Synthesis and maturation of recombinant human tumor necrosis factor in eukaryotic systems

Area of Science:

• Biochemistry
• Molecular Biology
• Immunology

Background:

• Human tumor necrosis factor (hTNF) is a key cytokine involved in inflammation and immunity.
• Understanding the biosynthesis and maturation of hTNF is crucial for its therapeutic applications and for studying inflammatory diseases.

Purpose of the Study:

• To investigate the post-translational processing and maturation of the human tumor necrosis factor (hTNF) precursor.
• To determine if hTNF maturation is restricted to specific cellular systems, such as macrophages.
• To elucidate the secretion mechanism of hTNF.

Main Methods:

• Cell-free system with dog pancreas microsomes to study N-terminal cleavage.
• Specific immunoprecipitation to detect hTNF polypeptides in the medium of TNF-producing CHO cells.
• Analysis of hTNF processing in Xenopus laevis oocytes injected with hTNF mRNA.

Main Results:

• The amino-terminal extension of the hTNF precursor was not cleaved in a cell-free system with microsomes.
• Multiple hTNF polypeptides (approx. 20 kDa, 18.5 kDa doublet, and 17 kDa) were detected in the medium of transformed CHO cells.
• The 17 kDa polypeptide corresponds to mature hTNF.
• Similar processing was observed in Xenopus oocytes, with the absence of the 20 kDa band.

Conclusions:

• hTNF maturation is not exclusive to macrophages and can occur in other cellular systems like CHO cells and Xenopus oocytes.
• The observed processing suggests a complex post-translational modification pathway for hTNF secretion.
• These findings provide insights into the secretion mechanism of hTNF.