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Tytuł:
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Increased in vitro cytotoxicity of TNF-alpha analog LK-805 is based on the interaction with cell surface heparan sulfate proteoglycan.
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Autorzy:
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Menart V; Lek d.d., Recombinant Biotechnology Department, Hajdrihova 19, Ljubljana, Slovenia. />Fonda I
Kenig M
Porekar VG
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Źródło:
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Annals of the New York Academy of Sciences [Ann N Y Acad Sci] 2002 Nov; Vol. 973, pp. 194-206.
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Typ publikacji:
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Journal Article; Research Support, Non-U.S. Gov't
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Język:
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English
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Imprint Name(s):
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Publication: 2006- : New York, NY : Malden, MA : New York Academy of Sciences ; Blackwell
Original Publication: New York, The Academy.
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MeSH Terms:
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Cell Survival/*drug effects
Heparan Sulfate Proteoglycans/*metabolism
Tumor Necrosis Factor-alpha/*toxicity
Animals ; Cattle ; Cell Membrane/drug effects ; Cell Membrane/physiology ; Cloning, Molecular ; Heparin Lyase/pharmacology ; Heparitin Sulfate/metabolism ; L Cells ; Mice ; Recombinant Proteins/toxicity ; Tumor Necrosis Factor-alpha/genetics ; Tumor Necrosis Factor-alpha/isolation & purification
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Substance Nomenclature:
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0 (Heparan Sulfate Proteoglycans)
0 (LK-805)
0 (Recombinant Proteins)
0 (Tumor Necrosis Factor-alpha)
9050-30-0 (Heparitin Sulfate)
EC 4.2.2.7 (Heparin Lyase)
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Entry Date(s):
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Date Created: 20021218 Date Completed: 20030213 Latest Revision: 20191210
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Update Code:
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20240104
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DOI:
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10.1111/j.1749-6632.2002.tb04632.x
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PMID:
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12485860
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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.