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J. Biol. Chem., Vol. 279, Issue 26, 27365-27375, June 25, 2004

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Loss of Ectodomain Shedding Due to Mutations in the Metalloprotease and Cysteine-rich/Disintegrin Domains of the Tumor Necrosis Factor- Converting Enzyme (TACE)^*

Xiaojin Li and Huizhou Fan

From the Department of Physiology and Biophysics, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854

Tumor necrosis factor- converting enzyme (TACE), a multidomain protease essential for development and disease, releases the ectodomains from many transmembrane proteins in a regulated fashion. To understand the mechanism underlying the regulation of TACE activity, we sought to identify the cause of ectodomain shedding deficiencies in two mutated CHO sublines designated M1 and M2. Transfection of expression vectors for human and mouse TACE restored ectodomain shedding of TNF- and TGF-, suggesting that defects in the TACE gene contribute to the phenotype of M1 and M2 cells. The overall levels of endogenous TACE forms in M1 cells were significantly lower than those found in their parental cells, whereas only TACE zymogen, but not its mature form, was detectable in M2 cells. Molecular analyses suggested that M1 cells contained only one expressible TACE allele encoding an M435I point mutation in the catalytic center of the protease, and M2 cells produced two TACE variants with distinct point mutations in the catalytic domain (C225Y) and the cysteinerich/disintegrin domain (C600Y). Overexpression of the C225Y and C600Y TACE by transient transfection largely compensated for maturation defects in the variants but failed to restore TNF- and TGF- release in the shedding-defective CHO cell lines and fibroblasts derived from TACE-null mouse embryo. Further mutagenesis and functional analyses demonstrated that Cys⁶⁰⁰ was absolutely essential for ectodomain shedding, suggesting that Cys⁶⁰⁰, similar to Cys²²⁵, participates in disulfide bonding, which is critical for both the processing and catalysis of TACE.

Received for publication, February 16, 2004 , and in revised form, April 8, 2004.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY313173, AY313174, AY313175, and AY313176.

^* This work was supported by grants from the UMDNJ Foundation, National Center of the American Heart Association, and New Jersey Commission on Cancer Research (to H. F.). Sequences of wild-type and mutated hamster TACE identified in this work were presented at the Matrix Metalloproteinase Gordon Research Conference held in Big Sky, Montana, August 2003. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 732-235-4607; Fax: 732-235-5038; E-mail: fanhu{at}umdnj.edu.

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