Consensus Analysis of Signal Peptide Peptidase and Homologous Human Aspartic Proteases Reveals Opposite Topology of Catalytic Domains Compared with Presenilins

Abstract

The human genome encodes seven intramembranecleaving GXGD aspartic proteases. These are the two presenilins that activate signaling molecules and are implicated in Alzheimer's disease, signal peptide peptidase (SPP), required for immune surveillance, and four SPP-like candidate proteases (SPPLs), of unknown function. Here we describe a comparative analysis of the topologies of SPP and its human homologues, SPPL2a, -2b, -2c, and -3. We demonstrate that their N-terminal extensions are located in the extracellular space and, except for SPPL3, are modified with N-glycans. Whereas SPPL2a, -2b, and -2c contain a signal sequence, SPP and SPPL3 contain a type I signal anchor sequence for initiation of protein translocation and membrane insertion. The hydrophilic loops joining the transmembrane regions, which contain the catalytic residues, are facing the exoplasm. The C termini of all these proteins are exposed toward the cytosol. Taken together, our study demonstrates that SPP and its homologues are all of the same principal structure with a catalytic domain embedded in the membrane in opposite orientation to that of presenilins. Other than presenilins, SPPL2a, -2b, -2c, and -3 are therefore predicted to cleave type II-oriented substrate peptides like the prototypic protease SPP.

The Fas ligand intracellular domain is released by ADAM10 and SPPL2a cleavage in T-cells

“…35 In contrast to presenilin, SPPL proteases catalyze the intramembrane proteolysis of type II anchored membrane proteins. 17 Our inhibitor and siRNA knockdown experiments demonstrated that intramembrane cleavage of the FasL remnant APL leads to the liberation of the 11-kDa SPA fragment, which consists of the FasL ICD. FasL processing by SPPL2a is further supported by co-immunoprecipitation experiments, which demonstrate a direct interaction between both molecules (data not shown).…”

“…26 Recently, a new group of proteases that belong to the signal peptide peptidase-like (SPPL) family of intramembrane-cleaving proteases (I-CliPs) has been described. 17 They display no particular sequence homology to PS, but all contain the same active-site motifs, YD and GxGD. The crucial difference, however, is the orientation of their catalytic domain, which suggests their propensity for type II transmembrane protein substrates.…”

“…The crucial difference, however, is the orientation of their catalytic domain, which suggests their propensity for type II transmembrane protein substrates. 17,26 To test the potential involvement of SPPL2a protease in FasL intramembrane cleavage, we co-expressed SPPL2a together with FLAG-FasL in 293T cells. Intriguingly, we observed that the 17-kDa FasL APL fragment is further cleaved by SPPL2a, generating a 13-kDa SPA fragment ( Figure 3a, lane 2; the SPA fragment produced from (a) 293 cells with stable FLAG-hFasL expression were incubated with or without the ADAM 10 inhibitor GI254023X (10 mM; 24 h), before cell lysates were subjected to immunoblotting.…”

“…Here, we describe how FasL is processed by the disintegrin and metalloprotease ADAM10 16 and by the intramembrane protease SPPL2a, 17 ultimately leading to the liberation of the FasL ICD into the cytosol. The released FasL fragment is highly unstable and capable of translocating into the nucleus and influencing gene transcription.…”

Export of a hyperexpressed mammalian globular cytochrome b5 precursor in Escherichia coli is dramatically affected by the nature of the amino acid flanking the secretory signal sequence cleavage bond

“…SS-like peptides, liberated from dormant, membrane-bound precursor proteins by the activities of intramembrane-cleaving proteases such as SS peptidase, are thought to play downstream functions in cell signaling and regulation, immune surveillance, and intercellular communication. 11 An antimicrobial peptide derivative of flesh fruit fly hemolymph mimics secretory SS and inhibited SPase in the export pathway. 12 In addition to the core hydrophobic region, the functionality of SS is critically determined by juxtaposition of the charge to the cleavage bond.…”