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
We determined the effect of low molecular weight components (LMWC) from healthy juvenile and adult Macrobrachium rosenbergii hemolymph on lectin activity and oxidative burst (OB) in hemocytes. In an attempt to identify the LMWC that affect the lectin's hemagglutinating activity or oxidative burst, we determined the hemolymph carbohydrates and free amino acids (FAA) concentration. The LMWC (<2000 Da) were obtained after dialysis of the hemolymph. Our results showed that LMWC from juveniles exerted a greater inhibition on lectin than LMWC from adult hemolymph. Production of superoxide radicals by hemocytes was lower in the presence of juvenile (p<0.05) as compared to adult LMWC. FAA composition of the hemolymph and of LMWC from adults showed higher proportion of alanine (which corresponded to 25% of total FAA) and proline (>20%); whereas, in juveniles, the main FAA identified were glycine (>40%) and alanine (26%). N-Acetyl-D-glucosamine (GlcNAc) was the main sugar residue in the hemolymph and LMWC from juveniles; its concentration was 2.4 times higher than glucose (Glc), whereas, in adults, Glc was the main free sugar residue. Our results suggest that the proportion of FAA and carbohydrates in the hemolymph of M. rosenbergii seems to be correlated with the maturation process; furthermore, the high proportion of free GlcNAc and glycine regulate, in the juvenile stage, lectin activity and cellular oxidative mechanisms, respectively.
Asunto(s)
Aminoácidos/sangre , Carbohidratos/sangre , Hemolinfa/metabolismo , Lectinas/metabolismo , Palaemonidae/metabolismo , Aminoácidos/química , Animales , Carbohidratos/química , Eritrocitos/fisiología , Agua Dulce , Hemaglutinación , Hemocitos/metabolismo , Ratas , Ratas Wistar , Estallido Respiratorio , Superóxidos/metabolismoRESUMEN
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.