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 [³H]mannose-labeled α-chain oligosaccharides identified after a 20-min pulse were Man₈GlcNAc₂ and Man₉GlcNAc₂. Following chase incubations, the major oligosaccharide accumulating intracellularly was Man₆GlcNAc₂, which was shown to contain a single α1,2-linked mannose residue. Conversion of Man₆GlcNAc₂ to complex-type oligosaccharides occurred at the time of secretion since appreciable amounts of Man₅GlcNAc₂ 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 Man₉GlcNAc₂ to Man₆GlcNAc₂. Furthermore, no large accumulation of Man₅GlcNAc₂ 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 Man₆GlcNAc₂ to Man₅GlcNAc₂ was greatly inhibited in the presence of monensin.