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The effects of tumor necrosis factor (TNF) derivatives on TNF receptors
H Tchorzewski1, K Zeman, E Paleolog
1Department of Pathophysiology and Immunology MMA, Lodz, Poland.
Abstract:
The pleiotropic cytokine TNF has been implicated in the regulation of many immune and inflammatory responses in vivo, and in addition exerts a wide range of effects on target cells in vitro. However, although two cell surface receptors for TNF have been identified, and their cDNAs cloned, the amino acid residues necessary for the biological activity of TNF have not been characterized. We have therefore constructed derivatives of TNF termed 'muteins', in which the first 3 to 7 amino acids of native TNF-alpha have been replaced, using synthetic cDNA expressed in E. coli. In the present study we compare the effects of native TNF-alpha and muteins III, IV, V and VI in several different in vitro systems and in one in vivo model. We observed binding to the p75 TNF receptor on Jijoye Burkitt lymphoma cells with native TNF-alpha and mutein III alone, whereas the p55 TNF receptor on the human epithelioid carcinoma cell line HeLa bound TNF-alpha, mutein III and mutein V. Muteins IV and VI failed to recognize either TNF receptor. WEHI 164 fibrosarcoma cells were killed by muteins III, V and VI. Human umbilical vein endothelial cells responded to native TNF-alpha and to muteins III, IV and V, but not to mutein VI, by increasing the surface expression of ICAM-1 antigen and secretion of the cytokines GM-CSF and IL-6. All four compounds were pro-inflammatory in a mouse in vivo model. The results presented in this report confirm that N-terminal amino acids are critical for both receptor binding and biological activity of TNF-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Tumor necrosis factor-alpha (TNF) muteins, altered in their N-terminal amino acids, show varied receptor binding and biological activity. These findings highlight the critical role of N-terminal residues in TNF-alpha
Area of Science:
- Immunology
- Molecular Biology
Background:
- Tumor necrosis factor-alpha (TNF) is a key cytokine in immune and inflammatory responses.
- Two TNF cell surface receptors are known, but the specific amino acid residues responsible for TNF's activity remain uncharacterized.
Purpose of the Study:
- To investigate the role of N-terminal amino acids in TNF-alpha's receptor binding and biological activity.
- To compare the effects of native TNF-alpha and its engineered derivatives (muteins) in vitro and in vivo.
Main Methods:
- Engineered TNF-alpha muteins by replacing the first 3-7 N-terminal amino acids using synthetic cDNA expressed in E. coli.
- Assessed receptor binding to p75 and p55 TNF receptors on different cell lines (Jijoye, HeLa).
- Evaluated cytotoxic effects on WEHI 164 fibrosarcoma cells and effects on ICAM-1 expression and cytokine secretion (GM-CSF, IL-6) in human umbilical vein endothelial cells.
- Tested pro-inflammatory effects in a mouse in vivo model.
Main Results:
- Native TNF-alpha and mutein III bound to the p75 receptor; TNF-alpha, mutein III, and mutein V bound to the p55 receptor.
- Muteins IV and VI did not bind to either TNF receptor.
- Muteins III, V, and VI exhibited cytotoxicity against WEHI 164 cells.
- Native TNF-alpha and muteins III, IV, and V induced ICAM-1 expression and cytokine secretion in endothelial cells; mutein VI did not.
- All four muteins demonstrated pro-inflammatory activity in vivo.
Conclusions:
- N-terminal amino acids of TNF-alpha are critical for its interaction with both p75 and p55 TNF receptors.
- Specific N-terminal modifications alter TNF-alpha's receptor binding profile and downstream biological activities, including cytotoxicity and cytokine induction.
- These findings provide insights into structure-activity relationships of TNF-alpha, crucial for understanding its role in inflammation and immunity.