PMID: 8344280

 

    Legend: Gene, Sites

Title : Biosynthesis and secretion of human interleukin 2 glycoprotein variants from baculovirus-infected Sf21 cells

Abstract :
  1. Characterization of polypeptides and posttranslational modifications
  2. 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
  3. Only approximately 25% of the total recombinant IL-2 protein synthesized by Sf21 cells was secreted into the culture medium
  4. Significant N-terminal truncations were detected in the secreted polypeptides (up to 85% of the molecules)
  5. 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
  6. The introduction of potential artificial O-glycosylation peptide sequences (
  8. GGKAPTPPPK
  9. .
  10. , 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
  11. 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
  12. 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
  13. 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
  14. 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
Output (sent_index, trigger, protein, sugar, site):
  • 0. glycoprotein, , interleukin 2 glycoprotein, -, -
  • 11. N-glycosylation, , -, -, site
  • 11. fucosylated, , -, truncated proximally fucosylated oligomannosidic glycans, -
  • 12. proteins, , proteins, complex oligosaccharide chains, -
  • 2. N-glycosylation, , proteins, -, -
  • 2. O-glycosylation, , -, -, sites
  • 9. attached, , -, the only carbohydrate constituents, Thr
  • 9. glycopeptide, , -, -, glycopeptide
Output(Part-Of) (sent_index, protein, site):
  • 11. -, position 4,
  • 11. interferon-beta, peptide
  • 11. interferon-beta, position 4,
  • 2. proteins, sites
*Output_Site_Fusion* (sent_index, protein, sugar, site):

 

 

Protein NCBI ID SENTENCE INDEX
Human interleukin 2 3558 2
IL-2 3558 2,10,11
interferon-beta 3456 13
IL-2 protein 3558 3
An IL-2 3558 13
interleukin 2 glycoprotein 3558 0