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Biosynthesis and Posttranslational Regulation of Human IL-12¹

Giuseppe Carra^2,*, Franca Gerosa^* and Giorgio Trinchieri^3,

^* Department of Pathology, Section of Immunology, University of Verona, Verona, Italy; and The Wistar Institute, Philadelphia, PA 19104

IL-12 is a heterodimeric proinflammatory cytokine consisting of a light -chain, formerly defined as p35, disulfide-linked to a heavier ß-chain, formerly defined as p40. The ß-chain is also produced in large excess in a free form, and disulfide-linked ß-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 and ß genes (P5-0.1). The IL-12 heterodimer and free ß-chain were immunoprecipitated from supernatants and cell lysates of metabolically labeled cells and resolved in SDS-PAGE. Whereas the ß-chain showed similar pI pattern whether in the free form or associated in the heterodimer, either in the secreted or intracellular form, the -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 -chain was extensively modified with sialic acid adducts to N-linked oligosaccharides before secretion. N-glycosylation inhibition by tunicamycin (TM) did not alter free ß-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. ß-chain disulfide-linked homodimers were observed in TM-treated P5-0.1 cells, but in neither TM-treated nor untreated monocytes.