Processing of MOPC 315 Immunoglobulin A Oligosaccharides: Evidence for Endoplasmic Reticulum and Trans Golgi α1,2-Mannosidase Activity

The processing of asparagine-linked oligosaccharides on the α-chains of an immunoglobulin A (IgA) has been investigated using MOPC 315 murine plasmacytoma cells. These cells secrete IgA containing complex-type oligosaccharides that were not sensitive to endo-β-N-acetylglucosaminidase H. In contrast, oligosaccharides present on the intracellular α-chain precursor were of the high mannose-type, remaining sensitive to endo-β-N-acetylglucosaminidase H despite a long intracellular half-life of 2-3 h. The major [3 H]mannose-labeled α-chain oligosaccharides identified after a 20-min pulse were Man8 GlcNAc2 and Man9 GlcNAc2. Following chase incubations, the major oligosaccharide accumulating intracellularly was Man6 GlcNAc2, which was shown to contain a single α1,2-linked mannose residue. Conversion of Man6 GlcNAc2 to complex-type oligosaccharides occurred at the time of secretion since appreciable amounts of Man5 GlcNAc2 or further processed structures could not be detected intracellularly. The subcellular locations of the α1,2-mannosidase activities were studied using carbonyl cyanide m-chlorophenylhydrazone and monensin. Despite inhibiting the secretion of IgA, these inhibitors of protein migration did not effect the initial processing of Man9 GlcNAc2 to Man6 GlcNAc2. Furthermore, no large accumulation of Man5 GlcNAc2 occurred, indicating the presence of two subcellular locations of α1,2-mannosidase activity involved in oligosaccharide processing in MOPC 315 cells. Thus, the first three α1,2-linked mannose residues were removed shortly after the α-chain was glycosylated, most likely in rough endoplasmic reticulum, since this processing occurred in the presence of carbonyl cyanide m-chlorophenylhydrazone. However, the removal of the final α1,2-linked mannose residue as well as subsequent carbohydrate processing occurred just before IgA secretion, most likely in the trans Golgi complex since processing of Man6 GlcNAc2 to Man5 GlcNAc2 was greatly inhibited in the presence of monensin.