DIFFERENTIAL N-GLYCAN PATTERNS OF SECRETED AND INTRACELLULAR IGG PRODUCED IN TRICHOPLUSIA NI CELLS

Structures of the N-linked oligosaccharide attached to the heavy chain of a heterologous murine IgG(2a) produced from Trichoplusia ni (TN-5B 1-4, High Five) insect cells were characterized. Coexpression of the c haperone immunoglobulin heavy chain-binding protein (BiP) in the bacul ovirus-infected insect cells increased the soluble intracellular and s ecreted IgG level. This facilitated the detailed analysis of N-glycans from both intracellular and secreted IgG. Following purification of t he immunoglobulins using Protein A-Sepharose, glycopeptides, prepared by trypsin-chymotrypsin digestion, were further digested with glycoami dase from sweet almond emulsin to obtain the oligosaccharide moieties. The resulting oligosaccharides were then reductively aminated with a- aminopyridine and the structures identified by two-dimensional high pe rformance liquid chromatography mapping (Tomiya, N., Awaya, J., Kurono , M., Endo, S., Arata, Y., and Takahashi, N. (1988) Anal. Biochem. 171 , 73-90). The N-glycans obtained from the secreted IgG contain 35% com plex type, some with terminal galactose residues at either alpha 1,3-M an or alpha 1,6-Man branches of the Man(3)GlcNAc(2) core. The remainin g oligosaccharides detected in the secreted IgG were principally hybri d (30%) and paucimannosidic (35%) type N-glycans. Most (84%) of these secreted glycoforms contained fucose alpha 1,6-linked to the innermost GlcNAc residue and the presence of a potentially allergenic fucose al pha 1,3-linked to the innermost GlcNAc residue was also detected. In c ontrast, the intracellular immunoglobulins included 50% high mannose-t ype N glycans with lower levels of complex, hybrid, and paucimannosidi c-type structures. Reverse phase one-dimensional high performance liqu id chromatography analysis of the IgG N-glycans in the absence of hete rologous BiP exhibited a similar distribution of intracellular and sec reted glycoforms. These studies indicate that Trichoplusia ni TN-5B1-4 cells are capable of terminal galactosylation. However, the processin g pathways in these cell lines appear to diverge from mammalian cells in the formation of paucimannosidic structures, in the presence of alp ha 1,3-fucose linkages, and in the absence of sialylation.