Differential N-Glycan Patterns of Secreted and Intracellular IgG Produced in Trichoplusia ni Cells

• Published in: The Journal of biological chemistry Vol. 272; no. 14; pp. 9062 - 9070
• Main Authors: Hsu, T A, Takahashi, N, Tsukamoto, Y, Kato, K, Shimada, I, Masuda, K, Whiteley, E M, Fan, J Q, Lee, Y C, Betenbaugh, M J
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 04-04-1997
• Subjects: Animals; Carbohydrate Sequence; Chromatography, High Pressure Liquid; Culture Media; Immunoglobulin G - chemistry; Lepidoptera; Mass Spectrometry; Molecular Sequence Data; Noctuidae; Oligosaccharides - chemistry; Polysaccharides - chemistry; Trichoplusia ni
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.14.9062

Structures of the N -linked oligosaccharide attached to the heavy chain of a heterologous murine IgG 2a produced from Trichoplusia ni (TN-5B1-4, High Five) insect cells were characterized. Coexpression of the chaperone immunoglobulin heavy chain-binding protein
(BiP) in the baculovirus-infected insect cells increased the soluble intracellular and secreted IgG level. This facilitated
the detailed analysis of N -glycans from both intracellular and secreted IgG. Following purification of the immunoglobulins using Protein A-Sepharose,
glycopeptides, prepared by trypsin-chymotrypsin digestion, were further digested with glycoamidase from sweet almond emulsin
to obtain the oligosaccharide moieties. The resulting oligosaccharides were then reductively aminated with 2-aminopyridine
and the structures identified by two-dimensional high performance 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% complex type, some with terminal galactose residues at either α1,3-Man
or α1,6-Man branches of the Man 3 GlcNAc 2 core. The remaining oligosaccharides detected in the secreted IgG were principally hybrid (30%) and paucimannosidic (35%)
type N -glycans. Most (84%) of these secreted glycoforms contained fucose α1,6-linked to the innermost GlcNAc residue and the presence
of a potentially allergenic fucose α1,3-linked to the innermost GlcNAc residue was also detected. In contrast, the intracellular
immunoglobulins included 50% high mannose-type N -glycans with lower levels of complex, hybrid, and paucimannosidic-type structures. Reverse phase one-dimensional high performance
liquid chromatography analysis of the IgG N -glycans in the absence of heterologous BiP exhibited a similar distribution of intracellular and secreted glycoforms. These
studies indicate that Trichoplusia ni TN-5B1-4 cells are capable of terminal galactosylation. However, the processing pathways in these cell lines appear to diverge
from mammalian cells in the formation of paucimannosidic structures, in the presence of α1,3-fucose linkages, and in the absence
of sialylation.