The study of the inhibition of the recombinant TACE prodomain to endotoxemia in mice.
OBJECTIVE
To demonstrate the inhibitory function of the prodomain of tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE) on TACE activity and to develop an approach to interfere with inflammation processes.


METHODS
The cDNA encoding the full-length ectodomain (T1300) and prodomain (T591) of TACE were amplified by RT-PCR. The expression plasmids (pET-28a (+)-T1300 and pET-28a (+)-T591) were constructed and transformed into E. coli BL21. After Ni(2+)-NTA resin affinity chromatography, the recombinant T591 protein was obtained and assayed. In order to detect its inhibiton of TACE activity, the mice in the LPS-induced endotoxemia model group were treated with the recombinant TACE prodomain protein prior to the injection of LPS. Murine peritoneal macrophages were isolated from mice abdominal cavity for FCM and the liver, kidney and lung were removed for traditionally histopathology sectioning.


RESULTS
The FCM results showed that the recombinant prodomain protein decreased the release of the sTNF-alpha, which mediated the accumulation of TNF-alpha on the surface of macrophage cells. HE staining proved that the recombinant protein can decrease the inflammatory response in internal organs of endotoxaemia mice.


CONCLUSIONS
The recombinant prodomain of TACE has the ability to inhibit sTNF-alpha release, which indicates that prodomain is an effective antagonist of TACE and might be useful in the molecular design of anti-inflammatory drugs.
Li
Xiaoou
X
Department of Pediatrics, Renming Hospital, Wuhan University, Wuhan 430044, China.
Yan
Yuan
Y
Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430030, China.
Huang
Wei
W
Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430030, China.
Yang
Yuzhen
Y
Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science & Technology, Wuhan 430030, China.
D051722:ADAM Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000072198:ADAM17 Protein; D000818:Animals; D019446:Endotoxemia / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D051379:Mice; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D051722:ADAM Proteins; D000072198:ADAM17 Protein; C000606463:Adam17 protein, mouse