N-glycan structures of human transferrin produced by Lymantria dispar (gypsy moth) cells using the LdMNPV expression system.
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(+/-Fucalpha6)GlcNAc2 (75.5%) and GlcNAcMan3(+/-Fucalpha6)GlcNAc2 (7.4%). There was only approximately 6% of high-mannose type glycans identified. Nearly half (49.8%) of the total N-glycans contained alpha(1,6)-fucosylation on the Asn-linked GlcNAc residue. However alpha(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.
Choi
One
O
Department of Biology, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
Tomiya
Noboru
N
Kim
Jung H
JH
Slavicek
James M
JM
Betenbaugh
Michael J
MJ
Lee
Yuan C
YC
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D003470:Culture Media; D015870:Gene Expression; D006801:Humans; D007915:Lepidoptera / Q000235:genetics*; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000096:biosynthesis* / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D014168:Transferrin / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D003470:Culture Media; D011134:Polysaccharides; D011994:Recombinant Proteins; D014168:Transferrin