RESUMEN
Aleuria aurantia lectin (AAL) is an L-fucose-specific lectin produced in the mycelia and fruit-bodies of the widespread ascomycete fungus Aleuria aurantia. It is extensively used in the detection of fucose, but its physiological role remains unknown. To investigate this, we analyzed the interaction between AAL and, a zygomycete fungus Mucor racemosus, which is assumed to contain fucose in its cell wall. AAL specifically bound to the hyphae of M. racemosus, because binding was inhibited by L-fucose but not by D-fucose. It inhibited the growth of the fungus at 1 µM, and the M. racemosus cells were remarkably disrupted at 7.5 µM. In contrast, two other fucose-specific lectins, Anguilla anguilla agglutinin and Ulex europaeus agglutinin, did not inhibit the growth of M. racemosus. These results suggest that the growth inhibition activity is unique to AAL, and that AAL could act as an antifungal protein in natural ecosystems.
Asunto(s)
Antifúngicos/farmacología , Ascomicetos/química , Cuerpos Fructíferos de los Hongos/química , Lectinas/farmacología , Mucor/efectos de los fármacos , Micelio/química , Antifúngicos/aislamiento & purificación , Antifúngicos/metabolismo , Pared Celular/química , Escherichia coli , Fucosa/metabolismo , Fucosa/farmacología , Lectinas/biosíntesis , Lectinas/aislamiento & purificación , Mucor/crecimiento & desarrollo , Plásmidos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , EstereoisomerismoRESUMEN
Protein glycosylation is a critical subject attracting increasing attention in the field of proteomics as it is expected to play a key role in the investigation of histological and diagnostic biomarkers. In this context, an enormous number of glycoproteins have now been nominated as disease-related biomarkers. However, there is no appropriate strategy in the current proteome platform to qualify such marker candidate molecules, which relates their specific expression to particular diseases. Here, we present a new practical system for focused differential glycan analysis in terms of antibody-assisted lectin profiling (ALP). In the developed procedure, (i) a target protein is enriched from clinic samples (e.g. tissue extracts, cell supernatants, or sera) by immunoprecipitation with a specific antibody recognizing a core protein moiety; (ii) the target glycoprotein is quantified by immunoblotting using the same antibody used in (i); and (iii) glycosylation difference is analyzed by means of antibody-overlay lectin microarray, an application technique of an emerging glycan profiling microarray. As model glycoproteins having either N-linked or O-linked glycans, prostate-specific antigen or podoplanin, respectively, were subjected to systematic ALP analysis. As a result, specific signals corresponding to the target glycoprotein glycans were obtained at a sub-picomole level with the aid of specific antibodies, whereby disease-specific or tissue-specific glycosylation changes could be observed in a rapid, reproducible, and high-throughput manner. Thus, the established system should provide a powerful pipeline in support of on-going efforts in glyco-biomarker discovery.
Asunto(s)
Anticuerpos/inmunología , Biomarcadores/análisis , Lectinas/análisis , Polisacáridos/análisis , Análisis por Matrices de Proteínas/métodos , Animales , Células CHO , Línea Celular Tumoral , Cricetinae , Cricetulus , Glicosilación , Humanos , Glicoproteínas de Membrana/metabolismo , Ratones , Ácido N-Acetilneuramínico/metabolismo , Agregación Plaquetaria , Antígeno Prostático Específico/metabolismo , Ratas , Reproducibilidad de los Resultados , Extractos de TejidosRESUMEN
A cDNA encoding tomato fruit lectin was cloned from an unripe cherry-tomato fruit cDNA library. The isolated lectin cDNA contained an open reading frame encoding 365 amino acids, including peptides that were sequenced. The deduced sequence consisted of three distinct domains: (i) an N-terminal short extensin-like domain; (ii) a Cys-rich carbohydrate binding domain composed of four almost identical chitin-binding domains; (iii) an internal extensin-like domain of 101 residues containing 15 SerPro(4) motifs inserted between the first and second chitin-binding domains. The molecular weight of the lectin was 65,633 and that of the deglycosylated lectin was 32,948, as determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). This correlated with the estimated molecular weight of the deduced sequence. Recombinant tomato lectin expressed in Pichia pastoris possessed chitin-binding but not hemagglutinating activity. These findings confirmed that the cDNA encoded tomato lectin.
Asunto(s)
Frutas/química , Frutas/metabolismo , Lectinas de Plantas/química , Lectinas de Plantas/metabolismo , Solanum lycopersicum/química , Secuencia de Aminoácidos , Secuencia de Bases , Cromatografía en Gel , Secuencia Conservada , ADN Complementario/genética , Frutas/genética , Expresión Génica , Glicosilación , Solanum lycopersicum/genética , Datos de Secuencia Molecular , Peso Molecular , Pichia/genética , Pichia/metabolismo , Lectinas de Plantas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Mucin-type O-glycans are the most typical O-glycans found in mammalian cells and assume many different biological roles. Here, we report a genetic engineered yeast strain capable of producing mucin-type sugar chains. Genes encoding Bacillus subtilis UDP-Gal/GalNAc 4-epimerase, human UDP-Gal/GalNAc transporter, human ppGalNAc-T1, and Drosophila melanogaster core1 beta1-3 GalT were introduced into Saccharomyces cerevisiae. The engineered yeast was able to produce a MUC1a peptide containing O-glycan and also a mucin-like glycoprotein, human podoplanin (hPod; also known as aggrus), which is a platelet-aggregating factor that requires a sialyl-core1 structure for activity. After in vitro sialylation, hPod from yeast could induce platelet aggregation. Interestingly, substitution of ppGalNAc-T1 for ppGalNAc-T3 caused a loss of platelet aggregation-inducing activity, despite the fact that the sialyl-core1 was detectable in both hPod proteins on a lectin microarray. Most of O-mannosylation, a common modification in yeast, to MUC1a was suppressed by the addition of a rhodanine-3-acetic acid derivative in the culture medium. The yeast system we describe here is able to produce glycoproteins modified at different glycosylation sites and has the potential for use in basic research and pharmaceutical applications.
Asunto(s)
Glicoproteínas/biosíntesis , Redes y Vías Metabólicas/genética , Ingeniería de Proteínas/métodos , Levaduras/metabolismo , Glicosilación , Glicosiltransferasas/genética , Humanos , Mucinas , Levaduras/enzimología , Levaduras/genéticaRESUMEN
Podoplanin (Aggrus) is a mucin-type sialoglycoprotein that plays a key role in tumor cell-induced platelet aggregation. Podoplanin possesses a platelet aggregation-stimulating (PLAG) domain, and Thr52 in the PLAG domain of human podoplanin is important for its activity. Endogenous or recombinant human podoplanin were purified, and total glycosylation profiles were surveyed by lectin microarray. Analyses of glycopeptides produced by Edman degradation and mass spectrometry revealed that the disialyl-corel (NeuAc alpha2-3Gal beta l-3(NeuAc alpha2-6)GalNAc alpha l-O-Thr) structure was primarily attached to a glycosylation site at residue Thr52. Sialic acid-deficient podoplanin recovered its activity after additional sialylation. These results indicated that the sialylated Corel at Thr52 is critical for podoplanin-induced platelet aggregation.
Asunto(s)
Glicoproteínas de Membrana/química , Polisacáridos/química , Secuencia de Aminoácidos , Animales , Plaquetas/efectos de los fármacos , Células CHO , Secuencia de Carbohidratos , Cricetinae , Cricetulus , Glicosilación , Humanos , Lectinas/química , Espectrometría de Masas , Glicoproteínas de Membrana/aislamiento & purificación , Glicoproteínas de Membrana/farmacología , Datos de Secuencia Molecular , Ácido N-Acetilneuramínico/análisis , Compuestos Organofosforados/química , Fragmentos de Péptidos/química , Agregación Plaquetaria , Polisacáridos/análisis , Análisis por Matrices de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Treonina/químicaRESUMEN
The mucin-type sialoglycoprotein, podoplanin (aggrus), is a platelet-aggregating factor on cancer cells. We previously described up-regulated expression of podoplanin in malignant astrocytic tumors including glioblastoma. Its expression was associated with tumor malignancy. In the present study, we investigated podoplanin expression and platelet-aggregating activities of glioblastoma cell lines. First, we established a highly reactive anti-podoplanin antibody, NZ-1, which inhibits podoplanin-induced platelet aggregation completely. Of 15 glioblastoma cell lines, LN319 highly expressed podoplanin and induced platelet aggregation. Glycan profiling using a lectin microarray showed that podoplanin on LN319 possesses sialic acid, which is important in podoplanin-induced platelet aggregation. Interestingly, NZ-1 neutralized platelet aggregation by LN319. These results suggest that podoplanin is a main reason for platelet aggregation induced by LN319. We infer that NZ-1 is useful to determine whether platelet aggregation is podoplanin-specific or not. Furthermore, podoplanin might become a therapeutic target of glioblastoma for antibody-based therapy.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/inmunología , Complejo Antígeno-Anticuerpo/inmunología , Glioblastoma/inmunología , Glicoproteínas de Membrana/inmunología , Agregación Plaquetaria/inmunología , Animales , Anticuerpos Monoclonales/química , Complejo Antígeno-Anticuerpo/química , Línea Celular Tumoral , Femenino , Glioblastoma/patología , Glicoproteínas de Membrana/química , Agregación Plaquetaria/efectos de los fármacos , Estructura Terciaria de Proteína , RatasRESUMEN
Since the involvement of Tyr residues in the fucose-binding of Aleuria aurantia lectin (AAL) was proved by chemical modification using the Tyr-specific reagent tetranitromethane, site-directed mutagenesis was attempted. Since the tertiary structure of AAL was determined recently to be a six-bladed beta-propeller fold, and five fucose-binding sites per subunit were found, based on positions of Tyr residues in the tertiary structure, three classes of mutants were constructed: 1) Tyr on the 2nd beta-strand of each blade (beta-2 mutants), 2) Tyr or Trp on the 3rd beta-strand (beta-3 mutants), and 3) Tyr outside of binding sites (other-Y mutants). The mutagenized cDNA was expressed in Escherichia coli as His-tag-AAL, and the hemagglutinating activity was assayed. Among 14 mutants, three beta-2 mutants (Y26A, Y79A, and Y181A), and three beta-3 mutants (Y92A, W149A, and Y241A) showed decreased activity. These mutated residues resided at Sites 1, 2, and 4, at the same locations relatively in the binding sites. Mutagenesis of Tyr or Trp at the corresponding locations in Sites 3 and 5 did not lead to a reduction in activity. Results indicate that the properties of Sites 1, 2, and 4 are different from those of Sites 3 and 5, and that the contribution of these two sites to the hemagglutination reaction was minor.
Asunto(s)
Ascomicetos , Fucosa/metabolismo , Lectinas/metabolismo , Mutagénesis Sitio-Dirigida/genética , Tirosina/metabolismo , Secuencia de Aminoácidos , Ascomicetos/química , Ascomicetos/genética , Ascomicetos/metabolismo , Sitios de Unión , ADN Complementario/genética , Hemaglutinación/efectos de los fármacos , Hemaglutinación/genética , Lectinas/química , Lectinas/genética , Lectinas/aislamiento & purificación , Datos de Secuencia Molecular , Mutación/genética , Mapeo Peptídico , Tetranitrometano/farmacología , Tirosina/genéticaRESUMEN
cDNA of a mycelial aggregate-specific lectin of Pleurotus cornucopiae was expressed in Pichia pastoris, and the expression product was purified and characterized. The product was functional, and the hemagglutinating activity was inhibited most strongly by the addition of N-acetyl-D-galactosamine as was the native lectin. The native lectin is a glycoprotein having five glycosylation recognition signals, and the expression product showed slightly larger molecular mass than that of the native one due to further glycosylation.
Asunto(s)
Lectinas/genética , Lectinas/metabolismo , Pichia/genética , Pleurotus/química , Metabolismo de los Hidratos de Carbono , ADN Complementario/genética , Glicosilación , Pruebas de Inhibición de Hemaglutinación , Lectinas/química , Lectinas/aislamiento & purificación , Pleurotus/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
A plasmid bearing a nucleotide sequence of fucose-specific lectin of Aleuria aurantia was constructed and expressed in a methylotrophic yeast, Pichia pastoris. The product showed almost the same hemagglutinating activity as the lectin produced in Escherichia coli, the properties of which were quite similar to the native one. Because of glycosylation of the product, the molecular mass was larger than that of the native one, and it acquired higher thermostability.
Asunto(s)
Clonación Molecular/métodos , Lectinas/genética , Pichia/genética , ADN Complementario , Glicosilación , Hemaglutinación , Lectinas/biosíntesis , Peso Molecular , TemperaturaRESUMEN
Fruiting-body lectin genes obtained from Pleurotus cornucopiae were expressed in Pichia pastoris Because of glycosylation of the products, their molecular mass was larger than that of the corresponding native lectins. They showed binding activity to porcine stomach mucin in the enzyme-linked lectin assay system, but did not agglutinate red blood cells.
RESUMEN
A lectin was isolated from an ascomycete mushroom, Ciborinia camelliae which was specific to N-acetyl-D-galactosamine. On SDS-polyacrylamide gel electrophoresis; this lectin gave a single band of approximately 17-kDa in the presence of 2-mercaptoethanol, but formed dimers, trimers and tetramers in its absence. Amino acid analysis revealed the lectin contained two cysteines and no methionine. The N-terminal sequence was determined up to residue 21, and no homologous proteins including other ascomycete lectins were found.