Differential Localization and Identification of a Critical Aspartate Suggest Non-redundant Proteolytic Functions of the Presenilin Homologues SPPL2b and SPPL3

Abstract

Signal peptide peptidase (SPP) is an unusual aspartyl protease that mediates clearance of signal peptides by proteolysis within the endoplasmic reticulum (ER). Like presenilins, which provide the proteolytically active subunit of the ␥-secretase complex, SPP contains a critical GXGD motif in its C-terminal catalytic center. Although SPP is known to be an aspartyl protease of the GXGD type, several presenilin homologues/SPP-like proteins (PSHs/SPPL) of unknown function have been identified by data base searches. We now investigated the subcellular localization and a putative proteolytic activity of PSHs/SPPLs in cultured cells and in an in vivo model. We demonstrate that SPPL2b is targeted through the secretory pathway to endosomes/lysosomes, whereas SPP and SPPL3 are restricted to the ER. As suggested by the differential subcellular localization of SPPL2b compared with SPP and SPPL3, we found distinct phenotypes upon antisense gripNA-mediated knockdown in zebrafish. spp and sppl3 knockdowns in zebrafish result in cell death within the central nervous system, whereas reduction of sppl2b expression causes erythrocyte accumulation in an enlarged caudal vein. Moreover, expression of D/A mutations of the putative C-terminal active sites of spp, sppl2, and sppl3 produced phenocopies of the respective knockdown phenotypes. Thus, our data suggest that all investigated PSHs/SPPLs are members of the novel family of GXGD aspartyl proteases. Furthermore, SPPL2b is shown to be the first member of the SPP/PSH/SPPL family that is not located within the ER but in endosomal/lysosomal vesicles.

Three-dimensional Structure of the Signal Peptide Peptidase

“…We also found that the N-terminal half is sufficient for the tetrameric assembly of SPP, although the precise mode of interaction still remains unknown. However, SPP/ SPPL proteins were never cofractionated with other member proteins of the SPP family (19,31), suggesting a specific mechanism of recognition and interaction behind homo-oligomerization. Very recently, 200-, 400-, and 600-kDa complexes containing SPP and its substrates were reported, in accordance with our results (46).…”

“…S1A) (22,40), suggesting that the ability of SPP to form a homo-oligomer is conserved beyond species irrespective of the formation of SDS-resistant dimer. In addition, DDM-solubilized SPPL2b, of which the mature form migrated at 90 kDa on SDS-PAGE (19), was detected at 400 kDa on BN-PAGE (supplemental Fig. S1, B and C).…”

“…Expression construct for SPPL2b in pEF4/myc-His (Invitrogen) was provided by Drs. Regina Fluhler and Christian Haass (Ludwig-Maximilians-University, München, Germany) (19). All constructs were sequenced using Thermo Sequenase (GE Healthcare) on an automated sequencer (LI-COR Biosciences, Lincoln, NE).…”

“…Moreover, some endoproteolytic products generated by SPP cleavage directly mediate signal transduction (15,16). In fact, loss-of-function studies of SPP in model animals resulted in severe developmental defects, inferring a vital role of SPP in metazoan development (17)(18)(19). Furthermore, a growing body of evidence indicates that SPP activity plays an important role in the maturation of several pathogens including the hepatitis C virus and the malaria parasite (7,20).…”

Signal Peptide Peptidase and γ-Secretase Share Equivalent Inhibitor Binding Pharmacology

“…Its activity is required for histocompatibility antigen E (HLA E) epitope formation (28); maturation of hepatitis C virus (29); and, in zebrafish, neuronal cell survival (30). Like presenilin, SPP can be photolabeled by a ␥-secretase transition state analog inhibitor, indicating conservation of active-site structure within the two enzymes (31,32).…”