Molecular characterization of interleukin 12 - PubMed (original) (raw)
Molecular characterization of interleukin 12
F J Podlaski et al. Arch Biochem Biophys. 1992 Apr.
Abstract
Interleukin 12 (IL-12), formerly known as cytotoxic lymphocyte maturation factor and natural killer cell stimulatory factor, is a cytokine secreted by a human B lymphoblastoid (NC-37) cell line when induced in culture with phorbol ester and calcium ionophore. This factor has been purified to homogeneity and shown to synergize with low concentrations of interleukin 2 in causing the induction of lymphokine-activated killer cells. In addition, purified IL-12 stimulated the proliferation of human phytohemagglutinin-activated lymphoblasts by itself and exerted additive effects when used in combination with suboptimal amounts of interleukin 2. The protein is a heterodimer composed of a 40- and a 35-kDa subunit. Amino acid sequence analysis confirmed predicted sequences from the cloned cDNAs of each subunit. Chemical and enzymatic deglycosylation of the heterodimer demonstrated that the 40- and 35-kDa subunits contain 10 and 20% carbohydrate, respectively. Structural analysis of IL-12 using site-specific chemical modification revealed that intact disulfide bonds are essential for bioactivity. The 40-kDa subunit of IL-12 was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and confirmed by immunoblotting as being present in NC-37 cell supernatant solutions in relatively large amounts uncomplexed to the 35-kDa subunit. Previously it had been shown that the 40-kDa subunit alone does not cause the proliferation of activated human T lymphocytes or enhance the cytolytic activity of human natural killer cells. However, results obtained by site-specific chemical modification suggesting that a tryptophan residue is at or near the active site of IL-12 may imply a direct role of the subunit in interacting with the IL-12 receptor. These data may support the recent proposal (D.P. Gearing and D. Cosman (1991) Cell 66, 9-10) that IL-12 consists of a complex of cytokine and soluble receptor.
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