Synthesis of glycosylated human tumor necrosis factor alpha coupled with N-acetylneuraminic acid.

In order to study the effect of glycosylation on its biological activities, and to develop TNFalpha with less deleterious effects, recombinant human TNFalpha was chemically coupled with N-acetylneuraminic acid (NeuAc). NeuAc with C9 spacer was coupled to TNFalpha by acyl azide method. Two glycosylated TNFalphas, designated L NeuAc-TNFalpha and H NeuAc-TNFalpha, were purified by anion-exchange chromatography. NeuAc coupling to TNFalpha was confirmed by lectin blotting. Average number of carbohydrate molecules introduced per molecule of L NeuAc-TNFalpha and H NeuAc-TNFalpha were estimated to be 1.0 and 1.5, respectively. We examined a variety of TNFalpha activities in vitro, including antiproliferative or cytotoxic activities to tumor cells, proliferative effect on fibroblast cells, stimulatory effects on IL-6 production by melanoma cells and NF-kappaB activation in hepatoma cells. L NeuAc-TNFalpha and H NeuAc-TNFalpha exhibited reduced activities about 1/3 and 1/10 as compared to native TNFalpha in all the activities performed in vitro.