STRUCTURAL CHARACTERIZATION OF THE RECOMBINANT P40 HEAVY-CHAIN SUBUNIT MONOMER AND HOMODIMER OF MURINE IL-12

Interleukin-12 (IL-12) is a heterodimeric cytokine that consists of tw o structurally unrelated subunits, P35 and P40. However, when expresse d alone in Chinese hamster ovary (CHO) cells, murine P40 showed two sp ecies of different molecular weights under nonreducing conditions, a m onomeric form of 45 kDa and a homodimer of >97 kDa. Under reducing con ditions the two forms migrated as an identical array of species of 40- 45 kDa. The monomer was separated from the homodimer under nonreducing conditions by heparin affinity chromatography and the disulfide bond structures of both species were determined by peptide mapping, Edman s equencing, and mass spectrometry. The peptide maps of the two species were identical except for a single peak that changed retention time. S equencing showed that this peak contained two peptides of identical se quences in both maps. Mass spectrometric analysis of the peak from the >97-kDa species revealed an ion of double the expected mass, thus ind icating that the peptide pair had dimerized. Mass analysis of the peak from the 40- to 45-kDa species showed that the peptide pair contained a mass difference that corresponded to that of an extra cysteine and which disappeared upon reduction. Amino acid analysis confirmed that t he monomeric form of rmP40 is modified by a reducible cysteine. Struct ural analysis of the remainder of the cysteine-containing peaks showed that both species of rmP40 contained the same set of intramolecular d isulfide bonds. The murine P40 homodimer arises from formation of a si ngle intermolecular disulfide bond at Cys(175). In the monomeric P40, however, this cysteine is capped by an additional cysteine. Purified r mP40 monomer and homodimer inhibited the IL-12-dependent induction of interferon-gamma, but neither appeared capable of inducing IL-12-like biological activity. (C) 1995 Academic Press, Inc.