Foamy virus envelope protein is a substrate for signal peptide peptidase-like 3 (SPPL3)

Abstract

Signal peptide peptidase (SPP), its homologs, the SPP-like proteases SPPL2a/b/c and SPPL3, as well as presenilin, the catalytic subunit of the γ-secretase complex, are intramembrane-cleaving aspartyl proteases of the GxGD type. In this study, we identified the 18-kDa leader peptide (LP18) of the foamy virus envelope protein (FVenv) as a new substrate for intramembrane proteolysis by human SPPL3 and SPPL2a/b. In contrast to SPPL2a/b and γ-secretase, which require substrates with an ectodomain shorter than 60 amino acids for efficient intramembrane proteolysis, SPPL3 cleaves mutant FVenv lacking the proprotein convertase cleavage site necessary for the prior shedding. Moreover, the cleavage product of FVenv generated by SPPL3 serves as a new substrate for consecutive intramembrane cleavage by SPPL2a/b. Thus, human SPPL3 is the first GxGD-type aspartyl protease shown to be capable of acting like a sheddase, similar to members of the rhomboid family, which belong to the class of intramembrane-cleaving serine proteases.

FIGURE 1.

SPPL-mediated proteolysis of FVenv. A, schematic overview of FVenv processing by PCs. The FVenv glycoprotein consists of a leader peptide (LP18), a surface subunit (SU), and C-terminal membrane-spanning domain (TM). PC and PC* cleavage site (arrowheads), the C-terminal ER retrieval signal (KKKNQ), and the epitope tags (FLAG and V5) are indicated. Glycosylation sites are also indicated (Y). B–E, FVenv proteolytic fragments were isolated from HEK293 cells coexpressing FVenv and the indicated SPPL proteases either as catalytically active (WT) or inactive (Asp-to-Ala (D/A)) variants. The open arrowheads indicate unglycosylated LP18. Coexpression of WT SPPL3 resulted predominantly in generation of ICD(L3) (black arrowheads), whereas catalytically active SPPL2a/b produced predominantly ICD(L2) (gray arrowheads). Upon coexpression of FVenv and SPPL2a/b Asp-to-Ala mutants, ICD(L3) and protein fragments (marked with asterisks) accumulated. Depending on the resolution of the gel system, ICD(L3) was sometimes detected as a doublet. IP, immunoprecipitation.

FIGURE 2.

SPPL3 cleavage of FVenv is independent of shedding. A, model depicting the proteolytic processing of FVenv and FVenv mut disregarding the C-terminal PC* cleavage site, which is hardly observed upon cellular expression of FVenv containing the ER retrieval signal. FVenv mut carries a R123A/R126A mutation to abolish cleavage at the N-terminal PC cleavage site. The respective cleavage products are denoted. Amino acids are depicted using the single-letter code. B, WT FVenv and FVenv mut were transiently transfected into HEK293 cells, and cleavage products were monitored. Note that processing of FVenv mut by endogenous PC was strongly reduced at the N-terminal PC cleavage site due to the mutation introduced and that, as expected, the C-terminal PC* cleavage site was hardly used at all. The IgG background signal is indicated (**). All other species are labeled as described in the legend to Fig. 1. C, WT FVenv or FVenv mut was transiently coexpressed in HEK293 cells stably expressing catalytically active SPPL2b or SPPL3. LP18 and ICD species generated by SPPL are indicated. The quantification depicts ICD levels relative to LP18. Ratios in cells expressing the indicated protease and WT FVenv were set to 1. Data represent means ± S.E. of three independent experiments. *, p < 0.049 (Student's unpaired t test). D, the samples shown in C were analyzed for high molecular mass fragments of FVenv. SPPL3-mediated cleavage of FVenv generated ICD(L3) and TMD/SU/TM. Note that TMD/SU/TM was detected exclusively in cells expressing SPPL3. The respective cleavage products in B and C are indicated according to A. E, CHAPSO lysates of cellular membranes from HEK293 cells coexpressing the catalytically inactive mutants of the indicated proteases and FVenv were subjected to immunoprecipitation (IP) against HA-tagged SPPL2b and SPPL3. Co-isolated FVenv fragments were detected as indicated. To determine the total amount of FVenv present in the lysate, 5% of the total lysate was applied (input). To trace unspecific binding, the respective CHAPSO lysates were incubated with protein A-Sepharose beads only (beads). Note that SPPL2b(D421A) preferentially co-immunoprecipitated LP18 and only minor amounts of FVenv, whereas SPPL3 selectively co-immunoprecipitated full-length FVenv.

FIGURE 3.

SPPL3 cleavage of FVenv is insensitive to GxGD protease inhibitors. HEK293 cells stably coexpressing FVenv and catalytically active SPPL2b (A) or SPPL3 (B) were treated with increasing concentrations of (Z-LL)₂ ketone, L-685,458, or DAPT. FVenv, LP18, and ICD levels were analyzed. Whereas L-685,458 affected ICD production in a similar manner to (Z-LL)₂ ketone, DAPT had no effect on intramembrane proteolysis of FVenv. Note that ICD(L3) generation was not decreased by any of the inhibitors. Upon treatment of cells coexpressing SPPL2b and FVenv, ICD(L3) and protein fragments (*) similar to those detected upon coexpression of SPPL2b(D421A) accumulated (compare with Fig. 1D). All species are labeled as described in the legend to Fig. 1. The IgG background signal is indicated (**). IP, immunoprecipitation.

FIGURE 4.

SPPL3 generates a substrate for consecutive SPPL2a/b cleavage. A, HEK293 cells stably coexpressing WT SPPL2b or WT SPPL3 and FVenv were immunoprecipitated (IP) using anti-FLAG antibody and separated on a Tris/Tricine gel, as in the previous figures (upper panels), or on a Tris/glycine gel (lower panels). Note that despite less starting material for the immunoprecipitation (IP input), a significant amount of ICD(L2) was generated from cells coexpressing SPPL3 and FVenv. The IgG background signal is indicated (**). untransf., untransfected. B, HEK293 cells stably coexpressing WT SPPL3 and FVenv were treated with the indicated inhibitors (10 μm) or the respective carrier (control (ctrl.)) for 16 h, and generation of ICD(L3) and ICD(L2) was analyzed in cell lysates. C, endogenous SPPL levels of cells described for B were reduced by siRNA as indicated, and ICD(L3) and ICD(L2) levels were assessed as described for B. Knockdown efficiency was verified by Western blotting (SPPL3) or TaqMan RT-PCR (SPPL2a/b, normalized to actin mRNA levels). D, HEK293 cells stably transfected with FVenv were transiently transfected with siRNAs targeting SPPL2a and SPPL2b or SPPL3 (10 nm) or the respective controls (Ctrl 1 and Ctrl 2). Knockdown efficiency was assessed by immunoblotting (SPPL3) or TaqMan RT-PCR (SPPL2a/b). ICD levels were determined by anti-FLAG immunoprecipitation following clearance of FVenv by anti-V5 immunoprecipitation. The asterisk indicated the degradation product of LP18 (see supplemental Fig. S2). E, cell lysates of HEK293 cells coexpressing SPPL3 and either FVenv or FVenv mut (compare with Fig. 2) were analyzed with regard to the indicated FVenv species. Note that ICD(L2) levels remained unchanged, although LP18 was strongly reduced. F, quantification of the experiment shown in D. Data represent means ± S.E. of three independent experiments. **, p < 0.004 (Student's unpaired t test); n.s., not significant. All species are labeled as described in the legend to Fig. 1.

FIGURE 5.

Intramembrane proteolysis of FVenv. Shown is a schematic overview of proteolytic FVenv processing. Glycosylation sites in FVenv are indicated (Y). Proteases and the respective FVenv fragments generated are indicated. FVenv is either processed by SPPL2a/b or SPPL3 following PC cleavage (A) or alternatively directly cleaved by SPPL3 and subsequently by SPPL2a/b (B).