RESUMEN
BACKGROUND: The Sda antigen and corresponding biosynthetic enzyme B4GALNT2 are primarily expressed in normal colonic mucosa and are down-regulated to a variable degree in colon cancer tissues. Although their expression profile is well studied, little is known about the underlying regulatory mechanisms. METHODS: To clarify the molecular basis of Sda expression in the human gastrointestinal tract, we investigated the transcriptional regulation of the human B4GALNT2 gene. The proximal promoter region was delineated using luciferase assays and essential trans-acting factors were identified through transient overexpression and silencing of several transcription factors. RESULTS: A short cis-regulatory region restricted to the -72 to +12 area upstream of the B4GALNT2 short-type transcript variant contained the essential promoter activity that drives the expression of the human B4GALNT2 regardless of the cell type. We further showed that B4GALNT2 transcriptional activation mostly requires ETS1 and to a lesser extent SP1. CONCLUSIONS: Results presented herein are expected to provide clues to better understand B4GALNT2 regulatory mechanisms.
Asunto(s)
N-Acetilgalactosaminiltransferasas/genética , Proteína Proto-Oncogénica c-ets-1/metabolismo , Colon , Células HT29 , Humanos , Mucosa Intestinal , N-Acetilgalactosaminiltransferasas/metabolismo , Oligosacáridos/biosíntesis , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/metabolismo , Activación TranscripcionalRESUMEN
TMEM165 is a Golgi protein whose deficiency causes a Congenital Disorder of Glycosylation (CDG). We have demonstrated that Mn2+ supplementation could suppress the glycosylation defects observed in TMEM165-deficient cells and that TMEM165 was a Mn2+-sensitive protein. In the Golgi, the other transmembrane protein capable to regulate Mn2+/Ca2+ homeostasis is SPCA1, encoded by the ATP2C1 gene. A loss of one copy of the ATP2C1 gene leads to Hailey-Hailey Disease (HHD), an acantholytic skin disorder in Humans. Our latest results suggest an unexpected functional link between SPCA1 and TMEM165. In order to clarify this link in case of partial SPCA1 deficiency, HHD fibroblasts were used to assess TMEM165 expression, subcellular localization and Mn2+-induced degradation. No differences were observed regarding TMEM165 expression and localization in HHD patients' fibroblasts compared to control fibroblasts. Nevertheless, we demonstrated both for fibroblasts and keratinocytes that TMEM165 expression is more sensitive to MnCl2 exposure in HHD cells than in control cells. We linked, using ICP-MS and GPP130 as a Golgi Mn2+ sensor, this higher Mn2+-induced sensitivity to a cytosolic Mn accumulation in MnCl2 supplemented HHD fibroblasts. Altogether, these results link the function of SPCA1 to the stability of TMEM165 in a pathological context of Hailey-Hailey disease.
Asunto(s)
Antiportadores/metabolismo , ATPasas Transportadoras de Calcio/metabolismo , Proteínas de Transporte de Catión/metabolismo , Fibroblastos/metabolismo , Queratinocitos/metabolismo , Pénfigo Familiar Benigno/metabolismo , Línea Celular , Fibroblastos/patología , Humanos , Queratinocitos/patología , Manganeso/metabolismoRESUMEN
Since 2012, the interest for TMEM165 increased due to its implication in a rare genetic human disease named TMEM165-CDG (Congenital Disorder(s) of Glycosylation). TMEM165 is a Golgi localized protein, highly conserved through evolution and belonging to the uncharacterized protein family 0016 (UPF0016). Although the precise function of TMEM165 in glycosylation is still controversial, our results highly suggest that TMEM165 would act as a Golgi Ca2+/Mn2+ transporter regulating both Ca2+ and Mn2+ Golgi homeostasis, the latter is required as a major cofactor of many Golgi glycosylation enzymes. Strikingly, we recently demonstrated that besides its role in regulating Golgi Mn2+ homeostasis and consequently Golgi glycosylation, TMEM165 is sensitive to high manganese exposure. Members of the UPF0016 family contain two particularly highly conserved consensus motifs E-φ-G-D-[KR]-[TS] predicted to be involved in the ion transport function of UPF0016 members. We investigate the contribution of these two specific motifs in the function of TMEM165 in Golgi glycosylation and in its Mn2+ sensitivity. Our results show the crucial importance of these two conserved motifs and underline the contribution of some specific amino acids in both Golgi glycosylation and Mn2+ sensitivity.
Asunto(s)
Antiportadores/fisiología , Proteínas de Transporte de Catión/fisiología , Aparato de Golgi/metabolismo , Manganeso/metabolismo , Calcio/metabolismo , Trastornos Congénitos de Glicosilación/metabolismo , Glicosilación , Células HEK293 , Humanos , Transporte IónicoRESUMEN
The Golgi ion homeostasis is tightly regulated to ensure essential cellular processes such as glycosylation, yet our understanding of this regulation remains incomplete. Gdt1p is a member of the conserved Uncharacterized Protein Family (UPF0016). Our previous work suggested that Gdt1p may function in the Golgi by regulating Golgi Ca2+/Mn2+ homeostasis. NMR structural analysis of the polymannan chains isolated from yeasts showed that the gdt1Δ mutant cultured in presence of high Ca2+ concentration, as well as the pmr1Δ and gdt1Δ/pmr1Δ strains presented strong late Golgi glycosylation defects with a lack of α-1,2 mannoses substitution and α-1,3 mannoses termination. The addition of Mn2+ confirmed the rescue of these defects. Interestingly, our structural data confirmed that the glycosylation defect in pmr1Δ could also completely be suppressed by the addition of Ca2+. The use of Pmr1p mutants either defective for Ca2+ or Mn2+ transport or both revealed that the suppression of the observed glycosylation defect in pmr1Δ strains by the intraluminal Golgi Ca2+ requires the activity of Gdt1p. These data support the hypothesis that Gdt1p, in order to sustain the Golgi glycosylation process, imports Mn2+ inside the Golgi lumen when Pmr1p exclusively transports Ca2+. Our results also reinforce the functional link between Gdt1p and Pmr1p as we highlighted that Gdt1p was a Mn2+ sensitive protein whose abundance was directly dependent on the nature of the ion transported by Pmr1p. Finally, this study demonstrated that the aspartic residues of the two conserved motifs E-x-G-D-[KR], likely constituting the cation binding sites of Gdt1p, play a crucial role in Golgi glycosylation and hence in Mn2+/Ca2+transport.
Asunto(s)
Canales de Calcio/fisiología , Calcio/metabolismo , Aparato de Golgi/metabolismo , Manganeso/metabolismo , Mananos/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/metabolismo , Secuencias de Aminoácidos , Sitios de Unión , Canales de Calcio/química , Canales de Calcio/genética , ATPasas Transportadoras de Calcio/metabolismo , Secuencia Conservada , Glicosilación , Transporte Iónico , Chaperonas Moleculares/metabolismo , Monosacáridos/metabolismo , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
TMEM165 deficiencies lead to one of the congenital disorders of glycosylation (CDG), a group of inherited diseases where the glycosylation process is altered. We recently demonstrated that the Golgi glycosylation defect due to TMEM165 deficiency resulted from a Golgi manganese homeostasis defect and that Mn2+ supplementation was sufficient to rescue normal glycosylation. In the present paper, we highlight TMEM165 as a novel Golgi protein sensitive to manganese. When cells were exposed to high Mn2+ concentrations, TMEM165 was degraded in lysosomes. Remarkably, while the variant R126H was sensitive upon manganese exposure, the variant E108G, recently identified in a novel TMEM165-CDG patient, was found to be insensitive. We also showed that the E108G mutation did not abolish the function of TMEM165 in Golgi glycosylation. Altogether, the present study identified the Golgi protein TMEM165 as a novel Mn2+-sensitive protein in mammalian cells and pointed to the crucial importance of the glutamic acid (E108) in the cytosolic ELGDK motif in Mn2+-induced degradation of TMEM165.
Asunto(s)
Aparato de Golgi/efectos de los fármacos , Lisosomas/efectos de los fármacos , Manganeso/farmacología , Proteínas de la Membrana/metabolismo , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Antiportadores , Western Blotting , ATPasas Transportadoras de Calcio/genética , ATPasas Transportadoras de Calcio/metabolismo , Proteínas de Transporte de Catión , Relación Dosis-Respuesta a Droga , Técnicas de Silenciamiento del Gen , Glutamatos/genética , Glutamatos/metabolismo , Glicosilación/efectos de los fármacos , Aparato de Golgi/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisosomas/metabolismo , Proteínas de la Membrana/genética , Microscopía Confocal , Mutación , Proteolisis/efectos de los fármacosRESUMEN
BACKGROUND: Defects in TMEM165 gene cause a type-II Congenital Disorder of Glycosylation affecting Golgi glycosylation processes. TMEM165 patients exhibit psychomotor retardation, important osteoporosis, scoliosis, irregular epiphyses and thin bone cortex. TMEM165 protein is highly conserved in evolution and belongs to the family of UPF0016 membrane proteins which could be an unique group of Ca2+/H+ antiporters regulating Ca2+ and pH homeostasis and mainly localized in the Golgi apparatus. METHODS: RT-PCR from human brain tissues revealed TMEM165 splice-transcript variants. mRNA expression was analyzed by RT-Q-PCR. Expression plasmids allowed us to visualize isoform proteins and their subcellular localization. Their functions on glycosylation were achieved by looking at the gel mobility of highly glycosylated proteins in cells overexpressing isoforms. RESULTS: In this study, we highlight, as previously shown for other ion channels, the existence of TMEM165 splice-transcripts isoforms, in particular the Short-Form (SF) and the Long-Form (LF) transcripts, leading to a 129 aa and 259 aa protein isoform, respectively. These proteins both localize in the endoplasmic reticulum and have different effects on glycosylation compared to the wild-type protein (324 aa). We also point out that the SF is expressed at low levels in all human cells and tissues checked, excepted in brain, and forms homodimer. The LF was only expressed in the temporal lobe of human brain. GENERAL SIGNIFICANCE: The finding of numerous splice variants could lead to a family of TMEM165 isoforms. This family of TMEM165 splice transcripts could participate in the fine regulation of TMEM165 isoforms' functions and localizations.
Asunto(s)
Empalme Alternativo/genética , Trastornos Congénitos de Glicosilación/genética , Variación Genética/genética , Proteínas de la Membrana/genética , Secuencia de Aminoácidos , Antiportadores , Encéfalo/metabolismo , Calcio/metabolismo , Proteínas de Transporte de Catión , Línea Celular Tumoral , Retículo Endoplásmico/genética , Glicosilación , Aparato de Golgi/genética , Células HeLa , Humanos , Isoformas de Proteínas/genética , ARN Mensajero/genéticaRESUMEN
We have previously shown that tumor necrosis factor (TNF) induced the up-regulation of the sialyltransferase gene ST3GAL4 (α2,3-sialyltransferase gene) BX transcript through mitogen- and stress-activated kinase 1/2 (MSK1/2), extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) signaling pathways. This up-regulation resulted in sialyl-Lewisx (sLex) overexpression on high-molecular-weight glycoproteins in inflamed airway epithelium and increased the adhesion of Pseudomonas aeruginosa PAO1 and PAK strains to lung epithelial cells. In the present study, we describe a TNF-responsive element in an intronic region of the ST3GAL4 gene, whose TNF-dependent activity is repressed by ERK/p38 and MSK1/2 inhibitors. This TNF-responsive element contains potential binding sites for ETS1 and ATF2 transcription factors related to TNF signaling. We also show that ATF2 is involved in TNF responsiveness, as well as in TNF-induced ST3GAL4 BX transcript and sLex overexpression in A549 lung epithelial cells. Moreover, we show that TNF induces the binding of ATF2 to the TNF-responsive element. Altogether, these data suggest that ATF2 could be a potential target to prevent inflammation-induced P. aeruginosa binding in the lung of patients suffering from lung diseases such as chronic bronchitis or cystic fibrosis.
Asunto(s)
Factor de Transcripción Activador 2/metabolismo , Células Epiteliales/metabolismo , Pulmón/metabolismo , Sistema de Señalización de MAP Quinasas , Oligosacáridos/biosíntesis , Mucosa Respiratoria/metabolismo , Elementos de Respuesta , Sialiltransferasas/biosíntesis , Factor de Necrosis Tumoral alfa/metabolismo , Células A549 , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Proteína Proto-Oncogénica c-ets-1/genética , Proteína Proto-Oncogénica c-ets-1/metabolismo , Pseudomonas aeruginosa/metabolismo , Antígeno Sialil Lewis X , Sialiltransferasas/genética , Factor de Necrosis Tumoral alfa/genética , beta-Galactosida alfa-2,3-SialiltransferasaRESUMEN
Natural and synthetically modified cytidine monophosphate activated sialic acids (CMP-Sias) are essential research assets in the field of glycobiology: among other applications, they can be used to probe glycans, detect sialylation defects at the cell surface or carry out detailed studies of sialyltransferase activities. However, these chemical tools are notoriously unstable because of hydrolytic decomposition, and are very time-consuming and costly to obtain. They are nigh impossible to store with satisfactory purity, and their preparation requires multiple laborious purification steps that usually lead to heavy product loss. Using in situ time-resolved 31P phosphorus nuclear magnetic resonance (31P NMR), we precisely established the kinetics of formation and degradation of a number of CMP-Sias including CMP-Neu5Ac, CMP-Neu5Gc, CMP-SiaNAl and CMP-SiaNAz in several experimental conditions. 31P NMR can be carried out in undeuterated solvents and is a sensitive and nondestructive technique that allows for direct in situ monitoring and optimization of chemo-enzymatic syntheses that involve phosphorus-containing species. Thus, we showed that CMP-sialic acid derivatives can be robustly obtained in high yields using the readily available Neisseria meningitidis CMP-sialic acid synthase. This integrated workflow takes less than an hour, and the freshly prepared CMP-Sias can be directly transferred to sialylation biological assays without any purification step.
Asunto(s)
Citidina Monofosfato/química , Sondas Moleculares/química , Polisacáridos/análisis , Ácidos Siálicos/química , Citidina Monofosfato/biosíntesis , Citidina Monofosfato/síntesis química , Sondas Moleculares/biosíntesis , Sondas Moleculares/síntesis química , N-Acilneuraminato Citidililtransferasa/metabolismo , Neisseria meningitidis/enzimología , Ácidos Siálicos/biosíntesis , Ácidos Siálicos/síntesis químicaRESUMEN
Congenital disorders of glycosylation (CDG) are severe inherited diseases in which aberrant protein glycosylation is a hallmark. From this genetically and clinically heterogenous group, a significant subgroup due to Golgi homeostasis defects is emerging. We previously identified TMEM165 as a Golgi protein involved in CDG. Extremely conserved in the eukaryotic reign, the molecular mechanism by which TMEM165 deficiencies lead to Golgi glycosylation abnormalities is enigmatic. AsGDT1 is the ortholog of TMEM165 in yeast, both gdt1Δ null mutant yeasts and TMEM165 depleted cells were used. We highlighted that the observed Golgi glycosylation defects due to Gdt1p/TMEM165 deficiency result from Golgi manganese homeostasis defect. We discovered that in both yeasts and mammalian Gdt1p/TMEM165-deficient cells, Mn(2+) supplementation could restore a normal glycosylation. We also showed that the GPP130 Mn(2+) sensitivity was altered in TMEM165 depleted cells. This study not only provides novel insights into the molecular causes of glycosylation defects observed in TMEM165-deficient cells but also suggest that TMEM165 is a key determinant for the regulation of Golgi Mn(2+) homeostasis.
Asunto(s)
Proteínas Fúngicas/genética , Aparato de Golgi/fisiología , Manganeso/farmacología , Proteínas de la Membrana/deficiencia , Mutación , Antiportadores , Proteínas de Transporte de Catión , Trastornos Congénitos de Glicosilación/genética , Proteínas Fúngicas/metabolismo , Glicosilación/efectos de los fármacos , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Células HEK293 , Células HeLa , Homeostasis , Humanos , Manganeso/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Complex interplays among proteins, lipids and carbohydrates can alter the phenotype and are suggested to have a crucial role in tumour metastasis. Our previous studies indicated that a complex of the GSLs (glycosphingolipids), AsGM1 (asialo-GM1), which lacks α2,3-linked sialic acid, and α2ß1 integrin receptors is responsible for the metastatic behaviour of C4-2B prostate cancer cells. Herein, we identified and addressed the functional significance of changes in sialylation during prostate cancer progression. We observed an increase in α2,3-linked sialic acid residues on α2 subunits of α2ß1 integrin receptors, correlating with increased gene expression of α2,3-STs (sialyltransferases), particularly ST3GAL3. Cell surface α2,3-sialylation of α2 subunits was required for the integrin α2ß1-dependent cell adhesion to collagen type I and the same α2,3-linked sialic acid residues on the integrin receptor were responsible for the interaction with the carbohydrate moiety of AsGM1, explaining the complex formation between AsGM1 and α2ß1 integrin receptors. These results provide novel insights into the role of sialic acids in the organization and function of important membrane components in invasion and metastatic processes.
Asunto(s)
Neoplasias Óseas/metabolismo , Gangliósido G(M1)/metabolismo , Integrina alfa2/metabolismo , Neoplasias de la Próstata/metabolismo , Ácidos Siálicos/metabolismo , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Neoplasias Óseas/secundario , Línea Celular Tumoral , Gangliósido G(M1)/genética , Humanos , Integrina alfa2/genética , Masculino , Metástasis de la Neoplasia , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Ácidos Siálicos/genéticaRESUMEN
The histo blood group carbohydrate Sd(a) antigen and its cognate biosynthetic enzyme B4GALNT2 show the highest level of expression in normal colon. Their dramatic down regulation previously observed in colon cancer tissues could play a role in the concomitant elevation of the selectin ligand sLe(x), involved in metastasis. However, down regulation of sLe(x) expression by B4GALNT2 has been so far demonstrated in vitro, but not in tissues. The human B4GALNT2 gene specifies at least two transcripts, diverging in the first exon, never studied in normal and cancer tissues. The long form contains a 253 nt exon 1L; the short form contains a 38 nt exon 1S. Using qPCR, we showed that cell lines and normal or cancerous colon, expressed almost exclusively the short form, while the long form was mainly expressed by the embryonic colon fibroblast cell line CCD112CoN. Immunochemistry approaches using colon cancer cells permanently expressing either B4GALNT2 cDNAs as controls, led to the observation of several protein isoforms in human normal and cancerous colon, and cell lines. We showed that tissues expressing B4GALNT2 protein isoforms were able to induce Sd(a) and to inhibit sLe(x) expression; both of which are expressed mainly on PNGase F-insensitive carbohydrate chains. Concomitant expression of B4GALNT2 and siRNA-mediated inhibition of FUT6, the major fucosyltransferase involved in sLe(x) synthesis in colon, resulted in a cumulative inhibition of sLe(x). In normal colon samples a significant relationship between sLe(x) expression and the ratio between FUT6/B4GALNT2 activities exists, demonstrating for the first time a role for B4GALNT2 in sLe(x) inhibition in vivo.
Asunto(s)
Proteínas de Ciclo Celular/biosíntesis , Neoplasias del Colon/genética , Fucosiltransferasas/biosíntesis , Antígeno Lewis X/biosíntesis , N-Acetilgalactosaminiltransferasas/biosíntesis , Proteínas Nucleares/biosíntesis , Adulto , Anciano , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Neoplasias del Colon/patología , Femenino , Fucosiltransferasas/metabolismo , Tracto Gastrointestinal/metabolismo , Regulación Neoplásica de la Expresión Génica , Glicosilación , Humanos , Antígeno Lewis X/metabolismo , Masculino , Persona de Mediana Edad , Proteínas Nucleares/metabolismo , Antígeno Sialil Lewis XRESUMEN
We have shown previously that the pro-inflammatory cytokine TNF (tumour necrosis factor) could drive sLe(x) (sialyl-Lewis(x)) biosynthesis through the up-regulation of the BX transcript isoform of the ST3GAL4 (ST3 ß-galactoside α-2,3-sialyltransferase 4) sialyltransferase gene in lung epithelial cells and human bronchial mucosa. In the present study, we show that the TNF-induced up-regulation of the ST3GAL4 BX transcript is mediated by MSK1/2 (mitogen- and stress-activated kinase 1/2) through the ERK (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) pathways, and increases sLe(x) expression on high-molecular-mass glycoproteins in inflamed airway epithelium. We also show that the TNF-induced sLe(x) expression increases the adhesion of the Pseudomonas aeruginosa PAO1 and PAK strains to lung epithelial cells in a FliD-dependent manner. These results suggest that ERK and p38 MAPK, and the downstream kinase MSK1/2, should be considered as potential targets to hamper inflammation, bronchial mucin glycosylation changes and P. aeruginosa binding in the lung of patients suffering from lung diseases such as chronic bronchitis or cystic fibrosis.
Asunto(s)
Adhesión Bacteriana/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Mucosa Respiratoria/efectos de los fármacos , Proteínas Quinasas S6 Ribosómicas 90-kDa/fisiología , Sialiltransferasas/genética , Factor de Necrosis Tumoral alfa/farmacología , Proteínas Bacterianas/fisiología , Bronquios/efectos de los fármacos , Bronquios/enzimología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Oligosacáridos/fisiología , Pseudomonas aeruginosa/fisiología , Mucosa Respiratoria/metabolismo , Antígeno Sialil Lewis X , Sialiltransferasas/metabolismo , Células Tumorales Cultivadas , Regulación hacia Arriba/efectos de los fármacos , beta-Galactosida alfa-2,3-SialiltransferasaRESUMEN
Bronchial mucins from severely infected patients suffering from lung diseases such as chronic bronchitis or cystic fibrosis exhibit increased amounts of sialyl-Lewis(x) (NeuAcα2-3Galß1-4[Fucα1-3]GlcNAc-R, sLe(x)) glycan structures. In cystic fibrosis, sLe(x) and its sulfated form 6-sulfo-sialyl-Lewis(x) (NeuAcα2-3Galß1-4[Fucα1-3](HO(3)S-6)GlcNAc-R, 6-sulfo-sLe(x)) serve as receptors for Pseudomonas aeruginosa and are involved in the chronicity of airway infection. However, little is known about the molecular mechanisms regulating the changes in glycosylation and sulfation of mucins in airways. Herein, we show that the pro-inflammatory cytokine TNF increases the expression of α2,3-sialyltransferase gene ST3GAL4, both in human bronchial mucosa and in A549 lung carcinoma cells. The role of sialyltransferase ST3Gal IV in sLe(x) biosynthesis was confirmed by siRNA silencing of ST3GAL4 gene. BX is the major transcript isoform expressed in healthy bronchial mucosa and in A549 cells, and is up-regulated by TNF in both models. Bioinformatics analysis and luciferase assays have confirmed that the 2 kb genomic sequence surrounding BX exon contains a promoter region regulated by TNF-related transcription factors. These results support further work aiming at the development of anti-inflammatory strategy to reduce chronic airway infection in diseases such as cystic fibrosis.
Asunto(s)
Antígeno Lewis X/metabolismo , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Oligosacáridos/metabolismo , Sialiltransferasas/genética , Factor de Necrosis Tumoral alfa/farmacología , Regulación hacia Arriba/efectos de los fármacos , Animales , Bronquios/citología , Bronquios/efectos de los fármacos , Bronquios/metabolismo , Línea Celular Tumoral , Clonación Molecular , Humanos , Antígeno Lewis X/biosíntesis , Luciferasas/genética , Membrana Mucosa/citología , Membrana Mucosa/efectos de los fármacos , Membrana Mucosa/metabolismo , Oligosacáridos/biosíntesis , Regiones Promotoras Genéticas/genética , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Antígeno Sialil Lewis X , Sialiltransferasas/deficiencia , beta-Galactosida alfa-2,3-SialiltransferasaRESUMEN
Abnormally elevated levels of sialylated tumor associated carbohydrate antigens are frequently described at the surface of cancer cells and/or secreted in biological fluids. It is now well established that this over-expression may result from deregulation in sialyltransferases enzymatic activity involved in their biosynthesis, but the precise molecular mechanisms remain unknown. Twenty different human sialyltransferases preside to the sialylation of glycoconjugates, either glycolipids or glycoproteins. This review summarizes the current knowledge on human sialyltransferases implicated in the altered expression of sialylated tumor associated antigens, the molecular basis of their regulated expression in cancer cells and the various tools developed by researchers and clinicians for their study in pathological samples.
Asunto(s)
Neoplasias/enzimología , Sialiltransferasas/metabolismo , Secuencia de Carbohidratos , Regulación Enzimológica de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Ácido N-Acetilneuramínico/metabolismo , Neoplasias/metabolismo , Sialiltransferasas/química , Sialiltransferasas/genéticaRESUMEN
Changes in cell surface glycosylation are common modifications that occur during oncogenesis, leading to the over-expression of tumour-associated carbohydrate antigens (TACA). Most of these antigens are sialylated and the increase of sialylation is a well-known feature of transformed cells. In breast cancer, expression of TACA such as sialyl-Lewis(x) or sialyl-Tn is usually associated with a poor prognosis and a decreased overall survival of patients. However, the specific role of these sialylated antigens in breast tumour development and aggressiveness is not clearly understood. These glycosylation changes result from the modification of the expression of genes encoding specific glycosyltransferases involved in glycan biosynthesis and the level of expression of sialyltransferase genes has been proposed to be a prognostic marker for the follow-up of breast cancer patients. Several human cellular models have been developed in order to explain the mechanisms by which carbohydrate antigens can reinforce breast cancer progression and aggressiveness. TACA expression is associated with changes in cell adhesion, migration, proliferation and tumour growth. In addition, recent data on glycolipid biosynthesis indicate an important role of G(D3) synthase expression in breast cancer progression. The aim of this review is to summarize our current knowledge of sialylation changes that occur in breast cancer and to describe the cellular models developed to analyze the consequences of these changes on disease progression and aggressiveness.
Asunto(s)
Antígenos/química , Antígenos/metabolismo , Neoplasias de la Mama/genética , Regulación Neoplásica de la Expresión Génica , Animales , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/patología , Secuencia de Carbohidratos , Humanos , Datos de Secuencia Molecular , Pronóstico , Sialiltransferasas/genéticaRESUMEN
The second human beta-galactoside alpha-2,6-sialyltransferase (hST6Gal II) differs from hST6Gal I, the first member of ST6Gal family, in substrate specificity and tissue expression pattern. While ST6GAL1 gene is expressed in almost all human tissues, ST6GAL2 shows a restricted tissue-specific pattern of expression, mostly expressed in embryonic and adult brain. In order to understand the mechanisms involved in the transcriptional regulation of ST6GAL2, we first characterized the transcription start sites (TSS) in SH-SY5Y neuroblastoma cells. 5' RACE experiments revealed multiple TSS located on three first alternative 5' exons, termed EX, EY and EZ, which are unusually close on the genomic sequence and are all located more than 42 kbp upstream of the first common coding exon. Using Taqman duplex Q-PCR, we showed that the ST6GAL2 transcripts initiated by EX or EY are mainly expressed in both brain-related cell lines and human cerebral cortex, testifying for the use of a similar transcriptional regulation in vivo. Furthermore, we also showed for the first time hST6Gal II protein expression in the different lobes of the human cortex. Luciferase reporter assays allowed us to define two sequences upstream EX and EY with a high and moderate promoter activity, respectively. Bioinformatics analysis and site-directed mutagenesis showed that NF-kappaB and NRSF are likely to act as transcriptional repressors, whereas neuronal-related development factors Sox5, Puralpha and Olf1, are likely to act as transcriptional activators of ST6GAL2. This suggests that ST6GAL2 transcription could be potentially activated for specific neuronal functions.
Asunto(s)
Corteza Cerebral/enzimología , Regulación Enzimológica de la Expresión Génica , Neuronas/enzimología , Sialiltransferasas/genética , Transcripción Genética , Regiones no Traducidas 5'/genética , Emparejamiento Base/genética , Secuencia de Bases , Sitios de Unión , Línea Celular Tumoral , Biología Computacional , Pruebas de Enzimas , Humanos , Luciferasas/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción/metabolismo , TransfecciónRESUMEN
Using a focused glycan-gene microarray, we compared the glycosyltransferase (GT) and sulfotransferase gene expression profiles of human monocytes, dendritic cells (DCs) and macrophages (Mphis), isolated or differentiated from the same donors. Microarray analysis indicated that monocytes express transcripts for a full set of enzymes involved in the biosynthesis of multi-multiantennary branched N-glycans, potentially elongated by poly-N-acetyl-lactosamine chains, and of mucin-type Core 1 and Core 2 sialylated O-glycans. Monocytes also express genes involved in the biosynthesis and modification of glycosaminoglycans, but display a limited expression of GTs implicated in glycolipid synthesis. Among genes expressed in monocytes (90 out of 175), one third is significantly modulated in DCs and Mphi respectively, most of them being increased in both cell types relative to monocytes. These changes might potentially enforce the capacity of differentiated cells to synthesize branched N-glycans and mucin-type O-glycans and to remodel cell surface proteoglycans. Stimulation of DCs and Mphis with lipopolysaccharide caused a general decrease in gene expression, mainly affecting genes found to be positively modulated during the differentiation steps. Interestingly, although a similar set of enzymes are modulated in the same direction in mature DCs and Mphis, cell specific genes are also differentially regulated during maturation, a phenomenon that may sustain functional specificities. Validation of this analysis was provided by quantitative real-time PCR and flow cytometry of cell surface glycan antigens. Collectively, this study implies an important modification of the pattern of glycosylation in DCs and Mphis undergoing differentiation and maturation with potential biological consequences.
Asunto(s)
Células Dendríticas/enzimología , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Glicosiltransferasas/genética , Macrófagos/enzimología , Monocitos/enzimología , Sulfotransferasas/genética , Biomarcadores/metabolismo , Diferenciación Celular/genética , Membrana Celular/metabolismo , Glicosiltransferasas/metabolismo , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sulfotransferasas/metabolismoRESUMEN
Bronchial mucins from patients suffering from CF (cystic fibrosis) exhibit glycosylation alterations, especially increased amounts of the sialyl-Lewis(x) (NeuAcalpha2-3Galbeta1-4[Fucalpha1-3]GlcNAc-R) and 6-sulfo-sialyl-Lewis(x) (NeuAcalpha2-3Galbeta1-4[Fucalpha1-3][SO(3)H-6]GlcNAc-R) terminal structures. These epitopes are preferential receptors for Pseudomonas aeruginosa, the bacteria responsible for the chronicity of airway infection and involved in the morbidity and early death of CF patients. However, these glycosylation changes cannot be directly linked to defects in CFTR (CF transmembrane conductance regulator) gene expression since cells that secrete airway mucins express no or very low amounts of the protein. Several studies have shown that inflammation may affect glycosylation and sulfation of various glycoproteins, including mucins. In the present study, we show that incubation of macroscopically healthy fragments of human bronchial mucosa with IL-6 (interleukin-6) or IL-8 results in a significant increase in the expression of alpha1,3/4-fucosyltransferases [FUT11 (fucosyltransferase 11 gene) and FUT3], alpha2-6- and alpha2,3-sialyltransferases [ST3GAL6 (alpha2,3-sialyltransferase 6 gene) and ST6GAL2 (alpha2,6-sialyltransferase 2 gene)] and GlcNAc-6-O-sulfotransferases [CHST4 (carbohydrate sulfotransferase 4 gene) and CHST6] mRNA. In parallel, the amounts of sialyl-Lewis(x) and 6-sulfo-sialyl-Lewis(x) epitopes at the periphery of high-molecular-mass proteins, including MUC4, were also increased. In conclusion, our results indicate that IL-6 and -8 may contribute to the increased levels of sialyl-Lewis(x) and 6-sulfo-sialyl-Lewis(x) epitopes on human airway mucins from patients with CF.
Asunto(s)
Bronquios/enzimología , Epítopos/biosíntesis , Fucosiltransferasas/metabolismo , Glicosiltransferasas/metabolismo , Interleucina-6/fisiología , Interleucina-8/fisiología , Antígeno Lewis X/biosíntesis , Sulfotransferasas/metabolismo , Fibrosis Quística/inmunología , Fucosiltransferasas/genética , Glicosiltransferasas/genética , Humanos , Antígeno Lewis X/metabolismo , Membrana Mucosa/enzimología , Reacción en Cadena de la Polimerasa , Sulfotransferasas/genéticaRESUMEN
The acceptor specificities of ST3Gal III, ST3Gal IV, ST6Gal I and ST6Gal II were investigated using a panel of beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O)(CH(2))(7)CH(3) analogues. Modifications introduced at either C2, C3, C4, C5, or C6 of terminal D-Gal, as well as N-propionylation instead of N-acetylation of subterminal D-GlcN were tested for their influence on the alpha-2,3- and alpha-2,6-sialyltransferase acceptor activities. Both ST3Gal enzymes displayed the same narrow acceptor specificity, and only accept reduction of the Gal C2 hydroxyl function. The ST6Gal enzymes, however, do not have the same acceptor specificity. ST6Gal II seems less tolerant towards modifications at Gal C3 and C4 than ST6Gal I, and prefers beta-D-GalpNAc-(1-->4)-beta-D-GlcpNAc (LacdiNAc) as an acceptor substrate, as shown by replacing the Gal C2 hydroxyl group with an N-acetyl function. Finally, a particularly striking feature of all tested sialyltransferases is the activating effect of replacing the N-acetyl function of subterminal GlcNAc by an N-propionyl function.
Asunto(s)
Oligosacáridos/metabolismo , Sialiltransferasas/metabolismo , Acetilglucosamina , Animales , Secuencia de Carbohidratos , Glucosamina/análogos & derivados , Humanos , Oligosacáridos/química , Ratas , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Sialyl-Tn is a carbohydrate antigen overexpressed in several epithelial cancers including breast cancer, and usually associated with poor prognosis. Sialyl-Tn is synthesized by a CMP-Neu5Ac: GalNAc alpha2,6-sialyltransferase: ST6GalNAc I, which catalyzes the transfer of a sialic acid residue in alpha2,6-linkage to the GalNAcalpha1-O-Ser/Thr structure. The resulting disaccharide (Neu5Acalpha2-6GalNAcalpha1-O-Ser/Thr) cannot be further elongated and sialyl-Tn expression results therefore in a shortening of the O-glycan chains. However, usual breast cancer cell lines express neither ST6GalNAc I nor sialyl-Tn antigen. We have previously shown that stable transfection of MDA-MB-231 cells with the hST6GalNAc I cDNA induces the sialyl-Tn antigen expression at the cell surface and leads to a decreased cell growth and an increased cell migration. We describe herein the generation of new T47-D clones expressing sialyl-Tn antigen after hST6GalNAc I cDNA stable transfection. sialyl-Tn antigen is carried by several high molecular weight membrane bound O-glycoproteins, including MUC1. We show that sialyl-Tn expression induces a decrease of cell growth and adhesion, and an increase of cell migration in sialyl-Tn positive clones compared to mock transfected cells. These observations show that the alteration of the O-glycans pattern is sufficient to modify the biological features of cancer cells. These T47-D sialyl-Tn expressing clones might allow further in vivo investigation to determine precisely the impact of such O-glycosylation modifications on breast cancer development.