Increasedin VitroCytotoxicity of TNF-α Analog LK-805 Is Based on the Interaction with Cell Surface Heparan Sulfate Proteoglycan

Autor: Viktor Menart, Irena Fonda, Maja Kenig, Vladka Gaberc Porekar
Rok vydání: 2002
Předmět:
Zdroj: Annals of the New York Academy of Sciences. 973:194-206
ISSN: 1749-6632
0077-8923
DOI: 10.1111/j.1749-6632.2002.tb04632.x
Popis: Our tumor necrosis factor-alpha (TNF-alpha) analog LK-805 (E107K) exhibited twofold higher specific cytotoxicity on the mouse fibroblast L-929 cell line than its native counterpart. In addition, significantly lowered systemic toxicity was observed in tumor-bearing mouse models treated with this analog. Due to a charge reversal and clustering of three lysines in the exposed tip region of LK-805, we assumed that additional ionic interactions between the positively charged TNF analog and the negatively charged components of the cell surface were created, which might contribute to improved properties of LK-805. To prove this hypothesis, we designed truncated forms of TNF-alpha and analog LK-805 and performed three independent sets of experiments: measurement of cytotoxic activity in the presence of excess heparan sulfate, determination of cytotoxic activity on heparinase-treated L-929 cells, and binding of various TNF-alpha proteins onto the heparin-sepharose affinity column. Cytotoxicity studies of both kinds confirmed the pivotal role of the E107K mutation for interaction with heparan sulfate proteoglycans on the cell surface of L-929 cells. However, heparin-binding studies revealed that intact, full-length N-termini of TNF-alpha or its analogs were necessary for high retention on the heparin affinity column, whereas the three-lysine containing tip of LK-805 by itself was not enough for binding. Obviously, immobilized heparin does not represent an adequate model for membrane-bound heparan sulfate proteoglycans of L-929 cells.
Databáze: OpenAIRE
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