellular ganglioside synthesis in the same cells, was unaffected by MDR1 inhibition, suggesting neutral and acid glycosphingolipids are synthesized from distinct precursor glycosphingolipid pools. Metabolic labeling in wild type and knock-out (MDR1a, 1b, MRP1) mouse fibroblasts showed the same loss of neutral glycosphingolipid (glucosyl ceramide, lactosyl ceramide) but not ganglioside (GM3) synthesis, confirming the proposed role for MDR1 translocase activity. Cryo-immunoelectron microscopy showed MDR1 was predominantly intracellular, largely in rab6-containing Golgi vesicles and Golgi cisternae, the site of glycosphingolipid synthesis. These studies identify MDR1 as the major glucosyl ceramide flippase required for neutral glycosphingolipid anabolism and demonstrate a previously unappreciated dichotomy between neutral and acid glycosphingolipid synthesis.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Glicoesfingolipídeos Acídicos/biossíntese , Glicoesfingolipídeos Neutros/biossíntese , Proteínas de Transferência de Fosfolipídeos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Animais , Astrocitoma , Proteínas de Transporte/metabolismo , Linhagem Celular , Ciclosporina/farmacologia , Cães , Expressão Gênica , Glucosilceramidas/metabolismo , Células HeLa , Humanos , Cetoconazol/farmacologia , Rim , Lactosilceramidas/biossíntese , Proteínas de Membrana/metabolismo , Meningioma , Camundongos , Camundongos Knockout , Microscopia Imunoeletrônica , Proteínas Recombinantes , Transfecção , Triexosilceramidas/biossíntese , Células Tumorais Cultivadas
16.
Biochem J ; 371(Pt 3): 1013-9, 2003 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-12578562

RESUMO

Although most glycosphingolipids (GSLs) are thought to be located in the outer leaflet of the plasma membrane, recent evidence indicates that GSLs and their precursor, ceramide, are also associated with intracellular organelles and, particularly, mitochondria. GSL biosynthesis starts with the formation of ceramide in the endoplasmic reticulum (ER), which is transported by controversial mechanisms to the Golgi apparatus, where stepwise addition of monosaccharides on to ceramides takes place. We now report the presence of GSL-biosynthetic enzymes in a subcompartment of the ER previously characterized and termed 'mitochondria-associated membrane' (MAM). MAM is a membrane bridge between the ER and mitochondria that is involved in the biosynthesis and trafficking of phospholipids between the two organelles. Using exogenous acceptors coated on silica gel, we demonstrate the presence of ceramide glucosyltransferase (Cer-Glc-T), glucosylceramide galactosyltransferase and sialyltransferase (SAT) activities in the MAM. Estimation of the marker-enzyme activities showed that glycosyltransferase activities could not be ascribed to cross-contamination of MAM by Golgi membranes. Cer-Glc-T was found to have a marked preference for ceramide bearing phytosphingosine as sphingoid base. SAT activities in MAM led to the synthesis of G(M3) ganglioside and small amounts of G(D3). G(M1) was also synthesized along with G(M3) upon incubation of the fraction with exogenous unlabelled G(M3), underlying the presence of other sphingolipid-specific glycosyltransferases in MAM. On the basis of our results, we propose MAM as a privileged compartment in providing GSLs for mitochondria.


Assuntos
Retículo Endoplasmático/enzimologia , Glicosiltransferases/metabolismo , Mitocôndrias Hepáticas/enzimologia , Esfingolipídeos/metabolismo , Animais , Compartimento Celular , Cromatografia em Camada Fina , Lactosilceramidas/biossíntese , Ratos , Ratos Sprague-Dawley , Especificidade por Substrato
17.
FASEB J ; 16(9): 1111-3, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12039850

RESUMO

Multidrug-resistant tumor cells display enhanced levels of glucosylceramide. In this study, we investigated how this relates to the overall sphingolipid composition of multidrug-resistant ovarian carcinoma cells and which mechanisms are responsible for adapted sphingolipid metabolism. We found in multidrug-resistant cells substantially lower levels of lactosylceramide and gangliosides in sharp contrast to glucosylceramide, galactosylceramide, and sphingomyelin levels. This indicates a block in the glycolipid biosynthetic pathway at the level of lactosylceramide formation, with concomitant accumulation of glucosylceramide. A series of observations exclude regulation at the enzyme level as the underlying mechanism. First, reduced lactosylceramide formation occurred only in intact resistant cells whereas cell-free activity of lactosylceramide synthase was higher compared with the parental cells. Second, the level of lactosylceramide synthase gene expression was equal in both phenotypes. Third, glucosylceramide synthase (mRNA and protein) expression and activity were equal or lower in resistant cells. Based on the kinetics of sphingolipid metabolism, the observation that brefeldin A does not restore lactosylceramide synthesis, and altered localization of lactosylceramide synthase fused to green fluorescent protein, we conclude that lactosylceramide biosynthesis is highly uncoupled from glucosylceramide biosynthesis in the Golgi apparatus of resistant cells.


Assuntos
Antígenos CD , Carcinoma/metabolismo , Resistencia a Medicamentos Antineoplásicos , Glicolipídeos/biossíntese , Complexo de Golgi/metabolismo , Neoplasias Ovarianas/metabolismo , Esfingolipídeos/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico , Carcinoma/tratamento farmacológico , Carcinoma/enzimologia , Resistência a Múltiplos Medicamentos , Feminino , Galactosilceramidas/biossíntese , Galactosiltransferases/metabolismo , Glucosilceramidas/biossíntese , Glucosilceramidas/metabolismo , Complexo de Golgi/enzimologia , Cinética , Lactosilceramidas/biossíntese , Modelos Biológicos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/enzimologia , Células Tumorais Cultivadas
18.
Hum Mol Genet ; 10(9): 927-40, 2001 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-11309366

RESUMO

A fatal infantile storage disorder with hepatosplenomegaly and severe neurological disease is described. Sphingolipids, including monohexosylceramides (mainly glucosylceramide), dihexosylceramides (mainly lactosylceramide), globotriaosyl ceramide, sulphatides, ceramides and globotetraosyl ceramide, were stored in the tissues. In general, cholesterol and sphingomyelin levels were unaltered. The storage process was generalized and affected a number of cell types, with histiocytes, which infiltrated a number of visceral organs and the brain, especially involved. The ultrastructure of the storage lysosomes was membranous with oligolamellar, mainly vesicular, profiles. Infrequently, there were Gaucher-like lysosomes in histiocytes. The neuropathology was severe and featured neuronal storage and loss with a massive depopulation of cortical neurons and pronounced fibrillary astrocytosis. There was a paucity of myelin and stainable axons in the white matter with signs of active demyelination. Immunohistochemical investigations indicated that saposins A, B, C and D were all deficient. The patient was homozygous for a 1 bp deletion (c.803delG) within the SAP-B domain of the prosaposin gene which leads to a frameshift and premature stop codon. In the heterozygous parents, mutant cDNA was detected by amplification refractory mutation analysis in the nuclear, but not the cytoplasmic, fraction of fibroblast RNA, indicating that the mutant mRNA was rapidly degraded. The storage process in the proband resembled that of a published case from an unrelated family. Saposins were also deficient in this case, leading to its reclassification as prosaposin deficiency, and her mother was found to be a carrier for the same c.803delG mutation. Both of the investigated families came from the same district of eastern Slovakia.


Assuntos
Antígenos CD , Glicoproteínas/deficiência , Glicoproteínas/genética , Lactosilceramidas/biossíntese , Mutação , Esfingolipidoses/genética , Sequência de Bases , Códon , Primers do DNA/química , Feminino , Fibroblastos/metabolismo , Gangliosídeos/metabolismo , Glicolipídeos/metabolismo , Glicoesfingolipídeos/metabolismo , Humanos , Recém-Nascido , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Saposinas , Esfingolipidoses/metabolismo , Esfingolipidoses/patologia , Sulfoglicoesfingolipídeos/metabolismo
19.
Neurochem Res ; 25(1): 145-52, 2000 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-10685614

RESUMO

It is known that ceramide (Cer), the precursor of sphingoglycolipids and of sphingomyelin, participates in events leading to activation of the apoptotic pathway, and per se or through conversion to glucosylceramide (GlcCer) modulates formation of neuritic processes in developing neurons. To learn about the fate of de novo synthesized Cer and GlcCer we examined, in Golgi membranes from chicken embryo neural retina cells, the metabolic relationships of endogenous Cer, GlcCer and lactosylceramide (LacCer). Incubation of the membranes with UDP-[3H]Glc revealed a pool of endogenous Cer useful for synthesis of GlcCer. Most of the GlcCer synthesized, however, was not used for synthesis of LacCer, indicating that it was functionally uncoupled from LacCer synthase. On the other hand, incubation with UDP-[3H]Gal revealed a pool of endogenous GlcCer that depending of the integrity of the membranes was functionally coupled to LacCer and ganglioside synthesis. These results indicate that most GlcCer formed in vitro from Cer is topologically segregated from the synthesis of LacCer. However, subfractionation in sucrose gradients of Golgi membranes labeled with both precursors failed to separate membranes enriched in [3H]GlcCer from those enriched in [3H]Gal-labeled LacCer. It is concluded that despite both transfer steps co-localize in the Golgi membranes, coupling of GlcCer synthesis to LacCer synthesis requires conditions not present in our in vitro assay. This suggests that a coupling activity exists that could be relevant for regulation of the cytoplasmic levels of Cer and GlcCer.


Assuntos
Antígenos CD , Ceramidas/metabolismo , Glucosilceramidas/biossíntese , Complexo de Golgi/metabolismo , Lactosilceramidas/biossíntese , Retina/embriologia , Animais , Centrifugação com Gradiente de Concentração , Embrião de Galinha , Gangliosídeo G(M1)/metabolismo , Gangliosídeo G(M3)/metabolismo , Gangliosídeos/metabolismo , Glucosamina/análogos & derivados , Glucosamina/metabolismo , Membranas Intracelulares/metabolismo , Retina/metabolismo , Retina/ultraestrutura , Trítio , Uridina Difosfato N-Acetilgalactosamina/análogos & derivados , Uridina Difosfato N-Acetilgalactosamina/metabolismo
20.
Biochim Biophys Acta ; 1472(3): 617-24, 1999 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-10564776

RESUMO

C-series gangliosides in rat hepatocytes and liver tissues were analyzed by thin-layer chromatographic (TLC) immunostaining with the specific monoclonal antibody A2B5. Primary cultures of hepatocytes isolated from adult rats were immunostained positively by A2B5. TLC immunostaining with A2B5 of gangliosides from the cells suggested that rat hepatocytes express c-series gangliosides including GT3, GT1c, GQ1c, and GP1c. Expression of c-series gangliosides in cultured hepatocytes was modulated by growth conditions of cells. The amount of GT3 was increased significantly by epidermal growth factor, while the contents of polysialo species such as GT1c, GQ1c, and GP1c were enhanced by higher cell density in culture. Examination of c-series gangliosides in rat liver tissues showed a unique developmental profile with a shift from GT3-dominant to polysialo species-dominant composition in late embryonic stages. These results suggest that the expression of c-series gangliosides in rat hepatocytes is regulated in a growth- and development-dependent manner.


Assuntos
Gangliosídeos/biossíntese , Lactosilceramidas/biossíntese , Fígado/metabolismo , Fatores Etários , Animais , Anticorpos Monoclonais , Células Cultivadas , Cromatografia em Camada Fina , Fator de Crescimento Epidérmico/farmacologia , Imunofluorescência , Gangliosídeos/química , Lactosilceramidas/química , Masculino , Oligossacarídeos/química , Ratos , Ratos Sprague-Dawley
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