The regulation of TACE catalytic function by its prodomain.
OBJECTIVE
To study the function of the prodomain of ADAM17 (TACE) and to develop an approach for interfering with inflammation processes.


METHODS
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 Ni(2+)-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-alpha secretion was detected by ELISA and flow cytometry (FCM).


RESULTS
The recombinant prodomain protein inhibited 57% of the TNF-alpha secretion and mediated an accumulation of TNF-alpha 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-alpha secretion by 61.09% and mediated an accumulation of mTNF-alpha on the surface of the U937 cells. The secretion of sTNF-alpha and the level of the mTNF-alpha in the pIRES2-EGFP-T57-T1824 transfected cells gave no difference when compared with the pIRES2-EGFP transfected cells. Also the secretion of sTNF-alpha from the cells transfected by the plasmid pIRES2-EGFP-T2472 increased, while the level of mTNF-alpha decreased, compared with the pIRES2-EGFP-transfected cells.


CONCLUSIONS
The prodomain has dual effects and might be useful in the molecular design of an anti-inflammatory drug.
Li
Xiaoou
X
Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science & Technology, Hongkong road 13#, Wuhan, China.
Yan
Yuan
Y
Huang
Wei
W
Yang
Yuzhen
Y
Wang
Hongwei
H
Chang
Liwen
L
D051722:ADAM Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000072198:ADAM17 Protein; D055162:Biocatalysis; D002460:Cell Line; D002942:Circular Dichroism; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary; D012995:Solubility; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D051722:ADAM Proteins; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human