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Increased in Vitro Cytotoxicity of TNF‐α Analog LK‐805 Is Based on the Interaction with Cell Surface Heparan Sulfate Proteoglycan
Author(s) -
MENART VIKTOR,
FONDA IRENA,
KENIG MAJA,
POREKAR VLADKA GABERC
Publication year - 2002
Publication title -
annals of the new york academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.712
H-Index - 248
eISSN - 1749-6632
pISSN - 0077-8923
DOI - 10.1111/j.1749-6632.2002.tb04632.x
Subject(s) - heparan sulfate , cytotoxicity , chemistry , heparin , proteoglycan , sepharose , glycosaminoglycan , cytotoxic t cell , tumor necrosis factor alpha , in vitro , perlecan , biophysics , biochemistry , microbiology and biotechnology , extracellular matrix , biology , immunology , enzyme
A bstract : Our tumor necrosis factor‐alpha (TNF‐α) 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‐α 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‐α 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‐α 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.