BISECTING GLCNAC STRUCTURES ACT AS NEGATIVE SORTING SIGNALS FOR CELL-SURFACE GLYCOPROTEINS IN FORSKOLIN-TREATED RAT HEPATOMA-CELLS

The bisecting N-acetylglucosamine residue is formed by mannoside-beta- 1,4-N-acetylglucosaminyltransferase III (GnT-III), a key branching enz yme for N-glycans. We found that forskolin, an adenylyl cyclase activa tor, markedly enhanced GnT-III at the transcriptional level in various hepatoma cells and hepatocytes, resulting in an increase of bisecting GlcNAc residues in various glycoproteins, as judged from the lectin b inding to erythroagglutinating phytohemagglutinin (E-PHA). In whole ce ll lysates, the E-PHA binding was increased, and leukoagglutinating ph ytohemagglutinin (L-PHA) binding was decreased at 12 h after forskolin treatment, by time, both GnT-III activity and mRNA had reached the ma ximum levels. In contrast, the binding capacity as to E-PHA, determine d by fluorescence-activated cell sorting on the cell surface, was decr eased, suggesting that bisecting GlcNAc structures in certain glycopro teins changed the expression levels of glycoproteins and decreased the ir sorting on the cell surface. Fractionated organelles of M31 cells s howed that the binding capacity as to E-PKA was mainly localized in Ge ls membranes and lysosomes. This was also supported by a fluorescence microscopy. In order to determine whether or not the bisecting GlcNAc residue acts as a sorting signal for glycoproteins, N-oligosaccharide structures of lysosomal-associated membrane glycoprotein 1 and beta-gl ucuronidase, gamma-glutamyltranspeptidase, and secretory glycoproteins such as ceruloplasmin and alpha-fetoprotein were measured by E-PHA an d L-PHA blotting after immunoprecipitation. The expression levels of l ysosomal membrane glycoprotein 1 and gamma-glutamyltranspeptidase on t he cell surface were decreased at 12 h after forskolin treatment, indi cating that the bisecting GlcNAc structure may act as a negative sorti ng signal for the cell surface glycoproteins and may alter the charact eristics of hepatoma cells. This is the first report on glycoprotein s orting related to a specific structure of oligosaccharides, bisecting GlcNAc.