Abstract
The bisecting N-acetylglucosamine residue is formed by UDP-N-acetylglucosamine:beta-D-mannoside-beta-1, 4-N-acetylglucosaminyltransferase III (GnT-III), a key branching enzyme for N-glycans. We found that forskolin, an adenylyl cyclase activator, 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 binding to erythroagglutinating phytohemagglutinin (E-PHA). In whole cell lysates, the E-PHA binding was increased, and leukoagglutinating phytohemagglutinin (L-PHA) binding was decreased at 12 h after forskolin treatment, by time, both GnT-III activity and mRNA had reached the maximum levels. In contrast, the binding capacity as to E-PHA, determined by fluorescence-activated cell sorting on the cell surface, was decreased, suggesting that bisecting GlcNAc structures in certain glycoproteins changed the expression levels of glycoproteins and decreased their sorting on the cell surface. Fractionated organelles of M31 cells showed that the binding capacity as to E-PHA was mainly localized in Golgi 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-glucuronidase, gamma-glutamyltranspeptidase, and secretory glycoproteins such as ceruloplasmin and alpha-fetoprotein were measured by E-PHA and L-PHA blotting after immunoprecipitation. The expression levels of lysosomal membrane glycoprotein 1 and gamma-glutamyltranspeptidase on the cell surface were decreased at 12 h after forskolin treatment, indicating that the bisecting GlcNAc structure may act as a negative sorting signal for the cell surface glycoproteins and may alter the characteristics of hepatoma cells. This is the first report on glycoprotein sorting related to a specific structure of oligosaccharides, bisecting GlcNAc.
Highlights
N-Linked glycosylation of proteins begins at the lumen of the rough endoplasmic reticulum, where a subset of Asn-X-Ser/Thr residues on newly synthesized proteins is subjected to addition with Glc3Man9GlcNAc2
We found that the enhanced GnT-III resulted in an increase of bisecting GlcNAc residues of glycoproteins in whole cell lysates but inhibited the glycoproteins sorting on the cell surface, such as that of Lamp-1 and ␥-GTP, while it did not affect the secretion levels of secretory proteins such as ceruloplasmin and ␣-fetoprotein, despite changing the oligosaccharide structures
While we were searching for some factors that induce GnT-III, we found that forskolin strongly enhanced the activity and mRNA expression of GnT-III in a rat hepatoma cell line, M31
Summary
N-Linked glycosylation of proteins begins at the lumen of the rough endoplasmic reticulum, where a subset of Asn-X-Ser/Thr residues on newly synthesized proteins is subjected to addition with Glc3Man9GlcNAc2. High GnT-III activity has been reported in many tumor cells, such as Novikoff ascites tumor cells [13], AH-66 hepatoma ascites cells [14], and Huh cells [15], and in both sera and livers of patients with liver cancer and cirrhosis [16]. To determine the correlation between the oligosaccharide structure of a specific glycoprotein and its distribution, especially after bisecting GlcNAc residues’ addition, we examined a number of glycoproteins that have different roles and expression sites, such as Lamp-1 and -glucuronidase as lysosomal proteins, ␥-GTP as a plasma membrane protein, ceruloplasmin, and ␣-fetoprotein as a secretory protein. Lamp-1 is expressed on the surface of many tumor cells [20, 21], the majority of this molecule resides in lysosomes [22, 23]
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