Biosynthesis and posttranslational regulation of human IL-12.
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.
Carra
G
G
Department of Pathology, Section of Immunology, University of Verona, Verona, Italy. abofcb@borgoroma.univr.it
Gerosa
F
F
Trinchieri
G
G
D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D019281:Dimerization; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005110:Extracellular Space / Q000737:chemistry / Q000276:immunology; D006031:Glycosylation; D006801:Humans; D018664:Interleukin-12 / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D007424:Intracellular Fluid / Q000737:chemistry / Q000276:immunology; D008262:Macrophage Activation / Q000187:drug effects; D009000:Monocytes / Q000737:chemistry / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D009439:Neuraminidase / Q000378:metabolism; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational / Q000276:immunology*; D013997:Time Factors; D014415:Tunicamycin / Q000494:pharmacology
D004220:Disulfides; D014415:Tunicamycin; D018664:Interleukin-12; D009439:Neuraminidase