Monocytes/macrophages and related cells are known to generate tissue factor (TF) , a membrane associated lipid-glycoprotein complex, following activation with LPS or other stimuli. Monkey (M. fuscata) mononuclear leukocytes (MNL, 3 × 106/ml) cultured with LPS (lµg/ml) in FCS-free RPMI medium were stimulated to produce the glycoprotein (TF-Apo). After a lag period of 2 h the TF-Apo production was initiated, and its accumulation reached the plateau after 12 h and then declined to approximately half of the maximum level after 24 h. A time course of the TF activity was strictly in accord with that of the TF-Apo accumulation. Tunicamycin, an antibiotic that blocks the first stage in formation of N-linked oligosaccharides of glycoprotein, affected to reduce the TF expression by 15 to 65 %, when monkey MNL (3 x 106/ml) were co-cultured with LPS (1 µg/ml) and the antibiotic (10 to 100 ng/ml) for 10 h. Similar reducing effect of tunicamycin to the TF expression was observed, when RET-1, a macrophage*related cell line that generates spontaneouly TF, was cultured with the antibiotic. Interestingly, leupeptin, an inhibitor to trypsin-type proteases including cathepsin B, protected completely the tunicamycin-induced reduction of the TF expression upon its addition to the culture medium at the concentration of 7 /iM. Chymostatin, an inhibitor to chymotrypsin-type proteases, also showed the protective effect. These results indicate that TF-Apo of monocytes and RET-1 has N-linked oligosaccharides and that defect of the oligosaccharide chain causes TF-Apo to be susceptible to proteolysis during intracellular processing. Thus, the N-glycosylated carbohydrate moiety of TF-Apo of these macrophage related cells has a roll to stabilize and/or protect it against proteolytic inactivation.
Turning the spotlight on the oligosaccharide chain of GM1 ganglioside
