Biosynthesis of complement C1̄ inhibitor by Hep G2 cells. Reactivity of different glycosylated forms of the inhibitor with C1̄s

Abstract

The biosynthesis of C1̄ Inh (C1̄ inhibitor) was studied in a human hepatoma cell line (Hep G2) by metabolic labelling, immunoprecipitation with anti-(C1̄ Inh) serum, analysis on SDS/polyacrylamide gel slabs and fluorography. Two forms of C1̄ Inh are secreted by Hep G2: a minor form of M(r) 90,000 and a major form of M(r) ~ 100,000. The latter form is also found in small amounts intracellularly in co-existence with an 80,000-M(r) form. Accumulation of the 80,000-M(r) C1̄ Inh is favoured when the cells are labelled at 23°C instead of 37°C or when they are treated with monensin. In the presence of tunicamycin, a compound that blocks the formation of N-asparagine-linked oligosaccharide chains, a decrease in M(r) of both secreted and intracellular major forms is observed, indicating that secreted and intracellular C1̄ Inh contain N-linked oligosaccharide units. The 100,000 M(r) secreted C1̄ Inh is sensitive to endoglycosidase F but resistant to endoglycosidase H, and it incorporates [3H]galactose, [3H]glucosamine and [3H]galactosamine, indicating the presence of both N-linked oligosaccharides of the complex type and O-linked oligosaccharides. The intracellular C1̄ Inh contains N-linked oligosaccharide units of the high-mannose type as demonstrated by endoglycosidase H-sensitivity. The functional activity of C1̄ Inh during its biosynthesis was tested by studying its reactivity towards C1̄s. Both secreted and intracellular C1̄ Inh form covalent-like complexes with purified plasma C1̄s. The underglycosylated C1̄ Inh secreted in presence of tunicamycin is still reactive with purified C1̄s. These results clearly show that sugars are not essential for this inhibitory activity of C1̄ Inh.