Calcium ionophore A 23 187 in the presence of phorbol ester PMA: a potent inducer of interleukin 2 and interferon-gamma synthesis by human blood cells.

Cultures of human blood mononuclear cells incubated with the calcium ionophore A 23 187 in the presence of the tumor promoter phorbol 12-myristate-13-acetate (PMA) produced 5-10 times more of the lymphokines interleukin 2 (IL 2) and interferon-gamma (IFN-gamma) than cultures which were stimulated with other combinations of inducing agents and PMA. Especially at low protein concentrations, the amount of ionophore which is necessary to induce maximal quantities of both lymphokines was determined by the protein content of the culture medium. The synthesis of the two lymphokines was inhibited by low doses of Mn++ which competes with Ca++ for binding to the ionophore. This suggests the importance of Ca++ in the induction process. The synthesis rates of IL 2 were maximal 10-12 h, and those of IFN-gamma 20-40 h after induction. Maximal titers of IL 2 were detected 48 h after the addition of A 23187 and PMA to the cultures, and the highest IFN-gamma levels 12-24 h later.

Posttranslational modification of interleukin-2 is a late event during activation of human T lymphocytes by ionophore A23187 and phorbol ester.

Human peripheral blood lymphocytes secrete high titers of interleukin-2 (IL-2) after stimulation by Ca2+-ionophore A23187/phorbol 12-myristate-13-acetate. During the first 30 hours of incubation cells secrete only the nonglycosylated IL-2 M form of the lymphokine, the glycosylated forms IL-2 N1,2 being detected only after prolonged culture times (30-48 h). After recultivation of cells for a second 48 h period (without additional mitogen), the glycosylated and nonglycosylated IL-2 forms are secreted at a constant ratio of 7:3 throughout. The detection of glycosylated IL-2 is parallelled by an increase in cellular glycosyltransferase activities involved in formation of sialylated oligosaccharides O-linked to proteins.

Enzymatic cleavage of the epsilon-peptide bond in alpha- and epsilon-substituted glycyl- and phenylalanyl-lysine peptides.

Lysine peptides, X-Lys-OH (Formula: see text) were synthesized, following classic or non-classic routes. Some bacterial and mammalian enzymes, endo- and exo-peptide hydrolases of the enzyme nomenclature type EC 3.4., were tested for their ability to split the epsilon-peptide bond in the above substrates. Kinetic constants (Km,kcat) were evaluated with leucine aminopeptidase from hog kidney and eye lens with aminopeptidase I from yeast. Aminopeptidase M (hog pancreas) and hog intestinal aminopeptidase were additionally examined for their Ki values with the above substrates in comparison to the classic protease substrate leucine p-nitroanilide. Especially the intestinal mucosa hydrolases are shown to be efficient in cleaving epsilon-peptide bonds.

Structures of the major carbohydrates of natural human interleukin-2.

Purified human interleukin-2 secreted by peripheral blood lymphocytes from healthy donors was found to exist in several forms. These forms were (partially) resolved by reversed-phase high-performance liquid chromatography and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Two major polypeptide species (interleukin-2 N1 and N2, 16.5 kDa) were shown to be glycosylated on the basis of [3H]galactose/[3H]glucosamine incorporation and determination of amino sugars after acid hydrolysis. A third component (interleukin-2 M, 14.5 kDa) represents a nonglycosylated form. The amino acid composition and the NH2-terminal sequence of both forms are consistent with the data deduced from the cDNA coding for interleukin-2 after removal of a leader peptide of 20 amino acids. Carbohydrates are O-linked to the IL-2 protein via threonine-3 of the polypeptide chain. The oligosaccharides were released by reductive beta-elimination and were purified by gel filtration and high-performance liquid chromatography. Applying methylation analysis, exoglycosidase digestion and fast atom bombardment mass spectrometry the following major carbohydrate structures were identified: N1, NeuAc(alpha 2-3)Gal(beta 1-3)GalNAc-ol; and N2, NeuAc(alpha 2-3)Gal(beta 1-3)[NeuAc(alpha 2-6)]GalNAc-ol.