RESUMEN
Metastasis accounts for most of cancer-related deaths. Paracrine signaling between tumor cells and the stroma induces changes in the tumor microenvironment required for metastasis. Transcription factor c-Myb was associated with breast cancer (BC) progression but its role in metastasis remains unclear. Here we show that increased c-Myb expression in BC cells inhibits spontaneous lung metastasis through impaired tumor cell extravasation. On contrary, BC cells with increased lung metastatic capacity exhibited low c-Myb levels. We identified a specific inflammatory signature, including Ccl2 chemokine, that was expressed in lung metastatic cells but was suppressed in tumor cells with higher c-Myb levels. Tumor cell-derived Ccl2 expression facilitated lung metastasis and rescued trans-endothelial migration of c-Myb overexpressing cells. Clinical data show that the identified inflammatory signature, together with a MYB expression, predicts lung metastasis relapse in BC patients. These results demonstrate that the c-Myb-regulated transcriptional program in BCs results in a blunted inflammatory response and consequently suppresses lung metastasis.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/patología , Quimiocina CCL2/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/secundario , Recurrencia Local de Neoplasia/patología , Proteínas Proto-Oncogénicas c-myb/metabolismo , Animales , Apoptosis , Biomarcadores de Tumor/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Proliferación Celular , Quimiocina CCL2/genética , Femenino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/metabolismo , Pronóstico , Proteínas Proto-Oncogénicas c-myb/genética , Células Tumorales Cultivadas , Microambiente Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Activation of hypoxia-inducible factor (HIF) is due to loss of von Hippel-Lindau protein (pVHL) function in most clear cell renal cell carcinomas (ccRCCs). Here we describe a novel pVHL-independent mechanism of HIF regulation and identify nuclear factor (NF)-κB essential modulator (NEMO) as a hitherto unknown oncogenic factor influencing human ccRCC progression. Over 60% of human ccRCCs (n=157) have negative or weak NEMO protein expression by immunohistochemistry. Moderate/strong NEMO protein expression is more frequent in VHL wild-type ccRCCs. We show that NEMO stabilizes HIFα via direct interaction and independently of NF-κB signaling in vitro. NEMO prolongs tumor cell survival via regulation of apoptosis and activation of epithelial-to-mesenchymal transition, facilitating tumor metastasis. Our findings suggest that NEMO-driven HIF activation is involved in progression of ccRCC. Therefore, NEMO may represent a clinically relevant link between NF-κB and the VHL/HIF pathways. Targeting NEMO with specific inhibitors in patients with metastatic ccRCC could be a novel treatment approach in patients with ccRCC expressing functional pVHL.
Asunto(s)
Carcinoma de Células Renales/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Quinasa I-kappa B/metabolismo , Neoplasias Renales/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Animales , Carcinoma de Células Renales/patología , Muerte Celular/fisiología , Movimiento Celular/fisiología , Progresión de la Enfermedad , Femenino , Xenoinjertos , Humanos , Neoplasias Renales/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , FN-kappa B/metabolismo , Transducción de Señal , Regulación hacia ArribaRESUMEN
Heparanase-1 (HPSE) plays a pivotal role in structural remodeling of the ECM and the glycocalyx, thus conferring protumorigenic, proangiogenic and prometastatic properties to many cancer entities. In addition to its extracellular function, recent studies suggest an intracellular activity of HPSE with a largely unknown significance during tumor progression. Therefore, we investigated the relevance of the dual functions of HPSE to malignant melanoma in vitro, as well as in different mouse melanoma models based on the intradermal or intravenous injection of melanoma cells. Consistent with its extracellular action, an HPSE deficiency led to a reduced shedding of the glycocalyx accompanied by a reduced availability of vascular endothelial growth factor, affecting tumor growth and vascularization. In contrast, we measured an elevated expression of the protumorigenic factors pentraxin-3, tissue factor, TNF-α and most prominently, MMP-9, upon HPSE knockdown. In vivo, an HPSE deficiency was related to increased lymph node metastasis. Since the inhibition of its extracellular function with heparin was unable to block the gene regulatory impact of HPSE, we proposed an intracellular mechanism. Immunostaining revealed a counter-staining of HPSE and NF-κB in the nucleus, suggesting a close relationship between both proteins. This finding was further supported by the discovery of a direct charge-driven molecular interaction between HPSE and DNA by using atomic force microscopy and a co-precipitation approach. Our findings are novel and point towards a dual function for HPSE in malignant melanoma with a protumorigenic extracellular activity and a tumor-suppressive nuclear action. The identification of molecular strategies to shuttle extracellular HPSE into the nuclei of cancer cells could provide new therapeutic options.
Asunto(s)
Núcleo Celular/metabolismo , ADN/metabolismo , Glucuronidasa/metabolismo , Melanoma/patología , Animales , Línea Celular Tumoral , Núcleo Celular/genética , Progresión de la Enfermedad , Perros , Humanos , Metástasis Linfática , Células de Riñón Canino Madin Darby , Melanoma/enzimología , Ratones , FN-kappa B/metabolismo , Neoplasias Experimentales , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The tumor microenvironment consists of stromal cells and leukocytes that contribute to cancer progression. Cross-talk between tumor cells and their microenvironment is facilitated by a variety of soluble factors, including growth factors and cytokines such as chemokines. Due to a wide expression of chemokine receptors on cells in the tumor microenvironment, including tumor cells, chemokines affect various processes such as leukocyte recruitment, angiogenesis, tumor cell survival, tumor cell adhesion, proliferation, vascular permeability, immune suppression, invasion and metastasis. Inflammatory chemokines are instrumental players in cancer-related inflammation and significantly contribute to numerous steps during metastasis. Recruitment of myeloid-derived cells to metastatic sites is mainly mediated by the inflammatory chemokines CCL2 and CCL5. Tumor cell homing and extravasation from the circulation to distant organs are also regulated by inflammatory chemokines. Recent experimental evidence demonstrated that besides leukocyte recruitment, tumor cell-derived CCL2 directly activated endothelial cells and together with monocytes facilitated tumor cell extravasation, in a CCL2- and CCL5-dependent manner. Furthermore, CX3CL1 expression in the bone facilitated metastasis of CX3CR1 expressing tumor cells to this site. Current findings in preclinical models strongly suggest that inflammatory chemokines have an important role during metastasis and targeting of the chemokine axis might have a therapeutic potential.
Asunto(s)
Quimiocinas/inmunología , Inflamación/inmunología , Neoplasias/inmunología , Neoplasias/patología , Microambiente Tumoral/inmunología , Animales , Humanos , Inflamación/patología , Metástasis de la NeoplasiaRESUMEN
BACKGROUND: Cancer-associated thrombosis and enduring inflammation are strongly associated with cancer progression and metastasis. Heparin is the mostly clinically used anticoagulant/antithrombotic drug, and has recently been shown to exhibit antimetastatic and anti-inflammatory activities that are linked to inhibition of P-selectin and/or L-selectin. P-selectin-mediated platelet-tumor cell and tumor cell-endothelium interactions facilitate the initial steps of metastasis. OBJECTIVES AND METHODS: The aim of the present study was to determine the capacity of dermatan sulfates to inhibit P-selectin and to test their potential to affect thrombosis, inflammation and metastasis in respective experimental mouse models. RESULTS: Two dermatan sulfates isolated from the ascidians Styela plicata and Phallusia nigra, composed of the same disaccharide core structure (IdoA2-GalNAc)(n) , but sulfated at carbon 4 or 6 of the GalNAc, respectively, have opposed heparin cofactor II (HCII) activities and are potent inhibitors of P-selectin. The ascidian dermatan sulfates effectively attenuated metastasis of both MC-38 colon carcinoma and B16-BL6 melanoma cells and the infiltration of inflammatory cells in a thioglycollate peritonitis mouse model. Moreover, both glycosaminoglycans reduced thrombus size in an FeCl(3) -induced arterial thrombosis model, irrespective of their HCII activities. The analysis of arterial thrombi demonstrated markedly reduced platelet deposition after dermatan sulfate treatment, suggesting that the glycosaminoglycan inhibited P-selectin and thereby the binding of activated platelets during thrombus formation. CONCLUSIONS: Collectively, these findings provide evidence that specific inhibition of P-selectin represents a potential therapeutic target in thrombosis, inflammation and metastasis, and that ascidian dermatan sulfates may serve as antiselectin agents.
Asunto(s)
Dermatán Sulfato/farmacología , Selectina-P/antagonistas & inhibidores , Urocordados/química , Animales , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/secundario , Dermatán Sulfato/aislamiento & purificación , Humanos , Inflamación/prevención & control , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/secundario , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Metástasis de la Neoplasia/prevención & control , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/secundario , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , Selectina-P/genética , Selectina-P/fisiología , Trombosis/prevención & controlRESUMEN
Hypoxia-elicited adaptations of tumor cells are essential for tumor growth and cancer progression. Although ample evidence exists for a positive correlation between hypoxia-inducible factors (HIFs) and tumor formation, metastasis and bad prognosis, the function of the HIF-α protein stability regulating prolyl-4-hydroxylase domain enzyme PHD2 in carcinogenesis is less well understood. In this study, we demonstrate that downregulation of PHD2 leads to increased tumor growth in a hormone-dependent mammary carcinoma mouse model. Tissue microarray analysis of PHD2 protein expression in 281 clinical samples of human breast cancer showed significantly shorter survival times of patients with low-level PHD2 tumors over a period of 10 years. An angiogenesis-related antibody array identified, amongst others, amphiregulin to be increased in the absence of PHD2 and normalized after PHD2 reconstitution. Cultivation of endothelial cells in conditioned media derived from PHD2-downregulated cells resulted in enhanced tube formation that was blocked by the addition of neutralizing anti-amphiregulin antibodies. Functionally, amphiregulin was regulated on the transcriptional level specifically by HIF-2 but not HIF-1. Our data suggest that PHD2/HIF-2/amphiregulin signaling has a critical role in the regulation of breast tumor progression and propose PHD2 as a potential tumor suppressor in breast cancer.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neoplasias de la Mama/metabolismo , Glicoproteínas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Anfirregulina , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular , Línea Celular Tumoral , Progresión de la Enfermedad , Familia de Proteínas EGF , Femenino , Regulación Neoplásica de la Expresión Génica , Glicoproteínas/genética , Humanos , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Immunoblotting , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular/genética , Estimación de Kaplan-Meier , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos , Ratones Desnudos , Persona de Mediana Edad , Procolágeno-Prolina Dioxigenasa/genética , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante HeterólogoRESUMEN
The adherence of sickle red blood cells (RBCs) to the vascular endothelium may contribute to painful vaso-occlusion in sickle cell disease. Sickle cell adherence involves several receptor-mediated processes and may be potentiated by the up-regulated expression of adhesion molecules on activated endothelial cells. Recent results showed that thrombin rapidly increases the adhesivity of endothelial cells for sickle erythrocytes. The current report presents the first evidence for the novel adhesion of normal and, to a greater extent, sickle RBCs to endothelial P-selectin. Studies of the possible interaction of erythrocytes with P-selectin revealed that either P-selectin blocking monoclonal antibodies or sialyl Lewis tetrasaccharide inhibits the enhanced adherence of normal and sickle cells to thrombin-treated endothelial cells. Both RBC types also adhere to immobilized recombinant P-selectin. Pretreating erythrocytes with sialidase reduces their adherence to activated endothelial cells and to immobilized recombinant P-selectin. Herein the first evidence is presented for the binding of normal or sickle erythrocytes to P-selectin. This novel finding suggests that P-selectin inhibition be considered as a potential approach to therapy for the treatment of painful vaso-occlusion in sickle cell disease.
Asunto(s)
Anemia de Células Falciformes/fisiopatología , Adhesión Celular , Endotelio Vascular/fisiopatología , Eritrocitos Anormales/fisiología , Selectina-P/fisiología , Anticuerpos Monoclonales/farmacología , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Eritrocitos/fisiología , Humanos , Neuraminidasa/farmacología , Oligosacáridos/farmacología , Selectina-P/inmunología , Antígeno Sialil Lewis X , Trombina/farmacologíaAsunto(s)
Imagenología Tridimensional/métodos , Neoplasias/sangre , Neoplasias/patología , Animales , Plaquetas/citología , Plaquetas/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Proteínas Fluorescentes Verdes , Heparina/farmacología , Heparina/uso terapéutico , Humanos , Selectina L/metabolismo , Proteínas Luminiscentes , Ratones , Microscopía Fluorescente , Metástasis de la Neoplasia/tratamiento farmacológico , Metástasis de la Neoplasia/patología , Neoplasias/tratamiento farmacológico , Selectina-P/metabolismoRESUMEN
Independent studies indicate that expression of sialylated fucosylated mucins by human carcinomas portends a poor prognosis because of enhanced metastatic spread of tumor cells, that carcinoma metastasis in mice is facilitated by formation of tumor cell complexes with blood platelets, and that metastasis can be attenuated by a background of P-selectin deficiency or by treatment with heparin. The effects of heparin are not primarily due to its anticoagulant action. Other explanations have been suggested but not proven. Here, we bring together all these unexplained and seemingly disparate observations, showing that heparin treatment attenuates tumor metastasis in mice by inhibiting P-selectin-mediated interactions of platelets with carcinoma cell-surface mucin ligands. Selective removal of tumor mucin P-selectin ligands, a single heparin dose, or a background of P-selectin deficiency each reduces tumor cell-platelet interactions in vitro and in vivo. Although each of these maneuvers reduced the in vivo interactions for only a few hours, all markedly reduce long-term organ colonization by tumor cells. Three-dimensional reconstructions by using volume-rendering software show that each situation interferes with formation of the platelet "cloak" around tumor cells while permitting an increased interaction of monocytes (macrophage precursors) with the malignant cells. Finally, we show that human P-selectin is even more sensitive to heparin than mouse P-selectin, giving significant inhibition at concentrations that are in the clinically acceptable range. We suggest that heparin therapy for metastasis prevention in humans be revisited, with these mechanistic paradigms in mind.
Asunto(s)
Plaquetas/metabolismo , Heparina/metabolismo , Mucinas/metabolismo , Neoplasias/fisiopatología , Selectina-P/metabolismo , Adenocarcinoma , Animales , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/inmunología , Metástasis de la Neoplasia , Neoplasias/metabolismo , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/metabolismo , Selectina-P/genética , Células Tumorales CultivadasRESUMEN
Surface glycosylation of endothelial cells is relevant to various processes including coagulation, inflammation, metastasis, and lymphocyte homing. One of the essential sugars involved in these processes is fucose linked alpha1-->3 to N-acetylglucosamine. A family of alpha1,3-fucosyltransferases (FucTs) called FucT-III, IV, V, VI, VII, and IX is able to catalyze such fucosylations. Reverse transcription-PCR analysis revealed that human umbilical vein endothelial cells express all of the FucTs except FucT-IX. The predominant activity, as inferred by acceptor specificity of enzyme activity in cell lysates, is compatible with the presence of FucT-VI. By using an antibody to recombinant soluble FucT-VI, the enzyme colocalized with beta4-galactosyltransferase-1 to the Golgi apparatus. By using a polyclonal antiserum raised against a 17-aa peptide of the variable (stem) region of the FucT-VI, immunocytochemical staining of FucT-VI was restricted to Weibel-Palade bodies, as determined by colocalization with P-selectin and von Willebrand factor. SDS/PAGE immunoblotting and amino acid sequencing of internal peptides confirmed the identity of the antigen isolated by the peptide-specific antibody as FucT-VI. Storage of a fucosyltransferase in Weibel-Palade bodies suggests a function independent of Golgi-associated glycosylation.
Asunto(s)
Fucosiltransferasas/análisis , Cuerpos de Weibel-Palade/enzimología , Animales , Células CHO , Cricetinae , Endotelio Vascular/citología , Técnica del Anticuerpo Fluorescente Indirecta , Fucosiltransferasas/genética , Expresión Génica , Humanos , Conejos , Análisis de SecuenciaRESUMEN
The major alpha1,3fucosyltransferase activity in plasma, liver, and kidney is related to fucosyltransferase VI which is encoded by the FUT6 gene. Here we demonstrate the presence of alpha1, 3fucosyltransferase VI (alpha3-FucT VI) in the human HepG2 hepatoma cell line by specific activity assays, detection of transcripts, and the use of specific antibodies. First, FucT activity in HepG2 cell lysates was shown to prefer sialyl-N-acetyllactosamine as acceptor substrate indicating expression of alpha3-FucT VI. RT-PCR analysis further confirmed the exclusive presence of the alpha3-FucT VI transcripts among the five human alpha3-FucTs cloned to date. alpha3-FucT VI was colocalized with beta1,4galactosyltransferase I (beta4-GalT I) to the Golgi apparatus by dual confocal immunostaining. Pulse/chase analysis of metabolically labeled alpha3-FucT VI showed maturation of alpha3-FucT VI from the early 43 kDa form to the mature, endoglycosidase H-resistant form of 47 kDa which was detected after 2 h of chase. alpha3-FucT VI was released to the medium and accounted for 50% of overall cell-associated and released enzyme activity. Release occurred by proteolytical cleavage which produced a soluble form of 43 kDa. Monensin treatment segregated alpha3-FucT VI from the Golgi apparatus to swollen peripheral vesicles where it was colocalized with beta4-GalT I while alpha2,6(N)sialyltransferase remained associated with the Golgi apparatus. Both constitutive secretion of alpha3-FucT VI and its monensin-induced relocation to vesicles analogous to beta4-GalT I suggest a similar post-Golgi pathway of both alpha3-FucT VI and beta4-GalT I.
Asunto(s)
Fucosiltransferasas/biosíntesis , Galactosiltransferasas/análisis , Aparato de Golgi/enzimología , Proteínas de Neoplasias/biosíntesis , Animales , Células CHO , Carcinoma Hepatocelular/patología , Cricetinae , Cricetulus , Espacio Extracelular/metabolismo , Fucosiltransferasas/química , Fucosiltransferasas/genética , Fucosiltransferasas/metabolismo , Humanos , Neoplasias Hepáticas/patología , Microscopía Confocal , Microscopía Fluorescente , Peso Molecular , Monensina/farmacología , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/enzimologíaRESUMEN
Selectins are adhesion molecules that mediate calcium-dependent cell-cell interactions among leukocytes, platelets, and endothelial cells. The naturally occurring vascular ligands for the selectins are mostly mucin-type glycoproteins. Increased expression and altered glycosylation of mucins are known to be prominent features of carcinoma progression. We have previously shown that all three selectins bind to colon carcinoma cell lines in a calcium-dependent fashion and that carcinoma growth and metastasis formation are attenuated in P-selectin-deficient mice. Here we show that the three recombinant soluble selectins recognize ligands within primary colon carcinoma tissue samples. Affinity chromatography showed that the ligands for all three selectins are O-sialoglycoprotease-sensitive mucins that are recognized in a calcium- and sialic acid-dependent manner. Furthermore, there are separate binding sites on the mucins for each selectin, allowing cross-binding of a single mucin molecule by more than one selectin. We also show that the selectin ligands on purified carcinoma mucins can mediate at least four different pathological interactions among platelets, leukocytes, and endothelial cells. These findings could explain some of the adhesive events of blood-borne tumor cells reported to occur with leukocytes, platelets, and endothelial cells, which are believed to play a part in modulating some early events in tumor metastases.
Asunto(s)
Plaquetas/patología , Comunicación Celular , Neoplasias del Colon/metabolismo , Endotelio Vascular/patología , Leucocitos/patología , Mucinas/metabolismo , Selectinas/fisiología , Animales , Plaquetas/metabolismo , Calcio/farmacología , Cromatografía de Afinidad , Neoplasias del Colon/patología , Relación Dosis-Respuesta a Droga , Selectina E/metabolismo , Endotelio Vascular/metabolismo , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/metabolismo , Humanos , Inmunohistoquímica , Selectina L/metabolismo , Leucocitos/metabolismo , Ligandos , Ratones , Ratones Noqueados , Modelos Biológicos , Mucinas/farmacología , Trasplante de Neoplasias , Selectina-P/metabolismo , Trombina/metabolismoRESUMEN
Selectins are adhesion receptors that normally recognize certain vascular mucin-type glycoproteins bearing the carbohydrate structure sialyl-Lewisx. The clinical prognosis and metastatic progression of many epithelial carcinomas has been correlated independently with production of tumor mucins and with enhanced expression of sialyl-Lewisx. Metastasis is thought to involve the formation of tumor-platelet-leukocyte emboli and their interactions with the endothelium of distant organs. We provide a link between these observations by showing that P-selectin, which normally binds leukocyte ligands, can promote tumor growth and facilitate the metastatic seeding of a mucin-producing carcinoma. P-selectin-deficient mice showed significantly slower growth of subcutaneously implanted human colon carcinoma cells and generated fewer lung metastases from intravenously injected cells. Three potential pathophysiological mechanisms are demonstrated: first, intravenously injected tumor cells home to the lungs of P-selectin deficient mice at a lower rate; second, P-selectin-deficient mouse platelets fail to adhere to tumor cell-surface mucins; and third, tumor cells lodged in lung vasculature after intravenous injection often are decorated with platelet clumps, and these are markedly diminished in P-selectin-deficient animals.
Asunto(s)
Neoplasias Colorrectales/patología , Selectina-P/fisiología , Animales , Secuencia de Bases , Plaquetas/patología , División Celular/fisiología , Cruzamientos Genéticos , Femenino , Humanos , Neoplasias Pulmonares/secundario , Masculino , Ratones , Mucinas/fisiología , Metástasis de la Neoplasia/genética , Trasplante de Neoplasias , Selectina-P/genética , Formación de RosetaRESUMEN
Carbohydrate-deficient glycoprotein syndromes (CDGS) type I are a group of genetic diseases characterized by a deficiency of N-linked protein glycosylation in the endoplasmic reticulum. The majority of these CDGS patients have phosphomannomutase (PMM) deficiency (type A). This enzyme is required for the synthesis of GDP-mannose, one of the substrates in the biosynthesis of the dolichol-linked oligosaccharide Glc3Man9GlcNAc2. This oligosaccharide serves as the donor substrate in the N-linked glycosylation process. We report on the biochemical characterization of a novel CDGS type I in fibroblasts of four related patients with normal PMM activity but a strongly reduced ability to synthesize glucosylated dolichol-linked oligosaccharide leading to accumulation of dolichol-linked Man9GlcNAc2. This deficiency in the synthesis of dolichol-linked Glc3Man9GlcNAc2 oligosaccharide explains the hypoglycosylation of serum proteins in these patients, because nonglucosylated oligosaccharides are suboptimal substrates in the protein glycosylation process, catalyzed by the oligosaccharyltransferase complex. Accordingly, the efficiency of N-linked protein glycosylation was found to be reduced in fibroblasts from these patients.
Asunto(s)
Trastornos Congénitos de Glicosilación/metabolismo , Dolicoles/metabolismo , Glucosa/metabolismo , Hexosiltransferasas , Proteínas de la Membrana , Oligosacáridos/metabolismo , Secuencia de Carbohidratos , Niño , Preescolar , Trastornos Congénitos de Glicosilación/clasificación , Consanguinidad , Femenino , Fibroblastos/metabolismo , Glucosiltransferasas/metabolismo , Glicosilación , Humanos , Masculino , Datos de Secuencia Molecular , Países Bajos , Fosfotransferasas (Fosfomutasas)/análisis , Sialoglicoproteínas/sangre , Transferasas/metabolismo , Transferrina/análisisRESUMEN
Peripheral alpha1,3-fucosylation of glycans occurs by the action of either one of five different alpha1,3-fucosyltransferases (Fuc-Ts) cloned to date. Fuc-TVI is one of the alpha1,3-fucosyltransferases which is capable to synthesize selectin ligands. The major alpha1, 3-fucosyltransferase activity in human plasma is encoded by the gene for fucosyltransferase VI, which presumably originates from liver cells. While the sequence, chromosomal localization, and kinetic properties of Fuc-TVI are known, immunocytochemical localization and trafficking studies have been impossible because of the lack of specific antibodies. Here we report on the development and characterization of a peptide-specific polyclonal antiserum monospecific to Fuc-TVI and an antiserum to purified soluble recombinant Fuc-TVI crossreactive with Fuc-TIII and Fuc-TV. Both antisera were applied for immunodetection in stably transfected CHO cells expressing the full-length form of this enzyme (CHO clone 61/11). Fuc-TVI was found to be a resident protein of the Golgi apparatus. In addition, more than 30% of cell-associated and released enzyme activity was found in the medium. Maturation and release of Fuc-TVI was analyzed in metabolically labeled CHO 61/11 cells followed by immunoprecipitation. Fuc-TVI occurred in two forms of 47 kDa and 43 kDa bands, while the secreted form was detected as a 43 kDa. These two different intracellular forms arose by posttranslational modification, as shown by pulse-chase experiments. Fuc-TVI was released to the supernatant by proteolytic cleavage as a partially endo-H resistant glycoform.
Asunto(s)
Células CHO/enzimología , Fucosiltransferasas/química , Animales , Cricetinae , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Fucosiltransferasas/inmunología , Aparato de Golgi/enzimología , Hexosaminidasas/metabolismo , Inmunohistoquímica , Isoenzimas/análisis , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa , Peso Molecular , Procesamiento Proteico-Postraduccional/fisiología , Proteínas Recombinantes/análisis , Transfección/genéticaRESUMEN
His6-tag technology has been introduced for easy purification of recombinant proteins expressed in Escherichia coli. Aiming at extending this technology to purification of glycoproteins expressed in Saccharomyces cerevisiae or in animal cells, respectively, we adapted this protocol to recombinant soluble beta-1,4-galactosyltransferase (rgal-T). A His6-tag was introduced to the N-terminus of the protein (hisGal-T). The Histagged enzyme expressed in yeast S. cerevisiae was enzymically active but could not be purified from the cell extract by virtue of the His6-tag. Binding efficiency of hisGal-T was found to be impaired by a bulky N-glycan close to the His-tag. Removal of the unique site of N-glycosylation using site-directed mutagenesis restored binding of hisGal-T to the Ni-NTA resin. In comparison N-glycosylated hisGal-T transiently expressed in COS cells was secreted as a soluble active enzyme and could be purified in one single step by virtue of the His6-tag.
Asunto(s)
Células COS/enzimología , N-Acetil-Lactosamina Sintasa/genética , N-Acetil-Lactosamina Sintasa/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Saccharomyces cerevisiae/genética , Animales , Western Blotting , Cromatografía en Agarosa , Enzimas Inmovilizadas , Expresión Génica , Glicosilación , Histidina , Mutagénesis Sitio-Dirigida , N-Acetil-Lactosamina Sintasa/metabolismo , Níquel/metabolismo , Plásmidos/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimología , Solubilidad , Transfección/genéticaRESUMEN
beta-1,4-Galactosyltransferase (Gal-T, EC 2.4.1.38) transfers galactose (Gal) from UDP-Gal to N-acetyl-D-glucosamine or a derivative GlcNAc-R. Soluble Gal-T, purified from human breast milk, was shown to be very heterogeneous by isoelectric focusing (IEF). In order to produce sufficient homogeneous enzyme for three-dimensional analysis, the human enzyme (hGal-T) has been expressed in Saccharomyces cerevisiae, production scaled up to 187 U recombinant Gal-T (rGal-T) and purified. The purification protocol was based on chromatography on concanavalin-A-Sepharose followed by affinity chromatographies on GlcNAc-Sepharose and alpha-lactalbumin-Sepharose. Analysis by SDS/PAGE revealed hyperglycosylation at the single N-glycosylation site, preventing recognition by antibodies. Analysis by IEF revealed considerable heterogeneity of rGal-T. The N-glycan could be removed by treatment with endoglycosidase H (endo H). The N-deglycosylated form of rGal-T retained full activity and showed only three isoforms by IEF analysis. Then we abolished the single N-glycosylation consensus sequence by site-directed mutagenesis changing Asn69-->Asp. The soluble mutated enzyme (N-deglycosylated rGal-T) was expressed in S. cerevisiae and its production scaled up to 60 U.N-deglycosylated rGal-T was purified to electrophoretic homogeneity. When analyzed by IEF, N-deglycosylated rGal-T was resolved in two bands. The O-glycans could be removed by jack bean alpha-mannosidase treatment and the completely deglycosylated Gal-T appeared homogeneous by IEF. The kinetic parameters of N-deglycosylated rGal-T were shown not to differ to any significant extent from those of the hGal-T. No significant changes in CD spectra were observed between hGal-T and N-deglycosylated rGal-T. Light-scattering analysis revealed dimerization of both enzymes. These data indicate that N-deglycosylated rGal-T was correctly folded, homogeneous and thus suitable for crystallization experiments.
Asunto(s)
Leche Humana/enzimología , N-Acetil-Lactosamina Sintasa/aislamiento & purificación , N-Acetil-Lactosamina Sintasa/metabolismo , Saccharomyces cerevisiae/enzimología , Sitios de Unión , Cromatografía de Afinidad , Dicroismo Circular , Clonación Molecular , ADN Complementario , Electroforesis en Gel de Poliacrilamida , Femenino , Fermentación , Glicosilación , Humanos , Focalización Isoeléctrica , Cinética , Lactalbúmina , Peso Molecular , N-Acetil-Lactosamina Sintasa/química , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
The fucosyltransferases constitute a family of glycosyltransferases incorporating fucose residues into glycoprotein or glycolipid glycans. They afford one of the possible termination steps of glycoconjugate biosynthesis creating the sialyl Lewisx or sialyl Lewisa determinant, which play an important role in cell-cell interaction. While cDNA, chromosomal localization and kinetic properties of a number of fucosyltransferases are known, immunocytochemical localization and trafficking studies have been delayed because of the lack of specific antibodies due to the pronounced homology of alpha 1, 3 fucolsyltransferases III, V and VI. Here we report development and characterization of monospecific polyclonal antibodies to alpha 1-3 fucosyltransferase V (FucT-V) and their application for immunodetection in transfected cells. Antisera against FucT-V were raised in two different ways: first by producing a fusion protein beta-galactosidase-FucT-V in Escherichia coli, and by synthesizing a peptide stretch specific for FucT-V. Polyclonal antisera were raised against each of both antigens and characterized by enzyme-linked immunosorbent assay, neutralization of activity, immunoblotting, immunofluorescence and immunoprecipitation of metabolically labeled COS cells, transiently transfected with cDNA encoding FucT-V. Both antibodies recognized only FucT-V. No cross-reactivity to FucT-III or FucT-VI was observed. FucT-V was localized mainly to the Golgi apparatus by colocalization with beta 1, 4-galactosyltransferase, and to the cell surface of COS, CHO and HeLa cells. Expression of FucT-V in COS cells revealed three enzyme forms of 58, 53 and 50 kDa, respectively. These size differences arose by post-translational modifications, as shown by pulse-chase experiments. Our results indicate that alpha 1-3 fucosyltransferase is a Golgi-associated enzyme and suggest its possible occurrence on the cell surface.
Asunto(s)
Fucosiltransferasas/análisis , Animales , Especificidad de Anticuerpos , Células CHO , Línea Celular , Cricetinae , Ensayo de Inmunoadsorción Enzimática , Fucosiltransferasas/inmunología , Células HeLa , Humanos , Inmunohistoquímica , Pruebas de PrecipitinaRESUMEN
Expression of recombinant full length human alpha 2,6(N)sialyltransferase has been scaled-up in S. cerevisiae in a 150-l bioreactor yielding 47 U at a concentration of 0.31 U/l. The protein specific activity as measured in reconstituted yeast lyophilisate was 0.8 mU/mg protein. The recombinant enzyme exhibited similar Michaelis constants as previously determined for the native rat enzyme. By immunoblotting the enzyme was shown to be heterogeneous by size (44-48 kD) and N-glycosylated. We conclude that recombinant alpha 2,6(N)sialyltransferase expressed in S. cerevisiae is retained in the endoplasmic reticulum as a fully active enzyme.