Open Access

Signal peptide peptidase-like (SPPL) proteases, members of the intramembrane aspartyl protease family, have attracted increased interest due to their involvement in immune cell differentiation and cellular glycan structure regulation. However, the enzymatic domain involved in substrate recognition remains enigmatic. Here we provide evidence that the N-terminal protease-associated (PA) domains of the SPPL2 subfamily are involved in substrate recognition and discrimination of substrates that differ slightly in their luminal/extracellular domain. Presence of the SPPL2c PA domain impairs SPPL2a/b mediated tumor necrosis factor α (TNFα) initial cleavage, kinetics, and processivity in cells and in vitro. In contrast, the SPPL2a PA domain enhances processing by SPPL2b. Additionally, we demonstrate non-canonical shedding activity of SPPL3 on full-length TNFα and that the ability for consecutive cleavage differs within the SPPL-family and is mainly based on the SPPL2a/b membrane spanning body. This provides the basis to finally understand the mechanistic differences of these homologous proteases.