title: Biosynthesis and secretion of human interleukin 2 glycoprotein variants from baculovirus-infected Sf21 cells. Characterization of polypeptides and posttranslational modifications.
abstract: Human interleukin 2 (IL-2) and human IL-2 mutant proteins, with artificially introduced N-glycosylation or O-glycosylation sites, have been expressed in a lepidopteran cell line (Sf21, Spodoptera frugiperda) using recombinant baculovirus vectors. Only approximately 25% of the total recombinant IL-2 protein synthesized by Sf21 cells was secreted into the culture medium. Significant N-terminal truncations were detected in the secreted polypeptides (up to 85% of the molecules). Alanine and proline were absent in the major truncated forms; the first 3-5 amino acids were also absent in a small proportion of the purified proteins. The introduction of potential artificial O-glycosylation peptide sequences (..GGKAPTPPPK..), to the C-terminus or between positions 80 and 81 of the IL-2 polypeptide chain, resulted in the secretion of unglycosylated and O-glycosylated variant forms. Fast atom bombardment mass spectrometry, compositional analysis and methylation analysis, of the tryptic glycopeptide APTPPPK, revealed the presence of either GalNAc or the disaccharide Gal(beta 1-3)GalNAc as the only carbohydrate constituents attached exclusively to Thr in this peptide, in a specific ratio for each individual IL-2 mutant protein. The Gal(beta 1-3)GalNAc protein forms could be partially altered in vitro to mammalian-type glycoforms by porcine liver beta-galactoside alpha-2,3-sialyltransferase in the presence of CMP-N-acetylneuraminic acid. An IL-2 mutant form, with an 11-amino-acid peptide of human interferon-beta at position 4, which includes its only N-glycosylation site, had exclusively truncated proximally fucosylated oligomannosidic glycans; Man3GlcNAc[Fuc(alpha 1-6)]GlcNAc or Man2GlcNAc[Fuc(alpha 1-6)]GlcNAc structures, in a ratio of 3:1, were detected in the secreted proteins. No evidence was obtained for the presence of secreted proteins with complex oligosaccharide chains, irrespective of the cell-culture conditions used or the harvesting time, for infected cells with recombinant baculovirus constructs.
authors.affiliation: Department of Cell Biology and Genetics, Gesellschaft für Biotechnologische Forschung, Braunschweig, Germany.
authors.forename: E
authors.initials: E
authors.lastname: Grabenhorst
authors.forename: B
authors.initials: B
authors.lastname: Hofer
authors.forename: M
authors.initials: M
authors.lastname: Nimtz
authors.forename: V
authors.initials: V
authors.lastname: Jäger
authors.forename: H S
authors.initials: HS
authors.lastname: Conradt
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D002240:Carbohydrate Sequence; D002460:Cell Line; D006031:Glycosylation; D006801:Humans; D007376:Interleukin-2 / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009036:Moths; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis*
chemical_list: D007376:Interleukin-2; D011994:Recombinant Proteins