Biosynthesis, processing, and secretion of glial cell line-derived neurotrophic factor in astroglial cells

Glial cell line-derived neurotrophic factor (GDNF) is synthesized as a precursor, proGDNF. However, the molecular mechanisms for the processing and secretion of GDNF are not fully characterized, since the amount of its biosynthesis and secretion in glial cells are below the detection limit of western blotting. We established stably GDNF-overexpressing C6 cells, and this enabled us to monitor its spontaneous secretion, as well as its processed forms in the cells. GDNF secretion was augmented by stimulation with high potassium, while it was inhibited by treatment with either tunicamycin, an inhibitor of protein glycosylation, or brefeldin A, a disturbing factor of ER-Golgi transport. Wild-type GDNF transfected cells secreted three forms of processed GDNF. After deglycosylation, the highest molecular weight of secreted GDNF showed the same mobility on electrophoresis as recombinant human GDNF without a whole pro-domain. Mutations in the pro-domain and two cysteines at the C-terminal of GDNF markedly diminished the secretion of resultant proteins into the culture medium. GDNF proteins having mutations in the putative furin-consensus sequence were secreted partly as unprocessed forms, and forms with lower molecular weights than a mature form were secreted from the C6 cells. Taking these observations together, we conclude that GDNF is likely secreted both with and without processing by furin-like proteases, and that the pro-domain and C-terminal cysteines of GDNF play important roles in its processing and secretion in cultured astrocytes and C6 cells.