Identification of N-acetyl-D-glucosamine-specific lectins from rat liver cytosolic and nuclear compartments as heat-shock proteins

Cytosolic and nuclear O-linked N-acetylglucosaminylation has been proposed to be involved in the nuclear transport of cytosolic proteins, We have isol ated nuclear and cytosolic N-acetyl-D-glucosamine (GlcNAc)-specific lectins from adult rat liver by affinity chromatography on immobilized GlcNAc and identified these lectins, by a proteomic approach, as belonging to the heat -shock protein (HSP)-70 family (one of them being heat-shock cognate 70 str ess protein). Two-dimensional electrophoresis indicated that the HSP-70 fra ction contained three equally abundant proteins with molecular masses of 70 , 65 and 55 kDa. The p70 and p65 proteins are phosphorylated and are themse lves O-linked GlcNAc (O-GlcNAc)-modified. The HSP-70 associated into high m olecular mass complexes that dissociated in the presence of reductive and c haotropic agents. The lectin(s) present in this complex was (were) able to recognize cytosolic and nuclear ligands, which have been isolated using whe at germ agglutinin affinity chromatography. These ligands are O-GlcNAc glyc osylated as demonstrated by [H-3]galactose incorporation and analysis of th e products released by reductive beta -elimination. The isolated lectins sp ecifically recognized ligands present in both the cytosol and the nucleus o f human resting lymphocytes. These results demonstrated the existence of en dogenous GlcNAc-specific lectins, identified as HSP-70 proteins, which coul d act as a shuttle for the nucleo-cytoplasmic transport of O-GlcNAc glycopr oteins between the cytosol and the nucleus.