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The regulation of TACE catalytic function by its prodomain

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Abstract

Aim To study the function of the prodomain of ADAM17 (TACE) and to develop an approach for interfering with inflammation processes. Method The expression plasmids of the TACE ectodomain (T1300), prodomain (T591), signal peptide and prodomain (T648), full length (T2472), and the turncated TACE without prodomain (T57-T1824) were constructed and designated as pET-28a-T300, pET-28a-T591, pIRES2-EGFP-648, pEGFP-N1-T648, pIRES2-EGFP-T2472, and pIRES2-EGFP-T57-T1824, respectively. After Ni2+-NTA resin-affinity chromatography, the recombinant T591 and T1300 proteins were obtained and assayed by western blotting and circular dichroism. The experiment was carried out on THP1 cell lines stimulated by LPS in vitro. The inhibition of recombinant protein T591 to TACE activity was detected by ELISA and immunohistochemical detection. The expression plasmids (pIRES2-EGFP-T648, pIRES2-EGFP-T2472, and pIRES2-EGFP-T57-T1824) were used to transfect the U937 cells. HeLa cells were also transfected with pEGFP-N1-T648. The transfected U937 cells were then stimulated by LPS and the effect of expression plasmids on TNF-α secretion was detected by ELISA and flow cytometry (FCM). Results The recombinant prodomain protein inhibited 57% of the TNF-α secretion and mediated an accumulation of TNF-α on the surface of THP1 cells. An intense green fluorescence was seen in the membranes of HeLa cells transfected with pEGFP-N1-T648. The plasmid pIRES2-EGFP-T648 inhibited TNF-α secretion by 61.09% and mediated an accumulation of mTNF-α on the surface of the U937 cells. The secretion of sTNF-α and the level of the mTNF-α in the pIRES2-EGFP-T57-T1824 transfected cells gave no difference when compared with the pIRES2-EGFP transfected cells. Also the secretion of sTNF-α from the cells transfected by the plasmid pIRES2-EGFP-T2472 increased, while the level of mTNF-α decreased, compared with the pIRES2-EGFP-transfected cells. Conclusion The prodomain has dual effects and might be useful in the molecular design of an anti-inflammatory drug.

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Acknowledgements

This work was supported by grants from The National Natural Sciences Foundation of China (No. 30070722, 30471963).

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science & Technology, Hongkong road 13#, Wuhan, China

    Xiaoou Li, Yuan Yan, Wei Huang & Yuzhen Yang

  2. Department of Pediatrics, Tongji Hospital, Tongji Medical College, Huazhong University of Science & Technology, Hongkong road 13#, Wuhan, China

    Xiaoou Li, Hongwei Wang & Liwen Chang

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  1. Xiaoou Li
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  2. Yuan Yan
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  3. Wei Huang
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  4. Yuzhen Yang
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Correspondence to Yuzhen Yang.

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Li, X., Yan, Y., Huang, W. et al. The regulation of TACE catalytic function by its prodomain. Mol Biol Rep 36, 641–651 (2009). https://doi.org/10.1007/s11033-008-9224-5

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