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.
Highlights
From the Departments of Gene Expression Sciences, Molecular Genetics, Macromolecular Sciencesa,nd Immunology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406
17-kDa carboxyl-terminal fragment, despite the fact these methods have their limitations which can affect interthat it containsa potential N-linkedglycosylation site pretation, and it is only via a combination of them that a near theNH2terminus (-Asn7-Cyss-Thrs-).cDNA coding for authentic mature IL-18 wfuased to the signal sequencefrom the Candidaalbicans glucoamylase gene, two amino acids downstream from the signal processing site
Interleukin-1 (IL-1)’ is a cytokine exhibiting a broad spectrum of proinflammatory and immunostimulatory activities
Summary
Integrative transformants were obtained according to the method of Beggs [29], involving the formation of spheroplasts generated by Strains andMedia-The following S. cereuisiae strains were used Glusulase (Du Pont Pharmaceuticals) treatment. Strain GL46 was grownin YEPD liquid medium plasmid constructions, and strainAR58 [21,22] was used for expres- at 30 "C to stationary phase (OD,,, = 8.1).For each construct, both sion.Transformation of E. coli cells was carried out according to cell and supernatant fractions from a t least six independent yeast. Of trichloroacetic acid-precipitable cpm wereimmunoprecipitated (see pYSK140 carries the yeast selectable marker TRPl aswell as partial below) and subjected to SDS-PAGE and fluorography. PGL103 was constructed by subcloning a with RIPA buffer (0.15 M NaCl, 0.1% SDS, 1%sodium deoxycholate, 1200-bp NcoI-XhoI fragment containing wild-type matureIL-10 1%Triton X-100,5mM EDTA, 20 mM Tris-HC1,pH 7.4) and blocked cDNA from pMGNcoIL-16 (see below) into the unique NcoI and Sal for 1 h at 37 "C in RIPA plus 1%gelatin (porcine skin, Sigma) and sites of pYSK142. Unbound material (ConA- fraction) was washed off the column with excess buffer A, and material bound to the column
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