Role of N-glycosylation in the synthesis, dimerization and secretion of human interferon-gamma.
Human interferon-gamma (IFN-gamma) is a secretory glycoprotein, which has two potential N-linked glycosylation sites at positions Asn-25 and Asn-97 of its 143 amino acid long mature polypeptide chain. In order to understand the role of glycan residues in the synthesis and secretion of human IFN-gamma, both or either one of the potential N-linked glycosylation sites were mutated to Gln. The mutant and the wild-type (Wt) polypeptides were expressed in insect cells using a baculovirus vector. Elimination of the N-glycosylation site at position Asn-97 (N97Q) resulted in secreted protein yields of 70-90% as compared with the Wt production, whereas only 10-25% (N25Q) and 1-10% (N25Q,N97Q) levels of protein production was observed when the first or both sites were mutated, respectively. Although there was a difference between extracellular levels of produced protein, the kinetics of secretion was similar for all different IFN-gamma molecules. The Wt and the N-glycosylation site mutants were all secreted as dimers. The formation of biologically active dimers was more efficient for IFN-gamma polypeptides that had the intact glycosylation site at Asn-25 as compared with the other two mutant forms of IFN-gamma. The extent of dimerization correlated well with the observed secretion. The specific antiviral activity was of the same order (1 x 10(7) i.u./mg of protein) for the glycosylated IFN-gamma molecules, whereas it was slightly lower (0.5 x 10(7) i.u./mg of protein) for the unglycosylated mutant form.
National Public Health Institute, Helsinki, Finland.
T
T
Sareneva
J
J
Pirhonen
K
K
Cantell
N
N
Kalkkinen
I
I
Julkunen
D000595:Amino Acid Sequence; D000818:Animals; D000998:Antiviral Agents / Q000378:metabolism / Q000494:pharmacology; D001216:Asparagine / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001704:Biopolymers; D002460:Cell Line; D003001:Cloning, Molecular; D006031:Glycosylation; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011134:Polysaccharides / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D018411:Spodoptera
D000998:Antiviral Agents; D001704:Biopolymers; D009838:Oligodeoxyribonucleotides; D011134:Polysaccharides; D011994:Recombinant Proteins; D001216:Asparagine; D007371:Interferon-gamma