Comparative study of high‐mannose‐type oligosaccharides in membrane glycoproteins of rat hepatocytes and different rat hepatoma cell lines

A comparative study was undertaken to characterize the oligosaccharides released by endo-beta-N-acetylglucosaminidase H (endo H) from the membrane glycoproteins of rat hepatocytes and three different Morris hepatoma cell lines (NA-MH 7777, HTC and MH1C1). It is shown that the membrane glycoproteins of hepatocytes and hepatoma cells contain markedly different quantities and forms of high-mannose-type carbohydrate chains. After radiolabelling of the cells with D-[2-3H]mannose, in the absence and presence of 1 mM 1,5-dideoxy-1,5-imino-D-mannitol (1-deoxymannojirimycin), high-mannose-type oligosaccharides were released from delipidated membrane glycoproteins by enzymic digestion with endo H. The carbohydrate chains were converted to their corresponding oligosaccharide alditols by reduction with sodium borohydride, then further analysed by HPLC using an APS-2 Hypersil column. In the absence of 1-deoxymannojirimycin, up to 10% of the radiolabelled oligosaccharides were released by endo H-treatment of the membrane glycoprotein fraction from rat hepatocytes. In contrast, the quantity of radiolabelled high-mannose-type carbohydrate chains released by endo H-treatment from tumour-cell membrane glycoproteins of hepatoma cell lines NA-MH 7777 (31.5%). MH1C1-MH 7795 (37.2%) and HTC-MH 7288c (48%) was increased up to fivefold. The formation of higher-mannosylated structures after oligosaccharide analysis was observed in all hepatoma cell lines, with Man8GlcNAcOH as the major component, whereas in hepatocytes Man5GlcNAcOH was the predominant high-mannose-type structure. In contrast, in the presence of the Golgi alpha-D-mannosidase I inhibitor, 1-deoxymannojirimycin, no significant differences were observed between the distribution of high-mannose-type oligosaccharides in the membrane glycoproteins of hepatocytes and hepatoma cells. However, in the presence of this inhibitor, the proportion of radiolabelled glycans sensitive to deglycosylation by endo H was greatly increased (> 85%) in all the cell lines investigated, the predominant structures being Man8-9-GlcNAcOH. This study shows that an increased content of high-mannose-type sugar chains is a general characteristic of membrane-bound glycoproteins for malignant transformed hepatocytes.