Processing of MOPC 315 immunoglobulin A oligosaccharides: evidence for endoplasmic reticulum and trans Golgi alpha 1,2-mannosidase activity.

Abstract

The processing of asparagine-linked oligosaccharides on the .alpha.-chains of an IgA was investigated using MOPC 315 murine plasmacytoma cells. These cells secrete IgA containing complex-type oligosaccharides that were not sensitive to endo-.beta.-N-acetylglucosaminidase H. Oligosaccharides present on the intracellular .alpha.-chain precursor were of the high mannose-type, remaining sensitive to endo-.beta.-N-acetylglucosaminidase H despite a long intracellular half-life of 2-3 h. The major [3H]mannose-labeled .alpha.-chain oligosaccharides identified after a 20-min pulse were Man8GlcNAc2 and Man9GlcNAc2. Following chase incubations, the major oligosaccharide accumulating intracellularly was Man6GlcNAc2, which contained a single .alpha.1,2-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. Subcellular locations of the .alpha.1,2-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. No large accumulation of Man5GlcNAc2 occurred, indicating the presence of 2 subcellular locations of .alpha.1,2-mannosidase activity involved in oligosaccharide processing in MOPC 315 cells. The first 3 .alpha.1,2-linked mannose residues were removed shortly after the .alpha.-chain was glycosylated, most likely in rough endoplasmic reticulum, since this processing occurred in the presence of carbonyl cyanide m-chlorophenylhydrazone. The removal of the final .alpha.1,2-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.