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

Abstract

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.

Increasing the sialylation of therapeutic glycoproteins: The potential of the sialic acid biosynthetic pathway

“…This type of in vitro glycosylation is suggested to be applicable to other glycoproteins containing terminal N ‐acetylglucosamine and galactose residues and could prove to be useful in increasing the serum half‐life and homogeneity of therapeutic glycoproteins. Recently, Hayashi et al66 reported the chemical sialylation of TNF‐alpha using an acyl azide method and proofed that also chemical methods can be used to sialylated glycoproteins.…”