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.
Hayashi
Akiko
A
Department of Molecular Health Sciences, Graduate School of Pharmaceutical Sciences, Nagoya City University, Tanabe, Mizuho, Nagoya 467-8603, Japan.
Chiba
Taku
T
Hayashi
Hidetoshi
H
Sasayama
Satoshi
S
Ishiguro
Toshiyuki
T
Onozaki
Kikuo
K
D000818:Animals; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D019158:N-Acetylneuraminic Acid / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000138:chemical synthesis* / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry / Q000378:metabolism*
D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D019158:N-Acetylneuraminic Acid