RESUMEN
The polyspermy occurrence is considerably lower under in vivo compared to in vitro embryo culture conditions, suggesting that the presence of some factors in the maternal environment is responsible for this. The α-L-fucosidase (FUCA) is a natural glycosidase present in the oviductal fluid, therefore, this study aimed at investigating the effect of adding FUCA to the hardening of the zona pellucida (ZP), polyspermy control, and embryonic yield and quality of bovine blastocysts produced in vitro. In the first experiment, the effect of FUCA (0.125 U/mL) was evaluated during the entire in vitro fertilization (IVF). However, it was demonstrated to be embryotoxic by completely inhibiting the blastocyst formation. In the second experiment, the FUCA (0.125 U/mL) was tested as short-term incubation before IVF (pre-fertilization step) for 30 min or 2 h, which demonstrated that FUCA treatment for 30 min resulted in ZP hardening. In the third experiment, a pre-fertilization FUCA treatment (1 h) at different concentrations (0, 0.0625, and 0.125 U/mL) showed that FUCA (0.0625 U/mL) improved pre-fertilization ZP hardening and tended to increase monospermic fertilization rates but did not improve embryo yield and quality. Together, it has been demonstrated that FUCA can induce oocyte pre-fertilization ZP hardening and might improve monospermic fertilization performance, and this effect is dependent on both variables (protein concentration and incubation time).
Asunto(s)
Zona Pelúcida , alfa-L-Fucosidasa , Bovinos , Animales , alfa-L-Fucosidasa/farmacología , Oocitos , Fertilización In Vitro/veterinaria , Fertilización In Vitro/métodos , FertilizaciónRESUMEN
Cancer-associated adipocytes (CAAs) have been suggested to promote tumor progression. Yet, the role of CAAs in triple-negative breast cancer (TNBC) is poorly investigated. We compared the expression of secretory protein-encoding genes in CAAs and control adipocytes. The effect of key secretory protein(s) on TNBC cell behaviors was explored. CAAs expressed and secreted FUCA2 at greater levels than control adipocytes. When FUCA2 activity was blocked with a neutralizing antibody, TNBC cell proliferation and migration induced by CAA-conditioned medium was impaired. In contrast, supplement of exogenous FUCA2 protein reinforced the proliferation, colony formation, and migration of TNBC cells. In vivo studies confirmed that FUCA2 exposure enhanced tumorigenesis and metastasis of TNBC cells. Mechanistic investigation revealed that FUCA2 induced TNBC aggressiveness through TM9SF3-dependent signaling. Depletion of TM9SF3 blocked CAA- and FUCA2-induced TNBC cell proliferation and migration. Compared to adjacent breast tissues, TNBC tissues had increased expression of TM9SF3. Moreover, high TM9SF3 expression was associated with advanced TNM stage, lymph node metastasis, and shorter overall survival of TNBC patients. Altogether, CAAs secrete FUCA2 to promote TNBC growth and metastasis through interaction with TM9SF3. Inhibition of TM9SF3 may represent a potential therapeutic strategy in the treatment of TNBC.
Asunto(s)
Neoplasias de la Mama Triple Negativas , Adipocitos/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Transducción de Señal , Neoplasias de la Mama Triple Negativas/metabolismo , alfa-L-Fucosidasa/genética , alfa-L-Fucosidasa/metabolismo , alfa-L-Fucosidasa/farmacologíaRESUMEN
Glycans containing α-L-fucose participate in diverse interactions between cells and extracellular matrix. High glycan expression on cell surface is often associated with neoplastic progression. The lysosomal exoenzyme, α-L-fucosidase-1 (FUCA-1) removes fucose residues from glycans. The FUCA-1 gene is down-regulated in highly aggressive and metastatic human tumors. However, the role of FUCA-1 in tumor progression remains unclear. It is speculated that its inactivation perturbs glycosylation of proteins involved in cell adhesion and promotes cancer. FUCA-1 expression of various thyroid normal and cancer tissues assayed by immunohistochemical (IHC) staining was high in normal thyroids and papillary thyroid carcinomas (PTC), whereas it progressively decreased in poorly differentiated, metastatic and anaplastic thyroid carcinomas (ATC). FUCA-1 mRNA expression from tissue samples and cell lines and protein expression levels and enzyme activity in thyroid cancer cell lines paralleled those of IHC staining. Furthermore, ATC-derived 8505C cells adhesion to human E-selectin and HUVEC cells was inhibited by bovine α-L-fucosidase or Lewis antigens, thus pointing to an essential role of fucose residues in the adhesive phenotype of this cancer cell line. Finally, 8505C cells transfected with a FUCA-1 containing plasmid displayed a less invasive phenotype versus the parental 8505C. These results demonstrate that FUCA-1 is down-regulated in ATC compared to PTC and normal thyroid tissues and cell lines. As shown for other human cancers, the down-regulation of FUCA-1 correlates with increased aggressiveness of the cancer type. This is the first report indicating that the down-regulation of FUCA-1 is related to the increased aggressiveness of thyroid cancer.
Asunto(s)
Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patología , alfa-L-Fucosidasa/genética , Anaplasia , Animales , Bovinos , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular , Selectina E/metabolismo , Activación Enzimática , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Humanos , Inmunohistoquímica , Invasividad Neoplásica , Metástasis de la Neoplasia , Unión Proteica , alfa-L-Fucosidasa/metabolismo , alfa-L-Fucosidasa/farmacologíaRESUMEN
The activity of α-L-fucosidase in oviductal fluid increases around the time of ovulation. α-L-fucosidase is also associated with the spermatozoal plasma membrane and its substrate, fucose, has been identified in the zona pellucida (ZP) and on the spermatozoal surface, suggesting a role in fertilisation. The aim of the present study was to investigate the role of exogenous α-L-fucosidase during fertilisation. Porcine oocytes were incubated with fucosidase and later subjected to in vitro fertilisation (IVF). No effect on the percentage of oocytes fertilised was observed, although there was a slight decrease in spermatozoa-ZP binding. Fucosidase was then added to IVF medium, and spermatozoa and oocytes were co-incubated for 15 min. A significant increase in spermatozoa-ZP binding and penetration was observed, suggesting a role of the enzyme in the fertilisation ability of spermatozoa. In addition, fluorescence intensity and the patterns of spermatozoa membrane-associated α-L-fucosidase distribution, as assessed by indirect immunofluorescence, were not affected by the presence or absence of exogenous enzyme, suggesting an independent role for the exogenous and spermatozoa-associated enzymes. Addition of exogenous α-L-fucosidase increased the spermatozoal intracellular ionised calcium concentration and tyrosine phosphorylation, suggesting a role in promoting capacitation and, at the same time, protecting spermatozoa from a premature acrosome reaction. Thus, α-L-fucosidase enhances capacitation-associated events in porcine spermatozoa.
Asunto(s)
Oocitos/fisiología , Capacitación Espermática/fisiología , Espermatozoides/fisiología , Porcinos/fisiología , alfa-L-Fucosidasa/farmacología , Animales , Fertilización In Vitro/veterinaria , Masculino , Interacciones Espermatozoide-Óvulo , Espermatozoides/efectos de los fármacos , Zona PelúcidaRESUMEN
Alterations in the glycosylation of few membrane proteins from human placenta during gestation have been documented, but data on N-glycome of placental membrane proteins are still missing. The primary goal of this study was to obtain N-glycan profiles of human placental membrane proteins using a reliable, simple and high-throughput method. The second goal was to examine whether the N-glycan profile alters during gestation. Placental membrane proteins were isolated from women of different ages after first and third trimesters of pregnancy. The N-glycan fingerprint of membrane proteins was obtained using DNA sequencer-assisted fluorophore-assisted carbohydrate electrophoresis (DSA-FACE). Lectin blotting was used to confirm DSA-FACE results. Observed gestation-related alterations were: greater abundance of core-fucosylated and multiantennary N-glycans, but lower amounts of bisected biantennary N-glycans together with a decrease in α2,3-sialylation. Age-related alterations were: more core Fuc and more α2,3-Sia in first trimester placentas from older women than in those from younger women; also less core Fuc and less α2,6-Sia in third trimester placentas from older women compared to those from younger women. This study represents the first N-glycan profiling of placental cell membrane proteins. These data represent a basis for future research on the N-glycome of placental proteins in different (patho)physiological conditions.
Asunto(s)
Placenta/metabolismo , Polisacáridos , Adulto , Electroforesis en Gel de Poliacrilamida/métodos , Femenino , Edad Gestacional , Humanos , Edad Materna , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/metabolismo , Placentación/fisiología , Polisacáridos/análisis , Polisacáridos/metabolismo , Embarazo , Proteínas Gestacionales/análisis , Proteínas Gestacionales/metabolismo , Trimestres del Embarazo/fisiología , Reproducibilidad de los Resultados , alfa-L-Fucosidasa/metabolismo , alfa-L-Fucosidasa/farmacologíaRESUMEN
Glycosylation drives critical processes important for mammalian cell-cell and cell-matrix interactions. Alpha-L-fucose (alpha-L-f) is a key monosaccharide component of oligosaccharides that has been found to be overexpressed during tumor progression. Modification of cell surface fucosylation, we hypothesized, alters tumor cell phenotype and function at the end of the neoplastic progression cascade including tumor invasion. Alpha-L-fucosidase (alpha-L-fase) is a glycosidase that specifically removes (alpha-L-f) from oligosaccharide sites. We first verified the effectiveness of the alpha-L-fase to specifically decrease the level of alpha-L-f on the cell surface of several human breast cancer cell lines and also examined the recovery time for these cells to repopulate their surfaces. To investigate the potential effect of defucosylation on tumor functions, we studied the proliferation, and invasion in vitro of human breast cancer MDA-MB-231 cells as the representative cell model. We further examined several fucose-associated molecules previously shown to be involved in tumor progression, including CD44 and CD15 (Lewis X antigen). We found that alpha-L: -fase pretreatment significantly decreased the invasive capability of breast cancer cells. Deoxyfuconojirimycin (DFJ), a specific alpha-L: -fase inhibitor, reversed this effect. After fucosidase treatment, the level of both CD15 and CD44 were found to be reduced as measured by flow cytometry. alpha-L-fase treatment, further, did not affect tumor cell proliferation in vitro under identical experimental conditions. Gelatin zymography of conditioned media from tumor cells treated with alpha-L-fase demonstrated no change in MMP-2 activity while MMP-9 was significantly reduced. In summary, fucose containing glycans were found widely distributed on the cell surface of breast cancer cells and could be effectively removed by alpha-L-fase treatment. This decreased fucosylation, in turn, was seen to impair the interaction between tumor cells and extracellular matrices, and thus affected key cell functions modulating tumor invasion. Further elucidation of the molecular pathways involved in the inhibition of tumor cell invasion may suggest a rationale for the use of glycobiologic therapeutics to deter tumor progression.
Asunto(s)
Neoplasias de la Mama/fisiopatología , Fucosa/metabolismo , Glicoproteínas de Membrana/metabolismo , alfa-L-Fucosidasa/metabolismo , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Progresión de la Enfermedad , Femenino , Humanos , Receptores de Hialuranos/metabolismo , Antígeno Lewis X/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Invasividad Neoplásica , Estadísticas no Paramétricas , Alcoholes del Azúcar/farmacología , Células Tumorales Cultivadas/metabolismo , alfa-L-Fucosidasa/antagonistas & inhibidores , alfa-L-Fucosidasa/farmacologíaRESUMEN
Glycosylation of proteins plays multiple roles in cell-cell and cell-matrix interactions. Fucose is a monosaccharide associated with glycosylation events and is known to be over-expressed in many malignant tumors. By using alpha-L-fucosidase (alpha-L-fase), a glycosidase that specifically removes alpha-L-fucose (alpha-L-f), we have examined the potential effects of defucosylation on tumor functions, focusing on tumor progression in the context of the interaction of tumor cells with the extracellular microenvironment. In this submission, we report that alpha-L-fase treatment decreases, in static assays, tumor cell adhesion to a wide variety of ECM components including fibronectin, laminin, collagen I, hyaluronic acid and the complex human biomatrix, HuBiogel(R). By immunofluorescence, co-localization of beta1 integrin and alpha-L-f was found to decrease accordingly. Sialyl Lewis X, an alpha-L-f-containing tetrasaccharide, which modulates the rolling of leukocytes and tumor cells on endothelium, was found to be diminished on human breast cancer cells after alpha-L-fase treatment. Using a dynamic flow chamber system, we were able to determine that defucosylation impaired the rolling of mammary cancer cells on human umbilical vein endothelial cells while significantly increasing their flow speed. Further, the rolling capability of these defucosylated tumor cells was also impaired on purified E and P-selectin matrices. Based on these data, we hypothesize that decreased fucosylation impairs the interaction between tumor cells and their external milieu, which in turn, affects key cell functions modulating tumor progression. Building on our previous studies which demonstrated alpha-L-fase decreased tumor cell invasion while significantly reducing MMP-9 activity, when added to the fact that decreased adhesion on HUVEC occurs in the presence of alpha-L-fase also leads us to propose that defucosylation may modulate metastasis, and thus provides a promising additional glycobiotic target for novel therapies.
Asunto(s)
Neoplasias de la Mama/patología , Fucosa/fisiología , Glicoproteínas/fisiología , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Selectina E/análisis , Células Endoteliales/fisiología , Femenino , Humanos , Molécula 1 de Adhesión Intercelular/análisis , Oligosacáridos/sangre , Selectina-P/análisis , Antígeno Sialil Lewis X , alfa-L-Fucosidasa/farmacologíaRESUMEN
Five sulfonium ion derivatives with 1,5-anhydro-5-thio-L-fucitol as a core structure were efficiently synthesized as potential alpha-L-fucosidase inhibitors. The key unit, the tri-O-benzyl derivative of L-fucitol, was readily synthesized from methyl alpha-D-mannopyranoside. Alkylation with methyl iodide or 5-methoxycarbonyl-1-pentyl iodide in acetonitrile containing AgBF4 afforded the corresponding alkylated sulfonium tetrafluoroborates. Alternatively, ring opening of three 1,3-cyclic sulfates in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) containing K2CO3 afforded the corresponding zwitterionic sulfonium sulfates.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Alcoholes del Azúcar/síntesis química , Alcoholes del Azúcar/farmacología , Compuestos de Sulfonio/síntesis química , alfa-L-Fucosidasa/farmacología , Inhibidores Enzimáticos/farmacología , Cinética , Espectroscopía de Resonancia Magnética , Compuestos de Sulfonio/farmacologíaRESUMEN
CD11b/CD18-mediated adhesive interactions play a key role in regulating polymorphonuclear leukocytes (PMN)) migration across intestinal epithelium. However, the identity of epithelial ligands for migrating PMN remains obscure. In this study we investigated the role of carbohydrates in mediating adhesive interactions between T84 intestinal epithelial cells and CD11b/CD18 purified from PMN. Fucoidin, heparin/heparin sulfate, N-acetyl-D-glucosamine, mannose-6-phosphate, and laminarin were found to inhibit adhesion of T84 cells to CD11b/CD18. The most potent inhibitory effects were observed with fucoidin (50% inhibition at 1-5 x 10(-8) M). Binding assays demonstrated that fucoidin directly bound to CD11b/CD18 in a divalent cation- and sulfation-dependent fashion that was blocked by anti-CD11b mAbs. Experiments employing CD11b/CD18 as a probe to blot T84 cell fucosylated proteins purified via fucose-specific lectin column revealed several candidate CD11b/CD18 binding proteins with molecular masses of 95, 50, 30, 25, and 20 kDa. Fucosidase treatment of T84 cells resulted in significantly reduced cell adhesion to CD11b/CD18, while no inhibition was observed after neuraminidase treatment. Finally, significant inhibition of T84 cell adhesion to CD11b/CD18 was observed after blocking cell proteoglycan synthesis with p-nitrophenyl-beta-D-xylopyranoside. These findings implicate epithelial cell surface proteoglycans decorated with sulfated fucose moieties as ligands for CD11b/CD18 during PMN migration across mucosal surfaces.
Asunto(s)
Antígeno CD11b/fisiología , Antígenos CD18/fisiología , Fucosa/fisiología , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Neutrófilos/inmunología , Neutrófilos/metabolismo , Proteoglicanos/fisiología , Unión Competitiva/inmunología , Biotinilación , Antígeno CD11b/metabolismo , Antígenos CD18/metabolismo , Metabolismo de los Hidratos de Carbono , Carbohidratos/farmacología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/inmunología , Fucosa/metabolismo , Humanos , Mucosa Intestinal/citología , Neutrófilos/fisiología , Polisacáridos/metabolismo , Unión Proteica/inmunología , Proteínas/metabolismo , Proteoglicanos/antagonistas & inhibidores , Proteoglicanos/biosíntesis , Proteoglicanos/metabolismo , Sulfatos/metabolismo , Células Tumorales Cultivadas , alfa-L-Fucosidasa/farmacologíaRESUMEN
Our laboratory recently demonstrated the pattern of cell surface glycosylation of nonsecretory central airway epithelium (Dorscheid DR, Conforti AE, Hamann KJ, Rabe KF, and White SR. Histochem J 31: 145-151, 1999), but the role of glycosylation in airway epithelial cell migration and repair is unknown. We examined the functional role of cell surface carbohydrates in wound repair after mechanical injury of 1HAEo(-) human airway epithelial and primary bronchial epithelial monolayers. Wound repair stimulated by epidermal growth factor was substantially attenuated by 10(-7) M tunicamycin (TM), an N-glycosylation inhibitor, but not by the inhibitors deoxymannojirimycin or castanospermine. Wound repair of 1HAEo(-) and primary airway epithelial cells was blocked completely by removal of cell surface terminal fucose residues by alpha-fucosidase. Cell adhesion to collagen matrix was prevented by TM but was only reduced ~20% from control values with prior alpha-fucosidase treatment. Cell migration in Blind Well chambers stimulated by epidermal growth factor was blocked by pretreatment with TM but alpha-fucosidase pretreatment produced no difference from control values. These data suggest that cell surface N-glycosylation has a functional role in airway epithelial cell adhesion and migration and that N-glycosylation with terminal fucosylation plays a role in the complex process of repair by coordination of certain cell-cell functions.
Asunto(s)
Fucosa/metabolismo , Proteínas de la Membrana/metabolismo , Mucosa Respiratoria/citología , Mucosa Respiratoria/metabolismo , Antibacterianos/farmacología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Línea Celular Transformada , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Glicosilación/efectos de los fármacos , Humanos , Lectinas , Tunicamicina/farmacología , Cicatrización de Heridas/fisiología , alfa-L-Fucosidasa/farmacologíaRESUMEN
We have used the lectin from Aleuria aurantia (AAL) which is highly specific for alpha(1-6)-linked fucose, to examine its effect on chicken retinogenesis in a reaggregation culture system. When dispersed cells of the embryonic chick retina are reaggregated to form histotypic retinospheroids, AAL elicits strong inhibition of spheroid growth. The action of AAL is specific, since its effect is dose-dependent, saturable, and inhibited by an excess of fucose. Fucosidase treatment entirely abolishes reaggregation. In contrast, Anguilla anguilla agglutinin (AAA) binding to fucose in alpha(1-2)-linkage does not show any effects. Incubation with CAB4-a specific monoclonal antibody for fucose in alpha(1-6)-linkage-reduces spheroid size and shape. AAL does not much affect primary aggregation, but rather subsequent processes of cell proliferation and histogenesis. In particular, AAL inhibits uptake of bromo-desoxyuridine (BrdU), most efficiently so during days in vitro 2 (div2) and div3. As a consequence, the histological differentiation is entirely disturbed, as evidenced by vimentin immunostaining; particularly, rosettes are not forming and the radial glia scaffold is disorganized. We conclude that glycoproteins exhibiting fucose in alpha(1-6)-linkage may play major roles in early processes of retinal tissue formation.
Asunto(s)
Fucosa/fisiología , Lectinas/farmacología , Organoides/metabolismo , Retina/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Bromodesoxiuridina/metabolismo , Conformación de Carbohidratos , Agregación Celular , Diferenciación Celular , División Celular/fisiología , Embrión de Pollo , Fucosa/inmunología , Organoides/efectos de los fármacos , Polisacáridos/metabolismo , Vimentina/análisis , alfa-L-Fucosidasa/farmacologíaRESUMEN
This study was aimed at characterizing the glycoconjugates produced by the horse sublingual gland and, in particular, at discriminating between the sialoderivatives by means of differential oxidation and saponification combined with lectin histochemistry and enzymatic degradation. The results showed a predominance of sialoglycoconjugates with beta-galactose as acceptor sugar in the salivary mucins produced by the sublingual gland. Besides being the most represented terminal residue, sialic acid was also expressed in a great variety of derivatives distinguishable on the basis of acceptor sugars to the penultimate beta-galactose as well as linkage and acetylation degree of the pyranose ring and the polyhydroxyl side chain. A role in the protection of mucous membranes from physical, chemical and pathogenic agents can be hypothesized for the horse sublingual mucins.
Asunto(s)
Glicoconjugados/metabolismo , Lectinas/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Glándula Sublingual/metabolismo , Acetilación/efectos de los fármacos , Animales , Femenino , Glicoconjugados/química , Histocitoquímica , Caballos , Inmunohistoquímica , Masculino , Muramidasa/metabolismo , Ácido N-Acetilneuramínico/análisis , Neuraminidasa/farmacología , alfa-L-Fucosidasa/farmacologíaRESUMEN
Inadequate knowledge of pathogenesis and pathophysiology has contributed to the high mortality and morbidity associated with neonatal Escherichia coli meningitis. We have shown previously that outer membrane protein A (OmpA) contributes to E. coli K1 membrane invasion of brain microvascular endothelial cells. In this study we report that this OmpA+ K1 E. coli invasion of brain microvascular endothelial cells was inhibited by wheat germ agglutinin and chitooligomers prepared from the polymer of 1,4-linked GlcNAc, chitin. The specificity of the interaction between OmpA and GlcNAc beta 1-4GlcNAc epitopes was verified by the demonstration that chitotriose-bound OmpA and wheat germ agglutinin-bound brain microvascular endothelial cell membrane proteins inhibit E. coli K1 invasion. Of interest, OmpA+ E. coli invasion into systemic endothelial cells did not occur, but invasion similar to that of brain microvascular endothelial cells was observed when systemic cells were treated with alpha-fucosidase, suggesting that the GlcNAc beta 1-4GlcNAc moieties might be substituted with L-fucose on these cells. More importantly, the chitooligomers prevented entry of E. coli K1 into the cerebrospinal fluid of newborn rats with experimental hematogenous E. coli meningitis, suggesting that the GlcNAc beta 1-4GlcNAc epitope of brain microvascular endothelial cells indeed mediates the traversal of E. coli K1 across the blood-brain barrier. A novel strategy with the use of soluble receptor analog(s) may be feasible in the prevention of devastating neonatal E. coli meningitis.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Barrera Hematoencefálica , Endotelio Vascular/microbiología , Escherichia coli/patogenicidad , Maltosa/análogos & derivados , Animales , Secuencia de Carbohidratos , Carbohidratos/farmacología , Bovinos , Sistema Nervioso Central/microbiología , Líquido Cefalorraquídeo/microbiología , Endotelio Vascular/química , Epítopos , Infecciones por Escherichia coli/etiología , Infecciones por Escherichia coli/microbiología , Glicoproteínas/farmacología , Maltosa/metabolismo , Meningitis Bacterianas/etiología , Meningitis Bacterianas/microbiología , Datos de Secuencia Molecular , Ratas , Ratas Sprague-Dawley , Virulencia/efectos de los fármacos , Aglutininas del Germen de Trigo/farmacología , alfa-L-Fucosidasa/farmacologíaRESUMEN
The entire surface of the cercarial stage of the human blood fluke Schistosoma mansoni is covered by a 1-microns thick, highly immunogenic, fucose-rich glycocalyx (GCX). Using strategies based on enzymatic, chemical, and mass spectrometric analysis, we have defined the structures of the major glycans released by reductive elimination from GCX. They comprise a heterogeneous population of multifocosylated complex oligosaccharides with the following nonreducing terminal sequences: [formula: see text] Our structural data suggest that these tri- to pentafucosylated epitopes are carried on type 1, R-->Gal beta-1-->3GalNAc, and type 2, R-->Gal beta 1-->3(R-->GlcNAc beta-1-->6)GalNAc, core structures via repeat units of (3GalNAc beta 1-->4(Fuc alpha 1-->2Fuc alpha 1-->2Fuc alpha 1-->3)GlcNAc beta-1-->3Gal alpha-->)n, where n is mainly 0 and 1, and all sugars are in the pyranose form. The proposed structure represents the first instance where an alpha-galactosylated beta-GalNAc(1-->4)-beta-GlcNAc sequence occurs as a repeating unit in a glycoprotein. It is also unique in being substituted with oligofucosyl appendages. The unusual oligosaccharide structures described here, particularly the potentially immunodominant oligofucosyl moieties, are most likely responsible for the known potency of GCX in modulating various immune responses including complement activation, B cell mitogenesis, and delayed type hypersensitivity in schistosomiasis.
Asunto(s)
Glicoproteínas/química , Polisacáridos/química , Schistosoma mansoni/química , Animales , Secuencia de Carbohidratos , Glicoproteínas/fisiología , Glicósido Hidrolasas/farmacología , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Polisacáridos/fisiología , alfa-L-Fucosidasa/farmacologíaRESUMEN
The porcine brush border glycoproteins of 210 and 240 kDa, recognized by Escherichia coli K88ac fimbriae, contained O-linked oligosaccharides. The carbohydrate moieties were analysed by deglycosylation, lectin-binding and agglutination assays. Neuraminidase susceptibility of the 210 kDa receptor suggested that a sialoglycoprotein may act as receptor for the K88ac fimbriae. In contrast, K88ac-binding to the 210 and 240 kDa glycoproteins totally disappeared after fucosidase treatment, indicating the critical role of fucosyl residues at the receptor sites. Among the oligosaccharides extracted from these O-glycoproteins, K88ac fimbriae showed affinity for neutral sugar chains while sialylated species were not recognized. Our data suggest a possible role of the polypeptide backbone in the definition of receptor sites. Specific agglutination by K88ac-fimbriated E. coli of the erythrocytes of the hamster Mesocricetus auratus was inhibited by the anti-T peanut lectin and the lectins of Datura stramonium, Aleuria aurantia and Maackia amurensis. Hence, we propose that Gal beta 1-3GalNAc- and Fuc alpha 1-2Gal beta 1-3/4GlcNAc- are the main sequences mediating K88ac fimbrial binding. These structures were not detected in the non-adhesive piglet brush borders characterized by a high carbohydrate content. Additional oligosaccharides probably masked the underlying receptor structures.
Asunto(s)
Fimbrias Bacterianas/metabolismo , Yeyuno/metabolismo , Polisacáridos/metabolismo , Animales , Adhesión Bacteriana/efectos de los fármacos , Secuencia de Carbohidratos , Cricetinae , Glicoproteínas/química , Glicoproteínas/metabolismo , Glicosilación , Hemaglutinación/efectos de los fármacos , Técnicas In Vitro , Yeyuno/microbiología , Lectinas/química , Lectinas/farmacología , Microvellosidades/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Neuraminidasa/farmacología , Oligosacáridos/química , Oligosacáridos/metabolismo , Polisacáridos/química , Porcinos , alfa-L-Fucosidasa/farmacologíaRESUMEN
The behaviour of a highly purified alpha-L-fucosidase (E.C. 3.2.1.51) extracted from octopus hepatopancreas was studied with phospholipid vesicles composed of phosphatidylcholine (PC) and phosphatidylserine (PS) containing the fucosylated ganglioside FucGM1, a potential natural substrate of the enzyme. The substrate recognition and hydrolysis take place only with PS/FucGM1 mixtures via an association process of the enzyme with the vesicles at acidic pH; the enzyme rapidly and stably binds to PS vesicles but not to PC vesicles. The data suggest that only the PS-associated enzyme is able to hydrolyse FucGM1 embedded in the same bilayer. The enzyme association with FucGM1/PS vesicles is a prerequisite for ganglioside hydrolysis but is followed by irreversible enzyme inactivation.
Asunto(s)
Sistema Digestivo/química , Gangliósido G(M1)/análogos & derivados , Membrana Dobles de Lípidos , Octopodiformes/química , Fosfolípidos/análisis , alfa-L-Fucosidasa/química , alfa-L-Fucosidasa/farmacología , Animales , Gangliósido G(M1)/análisis , Glicósidos/química , Espectrometría de Fluorescencia , Especificidad por Sustrato , alfa-L-Fucosidasa/aislamiento & purificaciónRESUMEN
Previously we have isolated a lysosomal enzyme binding receptor protein from monkey brain that exhibits protein kinase activity and undergoes phosphorylation on serine and tyrosine residues. Using the 32P-labelled receptor protein, we have found that the lysosomal enzyme fucosidase and mannose-6-phosphate, which are ligands for the receptor, stimulated a protein phosphatase activity associated with the receptor protein. Stimulation of protein phosphatase activity using the 32P-labelled receptor protein was demonstrated both by the loss in radioactivity of the receptor and by the release of 32P-phosphate. There was no stimulation by a non-lysosomal glycoprotein enzyme, or by the sugars mannose or glucose. Both serine-phosphate and tyrosine-phosphate residues were dephosphorylated. Stimulation of protein phosphatase activity by fucosidase and mannose-6-phosphate was also demonstrated using as substrate histone 32P-labelled, on serine/threonine or tyrosine residues. Insulin-like growth factor II, another known ligand for the lysosomal enzyme binding receptor, did not show any significant effect, either on the phosphorylation or dephosphorylation of the receptor protein. Our previous and present results suggest that a phosphorylation/dephosphorylation mechanism may be operative in the ligand binding and functions of the receptor.
Asunto(s)
Química Encefálica , Lisosomas/enzimología , Manosafosfatos/farmacología , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Citoplasmáticos y Nucleares , alfa-L-Fucosidasa/farmacología , Animales , Carbohidratos/farmacología , Activación Enzimática/efectos de los fármacos , Haplorrinos/metabolismo , Histonas/metabolismo , Factor II del Crecimiento Similar a la Insulina/farmacología , Ligandos , Nitrofenoles/farmacología , Compuestos Organofosforados/farmacología , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Receptor IGF Tipo 2 , Fluoruro de Sodio/farmacología , Estimulación Química , Vanadatos/farmacologíaRESUMEN
To determine if isolated galaptin, an endogenous galactoside-binding lectin, could serve as a transport vehicle of therapeutic agents to cells, galaptin and alpha-L-fucosidase were coupled using glutaraldehyde. The conjugates were incubated with alpha-L-fucosidase-deficient, EBV-immortalized lymphoid cells from a fucosidosis patient. Conjugates were effectively bound and internalized by the cells in a lactose inhibitable manner. Internalization of conjugate resulted in the reduced accumulation of alpha-L-fucosyl-N-acetylglucosaminylasparagine, a glycopeptide that accumulates in cells of fucosidosis patients, to levels found in lymphoid cells from a healthy individual. Thus, galaptin-alpha-L-fucosidase conjugates may be useful for enzyme replacement therapy of fucosidosis. The concept of using galaptin as a transport vehicle may be applied to the delivery of other compounds to cells bearing galaptin receptors.
Asunto(s)
Fucosidosis/metabolismo , Hemaglutininas/metabolismo , alfa-L-Fucosidasa/metabolismo , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Proteínas Portadoras/metabolismo , Línea Celular , Galectinas , Hemaglutininas/farmacología , Humanos , Cinética , Valores de Referencia , alfa-L-Fucosidasa/farmacologíaRESUMEN
We examined the distribution of blood group-related antigens using an indirect immunoperoxidase method with monoclonal antibodies (MAb) directed to A, B, H, Lewis a (Lea), Lewis b (Leb), Lewis x (Lex), and Lewis y (Ley) antigens and Type 1 precursor chain in human pancreas. Effects of prior digestion with exoglycosidases on MAb stainings were simultaneously investigated. A, B, H, Leb, and Ley antigens were detected in acinar cells and interlobular duct cells but not in centroacinar cells, intercalated duct cells, and islet of Langerhans cells. The expression of these antigens in acinar cells was not dependent on Lewis type and secretor status of the tissue donors, whereas that in interlobular duct cells was strictly dependent on secretor status. The distribution pattern of these antigens in acinar cells was not homogeneous, i.e., cells producing H antigens expressed both Leb and Ley antigens but not A or B antigens, whereas those producing A or B antigens did not secrete Leb and Ley as well as H antigens. Digestion with alpha-N-acetylgalactosaminidase or alpha-galactosidase resulted in the appearance of Leb and Ley antigens as well as H antigen in acinar cells producing A and/or B antigens. Type 1 precursor chain was not detected in pancreatic tissues from secretors but appeared in acinar cells producing H antigen after alpha-L-fucosidase digestion, which also disclosed Lex but not Lea antigen in acinar cells expressing both Leb and Ley. In some non-secretors, MAb against Type 1 precursor chain reacted with acinar cells without enzyme digestion. Although Lea antigen was not detected in acinar cells, it was found in centroacinar cells, intercalated duct cells, and interlobular duct cells from all individuals examined except two Le(a-b-) secretors. After sialidase digestion, Lex antigen appeared in centroacinar and intercalated duct cells from some individuals. Sialidase digestion also elicited reactivity with MAb against Type 1 precursor chain in islet of Langerhans cells from some individuals. These results demonstrate the complexity in the pattern of expression and regulation of blood group-related antigens in different cell types of human pancreas. Such complexity may largely be ascribed to differences in individual genotypes and in gene expression patterns of different cell types.
Asunto(s)
Sistema del Grupo Sanguíneo ABO/inmunología , Anticuerpos Monoclonales/inmunología , Antígenos/inmunología , Antígenos del Grupo Sanguíneo de Lewis/inmunología , Páncreas/inmunología , Acetilglucosaminidasa/farmacología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antígenos/metabolismo , Niño , Preescolar , Femenino , Glicósido Hidrolasas/farmacología , Humanos , Inmunohistoquímica/métodos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Neuraminidasa/farmacología , Páncreas/citología , Páncreas/metabolismo , alfa-L-Fucosidasa/farmacologíaRESUMEN
Myelin phagocytosis in nerves undergoing Wallerian degeneration has been shown to depend on their invasion by non-resident, hematogenous macrophages. This process can be studied in vitro using organ cultures of peripheral nerves exposed to cultured peritoneal macrophages. The present report concerns the role of cell surface carbohydrates in the invasion of degenerating nerves and in the recognition and ingestion of myelin by the phagocytic cells. Additional experiments explored the effect of pH, calcium and cytochalasin D on myelin phagocytosis. Organ cultures with peritoneal macrophages were treated with 14 simple or complex sugars or with eight sugar-splitting enzymes. Macrophage invasion was diminished by many simple or complex sugars, but exposure to sugars had no effect on the recognition or ingestion of myelin by the invading macrophages. Macrophage invasion was abolished upon treatment with beta-mannosidase. Exposure to L-fucosidase abolished the myelin phagocytic capacity of invading macrophages completely without affecting their capacity to ingest carbon or latex particles. The results indicate that the phagocytosis of myelin by macrophages is an L-fucosidase-sensitive process, probably by interaction with their complement receptor type C3.