Molecular characterization of interleukin 12.
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.
Podlaski
F J
FJ
Department of Protein Biochemistry, Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey 07110-1199.
Nanduri
V B
VB
Hulmes
J D
JD
Pan
Y C
YC
Levin
W
W
Danho
W
W
Chizzonite
R
R
Gately
M K
MK
Stern
A S
AS
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001402:B-Lymphocytes / Q000737:chemistry*; D001665:Binding Sites; D000001:Calcimycin / Q000494:pharmacology; D002460:Cell Line; D004357:Drug Synergism; D006031:Glycosylation; D006801:Humans; D018664:Interleukin-12; D007376:Interleukin-2 / Q000494:pharmacology; D007378:Interleukins / Q000737:chemistry* / Q000494:pharmacology; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000502:physiology; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010835:Phytohemagglutinins; D013329:Structure-Activity Relationship; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014364:Tryptophan
D000596:Amino Acids; D007376:Interleukin-2; D007378:Interleukins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010835:Phytohemagglutinins; D018664:Interleukin-12; D000001:Calcimycin; D014364:Tryptophan; D013755:Tetradecanoylphorbol Acetate