In our previous study, a galactose monosaccharide with C9 spacer was chemically coupled to recombinant human interleukin 1alpha (rhIL-1alpha) in order to study the effect of glycosylation on its activities, and to develop IL-1 with less deleterious effects. The glycosylated IL-la exhibited reduced activities in vitro by 10 to 10000-fold depending upon different aspects of activities addressed. The affinity to type I and II IL-1 receptors were also reduced. In this study we examined a variety of IL-1 activities in vivo, including upregulation of serum levels of IL-6, alpha1-acid glycoprotein, NOx, corticosterone, downregulation of serum level of glucose, and recovery of peripheral white blood cells (WBCs) from myelosuppression in 5-fluorouracil-treated mice. In contrast to the biological activities in vitro, these activities in vivo were uniformly reduced by only about 10 to 20-fold compared to untreated IL-1alpha.
In the previous study, N-acetylneuraminic acid (NANA) with C9 spacer was chemically coupled to human recombinant (rh) IL-1alpha in order to study the effect of glycosylation on its biological activities, and to develop IL-1 with less deleterious effects. In this study we examined a variety of IL-1 activities in vitro, including proliferative effect on T cells, antiproliferative effect on myeloid leukemic cells and melanoma cells, stimulatory effects on IL-6 synthesis by melanoma cells and PGE2 synthesis by fibroblast cells. NANA-introduced IL-1alpha (NANA-IL-1alpha) exhibited reduced activities about ten times compared with original IL-1alpha in all the activities performed in vitro. The competitive binding of 125I-IL-1alpha to mouse T cells and pre-B cells with unlabeled IL-1alphas suggests the decrease in binding affinities of NANA-IL-1alpha to both type I and type II IL-1 receptors. Therefore, reduced activities of NANA-IL-1alpha well correlated with the decrease in its receptor binding affinities.
In order to develop glycosylated cytokine, recombinant human IL-1alpha was chemically modified with N-acetylneuraminic acid (NANA). NANA with C9 spacer, 8-(hydrazinocarbonyl)octyl 5-acetamido-3, 5-dideoxy-D-glycero-alpha-D-galacto-2-nonulo-pyranosidonic acid potassium salt (6), was synthesized by glycosylation of C9 spacer, 8-[2-N-(benzyloxycarbonyl)hydrazinocarbonyl]octanol, with methylthio glycoside derivatives of NANA in the presence of molecular sieves 3A and methyl (methylthio)sulfonium trifrate in propionitrile, followed by separation of a and beta anomers with a column chromatography and deprotection. Compound 6 was coupled to IL-1alpha by the acyl azide method. The glycosylated IL-1 was purified by anion-exchange chromatography, and NANA coupled to IL-1 was confirmed by oxidation with NaIO4. Based on the molecular weight average number of carbohydrate molecules introduced per molecule of IL-1alpha was estimated to be 2.9.
In order to study the effect of glycosylation on its biological activities and to develop IL-1 with less deleterious effects, N-acetylneuraminic acid (NeuAc) with C9 spacer was chemically coupled to human recombinant IL-1alpha. NeuAc-coupled IL-1alpha (NeuAc-IL-1alpha) exhibited reduced activities in vitro and receptor-binding affinities by about ten times compared to IL-1alpha. In this study, we examined a variety of IL-1 activities in vivo. NeuAc-IL-1alpha exhibited a marked reduction in the activity to up-regulate serum IL-6, moderate reduction in the activities to up-regulate serum amyloid A and NOx. However, it exhibited comparable activities as IL-1alpha to down-regulate serum glucose and to improve the recovery of peripheral white blood cells from myelosuppression in 5-fluorouracil-treated mice. In addition, tissue level of NeuAc-IL-1alpha was high compared to IL-1alpha. These results indicate that coupling with NeuAc enabled us to develop neo-IL-1 with selective activities in vivo and enhanced tissue level.
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