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Published on: June 21, 2018
Molecular characterization of interleukin 12
F J Podlaski1, V B Nanduri, J D Hulmes
1Department of Protein Biochemistry, Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey 07110-1199.
Insights
Interleukin 12 (IL-12) is a cytokine that enhances immune responses by promoting lymphokine-activated killer cells and T-lymphoblast proliferation. Its structure, including disulfide bonds and subunits, is crucial for its bioactivity.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Interleukin 12 (IL-12), also known as cytotoxic lymphocyte maturation factor, is a cytokine.
- It is secreted by induced human B lymphoblastoid (NC-37) cells.
Purpose of the Study:
- To purify and characterize Interleukin 12 (IL-12).
- To investigate the synergistic effects of IL-12 with Interleukin 2 (IL-2) on immune cells.
- To elucidate the structural components and their role in IL-12 bioactivity.
Main Methods:
- Purification of IL-12 to homogeneity.
- Assays for lymphokine-activated killer cell induction and lymphoblast proliferation.
- Amino acid sequencing, deglycosylation, and site-specific chemical modification.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting.
Main Results:
- Purified IL-12 synergizes with IL-2 to induce lymphokine-activated killer cells.
- IL-12 alone stimulates lymphoblast proliferation and shows additive effects with IL-2.
- IL-12 is a heterodimer of 40- and 35-kDa subunits, with glycosylation on both.
- Intact disulfide bonds are essential for IL-12 bioactivity.
- The 40-kDa subunit is abundant but lacks activity alone, suggesting a role in receptor interaction.
Conclusions:
- IL-12 is a potent immune-stimulating cytokine with a complex heterodimeric structure.
- Specific structural features, including disulfide bonds and subunits, are critical for its function.
- The 40-kDa subunit may play a direct role in IL-12 receptor binding.
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.
