BIOSYNTHESIS AND SECRETION OF HUMAN INTERLEUKIN-2 GLYCOPROTEIN VARIANTS FROM BACULOVIRUS-INFECTED SF21 CELLS - CHARACTERIZATION OF POLYPEPTIDES AND POSTTRANSLATIONAL MODIFICATIONS

Human interleukin 2 (IL-2) and human IL-2 mutant proteins, with artificially introduced N-glycosylation or 0-glycosylation sites, have been expressed in a lepidopteran cell line (Sf21, Spodoptera frugiperda) using recombinant baculovirus vectors. Only approximately 25% of the total recombinant EL-2 protein synthesized by Sf21 cells was secreted into die 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 0-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 0-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(beta1-3)GalNAc as the only carbohydrate constituents attached exclusively to Thr in this peptide, in a specific ratio for each individual EL-2 mutant protein. The Gal(beta1-3)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(alpha1-6)]GlcNAc or Man2GlcNAc[Fuc(alpha1-6)]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.