N-glycan structures of human transferrin produced by Lymantria dispar (gypsy moth) cells using the LdMNPV expression system

• Published in: Glycobiology (Oxford) 2003-07, Vol.13 (7), p.539-548
• Main Authors: Choi, One, Tomiya, Noboru, Kim, Jung H., Slavicek, James M., Betenbaugh, Michael J., Lee, Yuan C.
• Format: Article
• Language: English
• Subjects: AcMNPV; Animals; Autographa californica multicapsid nucleopolyhedrovirus; Baculovirus; Carbohydrate Conformation; Carbohydrate Sequence; Culture Media; FBS; fetal bovine serum; Gene Expression; glucose unit; Gypsy moth; high-performance anion-exchange chromatography with pulsed amperometric detection; high-performance liquid chromatography; HPAEC-PAD; HPLC; hTf; human transferrin; Humans; Insect cells; LdMNPV; Lepidoptera - genetics; Lymantria dispar; Lymantria dispar multicapsid nucleopolyhedrovirus; Lymantria dispar nucleopolyhedrovirus; MALDI-TOF; mass spectrometry; matrix-assisted laser desorption/ionization time of flight; Molecular Sequence Data; N-glycan; Nuclear polyhedrosis virus; Polysaccharides - biosynthesis; Polysaccharides - chemistry; pyridylamino; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; SDS–PAGE; sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TCID50; tissue culture infectious dose; Transferrin - biosynthesis; Transferrin - chemistry; Transferrin - genetics; Transferrin - isolation & purification
• ISSN: 0959-6658; 1460-2423
• DOI: 10.1093/glycob/cwg071

N-glycan structures of recombinant human serum transferrin (hTf) expressed by Lymantria dispar (gypsy moth) 652Y cells were determined. The gene encoding hTf was incorporated into a Lymantria dispar nucleopolyhedrovirus (LdMNPV) under the control of the polyhedrin promoter. This virus was then used to infect Ld652Y cells, and the recombinant protein was harvested at 120 h postinfection. N-glycans were released from the purified recombinant human serum transferrin and derivatized with 2-aminopyridine; the glycan structures were analyzed by a two-dimensional HPLC and MALDI-TOF MS. Structures of 11 glycans (88.8% of total N-glycans) were elucidated. The glycan analysis revealed that the most abundant glycans were Man1–3(±Fucα6)GlcNAc2 (75.5%) and GlcNAcMan3(±Fucα6)GlcNAc2 (7.4%). There was only ∼6% of high-mannose type glycans identified. Nearly half (49.8%) of the total N-glycans contained α(1,6)-fucosylation on the Asn-linked GlcNAc residue. However α(1,3)-fucosylation on the same GlcNAc, often found in N-glycans produced by other insects and insect cells, was not detected. Inclusion of fetal bovine serum in culture media had little effect on the N-glycan structures of the recombinant human serum transferrin obtained.