Abstract
Objective:To demonstrate the inhibitory function of the prodomain of tumor necrosis factor-α (TNF-α) 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 Ni2+-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-α, which mediated the accumulation of TNF-α 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-α release, which indicates that prodomain is an effective antagonist of TACE and might be useful in the molecular design of anti-inflammatory drugs.
Highlights
The ADAMs are a family of membrane-anchored glycoproteins that play an important role in development, cell-cell interaction, and protein ectodomain shedding [1].ADAMs are usually comprised of several different proteins modules as follows: an N-terminal signal sequence, prodomain, a metalloprotease domain, a disintergrin domain, a cysteine-rich domain, an EGF-like domain, a transmembrane domain, and a cytoplasmic tail [2,3]
We investigated the inhibitory function of the prodomain on TACE catalytic activity, using a well-established LPS-induced endotoxemia model
The results demonstrate that the prodomain protein mediated an accumulation of mTNF-α on the cell surface and led to a decrease of sTNF-α release from cells
Summary
The ADAMs (a disintergrin and metalloprotease) are a family of membrane-anchored glycoproteins that play an important role in development, cell-cell interaction, and protein ectodomain shedding [1]. A conserved cysteine residue in the prodomain preferentially coordinates the essential active site zinc atom of the metalloprotease domain, keeping ADAMs in the latent state [4,5]. We investigated the inhibitory function of the prodomain on TACE catalytic activity, using a well-established LPS-induced endotoxemia model. With this model, we hope to develop a new approach to interfere inflammation processes
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