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A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells
07:09

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Published on: July 31, 2012

Biosynthesis and posttranslational regulation of human IL-12

G Carra1, F Gerosa, G Trinchieri

  • 1Department of Pathology, Section of Immunology, University of Verona, Verona, Italy. abofcb@borgoroma.univr.it

Journal of Immunology (Baltimore, Md. : 1950)
|April 26, 2000
PubMed

IL-12 is a heterodimeric proinflammatory cytokine consisting of a light alpha-chain, formerly defined as p35, disulfide-linked to a heavier beta-chain, formerly defined as p40. The beta-chain is also produced in large excess in a free form, and disulfide-linked beta-chain homodimers with anti-inflammatory effects are produced in the mouse. We analyzed the biosynthesis and glycosylation of IL-12 in human monocytes, and in a cell line stably transfected with IL-12 alpha and beta genes (P5-0.1). The IL-12 heterodimer and free beta-chain were immunoprecipitated from supernatants and cell lysates of metabolically labeled cells and resolved in SDS-PAGE. Whereas the beta-chain showed similar pI pattern whether in the free form or associated in the heterodimer, either in the secreted or intracellular form, the alpha-chain in the secreted heterodimer was much more acidic than that present in the intracellular heterodimer. Deglycosylation experiments with neuraminidase and Endo-F combined with two-dimensional PAGE of single bands of the intracellular vs extracellular IL-12 heterodimer revealed that the alpha-chain was extensively modified with sialic acid adducts to N-linked oligosaccharides before secretion. N-glycosylation inhibition by tunicamycin (TM) did not alter free beta-chain secretion, while preventing the IL-12 heterodimer assembling and secretion. Pulse-chase experiments indicated that IL-12 persists intracellularly for a long period as an immature heterodimer, and that glycosylation is the regulatory step that determines its secretion. beta-chain disulfide-linked homodimers were observed in TM-treated P5-0.1 cells, but in neither TM-treated nor untreated monocytes.

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