title: Increased in vitro cytotoxicity of TNF-alpha analog LK-805 is based on the interaction with cell surface heparan sulfate proteoglycan.
abstract: 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.
authors.affiliation: Lek d.d., Recombinant Biotechnology Department, Hajdrihova 19, Ljubljana, Slovenia. viktor.menart@ki.si
authors.forename: Viktor
authors.initials: V
authors.lastname: Menart
authors.forename: Irena
authors.initials: I
authors.lastname: Fonda
authors.forename: Maja
authors.initials: M
authors.lastname: Kenig
authors.forename: Vladka Gaberc
authors.initials: VG
authors.lastname: Porekar
mesh_terms: D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects*; D003001:Cloning, Molecular; D019812:Heparan Sulfate Proteoglycans / Q000378:metabolism*; D019763:Heparin Lyase / Q000494:pharmacology; D006497:Heparan Sulfate / Q000378:metabolism; D007739:L Cells; D051379:Mice; D011994:Recombinant Proteins / Q000633:toxicity; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000302:isolation & purification / Q000633:toxicity*
chemical_list: D019812:Heparan Sulfate Proteoglycans; D019763:Heparin Lyase; D006497:Heparan Sulfate; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; C471050:LK-805