IL-12 is a heterodimeric proinflammatory cytokine consisting of a light alp
ha-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-inflam
matory effects are produced in the mouse. We analyzed the biosynthesis and
glycosylation of IL-12 in human monocytes, and in a cell line stably transf
ected with IL-12 alpha and beta genes (P5-0.1), The IL-12 heterodimer and f
ree beta-chain were immunoprecipitated from supernatants and cell lysates o
f metabolically labeled cells and resolved in SDS-PAGE, Whereas the beta-ch
ain 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 a
nd Endo-F combined with two-dimensional PAGE of single bands of the intrace
llular 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 a
lter free beta-chain secretion, while preventing the IL-12 heterodimer asse
mbling and secretion. Pulse-chase experiments indicated that IL-12 persists
intracellularly for a long period as an immature heterodimer, and that gly
cosylation 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.