Biosynthesis and posttranslational regulation of human IL-12

Citation
G. Carra et al., Biosynthesis and posttranslational regulation of human IL-12, J IMMUNOL, 164(9), 2000, pp. 4752-4761
Citations number
31
Categorie Soggetti
Immunology
Journal title
JOURNAL OF IMMUNOLOGY
ISSN journal
00221767 → ACNP
Volume
164
Issue
9
Year of publication
2000
Pages
4752 - 4761
Database
ISI
SICI code
0022-1767(20000501)164:9<4752:BAPROH>2.0.ZU;2-P
Abstract
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.