title: Secretion of N-glycosylated interleukin-1 beta in Saccharomyces cerevisiae using a leader peptide from Candida albicans. Effect of N-linked glycosylation on biological activity.
abstract: Human interleukin-1 beta (IL-1 beta) is expressed in activated monocytes as a 31-kDa precursor protein which is processed and secreted as a mature, unglycosylated 17-kDa carboxyl-terminal fragment, despite the fact that it contains a potential N-linked glycosylation site near the NH2 terminus (-Asn7-Cys8-Thr9-). cDNA coding for authentic mature IL-1 beta was fused to the signal sequence from the Candida albicans glucoamylase gene, two amino acids downstream from the signal processing site. Upon expression in Saccharomyces cerevisiae, approximately equimolar amounts of N-glycosylated (22 kDa) and unglycosylated (17 kDa) IL-1 beta protein were secreted. The N-glycosylated yeast recombinant IL-1 beta exhibited a 5-7-fold lower specific activity compared to the unglycosylated species. The mechanism responsible for inefficient glycosylation was also studied. We found no differences in secretion kinetics or processing between the two extracellular forms of IL-1 beta. The 17-kDa protein, which was found to lack core sugars, does not result from deglycosylation of the 22-kDa protein in vivo and does not result from saturation of the glycosylation enzymatic machinery through overexpression. Alteration of the uncommon Cys8 residue in the -Asn-X-Ser/Thr-glycosylation site to Ser also had no effect. However, increasing the distance between Asn7 and the signal processing site increased the extent of core N-linked glycosylation, suggesting a reduction in glycosylation efficiency near the NH2 terminus.
authors.affiliation: Department of Gene Expression Sciences, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406.
authors.forename: G P
authors.initials: GP
authors.lastname: Livi
authors.forename: J S
authors.initials: JS
authors.lastname: Lillquist
authors.forename: L M
authors.initials: LM
authors.lastname: Miles
authors.forename: A
authors.initials: A
authors.lastname: Ferrara
authors.forename: G M
authors.initials: GM
authors.lastname: Sathe
authors.forename: P L
authors.initials: PL
authors.lastname: Simon
authors.forename: C A
authors.initials: CA
authors.lastname: Meyers
authors.forename: J A
authors.initials: JA
authors.lastname: Gorman
authors.forename: P R
authors.initials: PR
authors.lastname: Young
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002176:Candida albicans / Q000378:metabolism*; D003001:Cloning, Molecular; D004271:DNA, Fungal; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D007375:Interleukin-1 / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D021382:Protein Sorting Signals / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014415:Tunicamycin / Q000494:pharmacology
chemical_list: D004271:DNA, Fungal; D007375:Interleukin-1; D021382:Protein Sorting Signals; D014415:Tunicamycin