Substrate specificity is a determinant of ectodomain shedding by tumor necrosis factor alpha converting enzyme (TACE)

Ectodomain shedding is a proteolytic mechanism by which the extracellular domain of a transmembrane protein is cleaved in a regulated fashion and released into the extracellular space. Several studies have implicated Tumor Necrosis Factor Alpha Converting Enzyme (TACE) in the shedding of a wide variety of transmembrane molecules. However, the mechanism by which TACE potentially processes such a wide variety of molecules is not understood. The “lawnmower” sheddase model postulates that TACE is an undiscriminating enzyme that cleaves juxtamembrane stalks of all target proteins, simply and nonspecifically at a certain distance from the membrane. We studied the relative importance of substrate cleavage site sequence in directing the activity of TACE towards its substrates. We used model peptides corresponding to the cleavage sites of several transmembrane proteins and tested their ability to be cleaved by recombinant, purified TACE. Interestingly, TACE was shown to have high selectivity towards TNFα and N-terminal TGFα cleavage site peptides only. We reproduced these results with native TACE-enriched cellular extracts. We proceeded to test native-TACE mediated cleavage of substrate cleavage sites by swapping them into the context of the well characterized TACE substrate, TNFα. Our results indicated that TACE has distinct sequence preferences, both in terms of the amino acid sequence and of the directionality of the cleavage site. We also examined the role of TACE in TGFα processing. Results from cell based assays showed that TACE has significant affinity for both the C-terminal and N-terminal TGFα processing site. Nevertheless, TACE is not the only physiological sheddase for the C-terminal site of TGFα. We demonstrated the existence of a novel, C-terminal TGFα processing activity in rat liver epithelial cells that is neither TACE, nor ADAM10 (a close relative of TACE). Our results suggest that, rather than being a “lawnmower” sheddase, TACE is an enzyme with high substrate selectivity and may operate in conjunction with other proteases to regulate the processing of a target transmembrane molecule