Inactivating mutations block the tumor necrosis factor-α-converting enzyme in the early secretory pathway

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Abstract

The ectodomain of different transmembrane molecules is released by a proteolytic event known as shedding. The metalloprotease disintegrin proTNF-α converting enzyme (TACE) is responsible for the shedding of various proteins, including protransforming growth factor-α (proTGF-α) and amyloid-β precursor protein (APP). Inactive TACE accumulates in the early secretory pathway of cell mutants (M1 and M2) defective in proTGF-α and APP shedding. Although previous evidences indicated that the component mutated in M1 and M2 cells is different from TACE, recent results show the existence of two heterozygous point mutations in TACE from M2 cells. Here, we show that wild-type TACE stably transfected in M2 cells is processed, transported to the cell surface, and rescues the proTGF-α and APP shedding-defective phenotype. Furthermore, M1 cells also express mutant TACE and transfection with wild-type TACE restores the wild-type phenotype. Therefore, different inactivating mutations result in the accumulation of TACE in the early secretory pathway, emphasizing the importance of the initial steps in the biosynthesis of TACE.

Section snippets

Materials and methods

Antibodies and reagents. Polyclonal antibodies against the extracellular domain of TACE were kindly provided by Dr. Carl Blobel. The polyclonal antibodies directed against the cytoplasmic domain of TACE have been previously described [26]. The monoclonal anti-βAPP antibodies 22C11, mouse IgM anti-actin (Ab-1), and mouse anti-HER2 (CB-11) were from Chemicon International, Oncogene research products, and Biogenex, respectively. Sulfo-NHS-LC-Biotin, immobilized neutravidin, and the SuperSignal

Permanently transfected TACE is processed and transported to the cell surface in M2 cells

Although automatic sequencing of the cDNA encoding TACE from M2 cells did not show the presence of mutations [16], a subsequent systematic scrutiny of the electropherograms showed that M2 mutants express two mutant forms of TACE that result in Cys 225 to Tyr and Cys 600 to Tyr substitutions, respectively (see correction to [16] and Fig. 1). According to the predicted structure of TACE, Cys 225 and Cys 600 form disulfide bonds within the metalloprotease and cysteine-rich domains of TACE,

Discussion

Given the practical importance of protein ectodomain shedding, several years ago we isolated and characterized somatic cell mutants defective in the PMA-activated shedding of the transmembrane growth factor proTGF-α [23], [24]. Subsequently, the ADAM protease TACE was found to be responsible for the shedding of proTGF-α [11]. Previous transient transfection and cell fusion experiments indicated that the component mutated in the shedding-defective cell lines M1 and M2 was different from TACE [25]

Acknowledgements

We thank Dr. Huizhou Fan for helpful discussions on the sequence of TACE from M2 cells. We are also grateful to Dr. Carl Blobel for reagents and constructive suggestions and Drs. Anna Merlos-Suárez and Aldo Borroto Revuelta for critically reading the manuscript. T.V.-T. is the recipient of fellowships from the Instituto de Salud Carlos III. This work was supported by grants from La Marató de TV3, Fondo de Investigación Sanitaria (PI021003), and the EMBO Young Investigator Programme (YIP) to

References (33)

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Supplementary data associated with this article can be found, in the online version, at 10.1016/j.bbrc.2003.12.186.

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