RESUMEN
Neutrophils participate in host protection and central to this process is the regulation of oxidative mechanisms. We purified by affinity chromatography the receptor for the GlcNAc-specific WGA from CD14+ CD16+ cell lysates (WGAr). The receptor is a 141 kDa glycoprotein constituted by two subunits of 78 and 63 kDa. It is mainly composed of Ser, Asx, and Gly, and, in a minor proportion, His, Cys, and Pro. Its glycan portion contains GlcNAc, Gal, and Man; NeuAc and GalNAc were identified in a minor proportion. The amino acid sequence of the WGA receptor was predicted from tryptic peptides by MALDI-TOF, both subunits showed homology with cytokeratin type II (26 and 29% for the 78 and 63 kDa subunits, respectively); the 78 kDa subunit showed also homology with the human transferrin receptor (24%). Antibodies against WGAr induce higher oxidative burst than WGA, determined by NBT reduction; however, this effect was inhibited (p < 0.05) with GlcNAc suggesting that WGAr participates as mediator in signal transduction in neutrophils.
Asunto(s)
Neutrófilos/metabolismo , Receptores Mitogénicos/sangre , Aglutininas del Germen de Trigo/metabolismo , Secuencia de Aminoácidos , Animales , Granulocitos/metabolismo , Humanos , Técnicas In Vitro , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/aislamiento & purificación , Mapeo Peptídico , Subunidades de Proteína , Receptores Mitogénicos/química , Receptores Mitogénicos/genética , Receptores Mitogénicos/aislamiento & purificación , Estallido RespiratorioRESUMEN
Amaranthus leucocarpus lectin (ALL) is specific for GalNAc, and recognizes human T cells. The receptor for ALL was purified from T cells using biotin-labeled lectin and avidin-agarose as affinity matrix. It is a 70-kDa glycoprotein, constituted mainly by serine, glycine, and glutamic acid; its glycosidic portion contains mainly GalNAc; galactose, sialic acid, mannose, and GlcNAc were identified at a lower proportion. By ionic strength chromatography, as well as double dimension electrophoresis, we identified four isoforms of the ALL-receptor. N-terminal amino acid was blocked both in the ALL-receptor and its isoforms, therefore, tryptic peptides of ALL-receptor, analyzed through MALDI-TOF, were compared with the relative values obtained from the NCBInr (ProFound 2004/06/01) database. Our results indicated that the tryptic peptides obtained showed 54% homology with a DnaK-core molecular chaperone, 47% with human KIAA protein, and 44% with heat shock protein 8. The most frequent phenotype of the CD4 or CD8 ALL+ T cells was CD45RA+ CD27+; 26% of ALL+ T cells were CD25+ and 13% were CD69+, indicating that the glycoprotein recognized by ALL is present mainly on naive or quiescent T cells.
Asunto(s)
Glicoproteínas/química , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/aislamiento & purificación , Lectinas de Plantas/química , Receptores Mitogénicos/química , Receptores Mitogénicos/aislamiento & purificación , Linfocitos T/metabolismo , Amaranthus/metabolismo , Secuencia de Aminoácidos , Antígenos CD/análisis , Glicoproteínas/metabolismo , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fenotipo , Lectinas de Plantas/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/aislamiento & purificación , Linfocitos T/inmunologíaRESUMEN
We purified a 70 kDa O-glycoprotein that binds to the GalNAc specific lectin from Amaranthus leucocarpus (ALLr) and determined its expression pattern on T lymphocytes from different murine lymphoid organs. High level of ALLr expression was demonstrated in 95-98% of both CD4(+)8(+) and CD4(-)8(+) thymocytes, and in 80-95% of CD8(+) T cells from peripheral blood, lymph nodes, and spleen, whereas a minor fraction of CD4(+)8(-) thymocytes (46-67%) and peripheral CD4(+) T cells (9-40%) showed low ALLr expression. Peripheral CD19(+) B cells were ALLr negative and most of the peripheral ALL(+) T cells showed a CD62L(hi)CD45RB(hi)CD44(lo/-) phenotype, indicating features of naive cells. Mitogenic activation of peripheral T cells increased 3-fold the number of ALL(+)CD4(+) T cells 24 h after stimulation, as opposed to a >80% decrease in CD8(+) T cells 72 h after stimulation. Our results suggest that ALL detects a non-described surface O-glycoprotein selectively expressed by naive CD8(+) T cells and by early activated CD4(+) T cells.
Asunto(s)
Glicoproteínas/metabolismo , Activación de Linfocitos , Glicoproteínas de Membrana/aislamiento & purificación , Lectinas de Plantas/metabolismo , Receptores Mitogénicos/aislamiento & purificación , Subgrupos de Linfocitos T/química , Animales , Antígenos CD/análisis , Linfocitos T CD4-Positivos/química , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/química , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular , Linaje de la Célula , Cromatografía de Afinidad , Regulación de la Expresión Génica , Glicosilación , Inmunofenotipificación , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Peso Molecular , Procesamiento Proteico-Postraduccional , Receptores Mitogénicos/metabolismo , Ácidos Siálicos/análisis , Subgrupos de Linfocitos T/metabolismoRESUMEN
Crithidia oncopelti, C. deanei, and C. desouzai are flagellates of the Trypanosomatidae family that present bacterium-like endosymbionts in their cytoplasm. Direct and indirect lectin-gold labeling techniques were used at the electron microscopic level in Lowicryl K4M-embedded cells to demonstrate the presence of intracellular lectin-binding sites. We used the lectins Ulex europaeus I, Griffonia simplicifolia II, Ricinus communis I, Arachis hypogaea, G. simplicifolia I, Wistaria floribunda, Limulus polyphemus, and Canavalia ensiformis, which recognize alpha-L-fucose, alpha- and beta-N-acetylglucosamine, beta-galactose and beta-N-acetylgalactosamine, beta-galactose, alpha-galactose, beta-N-acetylgalactosamine, sialic acid and alpha-D-mannose, and alpha-D-glucose residues, respectively. The nucleus was the cellular structure most frequently labeled by the lectins. The Golgi complex was seldom labeled, whereas the endoplasmic reticulum and the flagellar pocket presented a large number of binding sites. Symbionts had their two unit membranes weakly labeled by the different lectins but displayed no labeling of the space between the membranes.