Title : Biosynthesis and secretion of human
interleukin 2 glycoprotein variants from baculovirus-infected
Sf21 cells
Abstract :
- Characterization of polypeptides and posttranslational modifications
- 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, com position al 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