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(The FASEB Journal. 2006;20:1671-1679.)
© 2006 FASEB

Intramembrane proteolytic cleavage by human signal peptide peptidase like 3 and malaria signal peptide peptidase

Andrew C. Nyborg, Thomas B. Ladd, Karen Jansen, Thomas Kukar and Todd E. Golde1

Department of Neuroscience, Mayo Clinic Jacksonville, Mayo Clinic College of Medicine, Jacksonville, Florida, USA

1Correspondence: Department of Neuroscience, Mayo Clinic Jacksonville, Mayo Clinic College of Medicine, 4500 San Pablo Rd., Jacksonville, Florida 32224, USA. E-mail: tgolde{at}mayo.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Signal peptide peptidase (SPP) is an intramembrane cleaving protease (I-CLiP) identified by its cleavage of several type II membrane signal peptides. To date, only human SPP has been directly shown to have proteolytic activity. Here we demonstrate that the most closely related human homologue of SPP, signal peptide peptidase like 3 (SPPL3), cleaves a SPP substrate, but a more distantly related homologue, signal peptide peptidase like 2b (SPPL2b), does not. These data provide strong evidence that the SPP and SPPL3 have conserved active sites and suggest that the active sites SPPL2b is distinct. We have also synthesized a cDNA designed to express the single SPP gene present in Plasmodium falciparum and cloned this into a mammalian expression vector. When the malaria SPP protein is expressed in mammalian cells it cleaves a SPP substrate. Notably, several human SPP inhibitors block the proteolytic activity of malarial SPP (mSPP). Studies from several model organisms that express multiple SPP homologs demonstrate that the silencing of a single SPP homologue is lethal. Based on these data, we hypothesize that mSPP is a potential a novel therapeutic target for malaria.—Nyborg, A. C., Ladd, T. B., Jansen, K., Kukar, T., Golde, T. E. Intramembrane proteolytic cleavage by human signal peptide peptidase like 3 and malaria signal peptide peptidase.


Key Words: {gamma}-secretase • aspartyl protease • hepatitis C virus • MHC class I • Plasmodium falciparum


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PRESENILIN 1 AND 2 AND signal peptide peptidase (SPP) are the prototypic members of a large family of putative aspartyl proteases that cleave a variety of transmembrane substrates. Intramembrane cleaving proteases (I-CLiP) play a variety of important roles in cell signaling (1) and regulation (2) , cell surveillance (3) , and intracellular communication (4) . Each contains a conserved 1) active site motif of YD and GXGD in adjacent transmembrane domains (5 , 6) and 2) a PAL motif near the COOH terminus (7) that have been shown to be critical for activity. Presenilins and SPP differ in that presenilins cleave type I membrane proteins and SPP cleaves type II membrane proteins (5 , 6 , 8 9 10) . In addition, SPP and presenilin have inverted active site topologies (11 12 13) and presenilins seem to require three additional proteins to function as {gamma}-secretase (14 15 16 17 18 19) whereas SPP appears to function as a homodimer (11 , 12 , 20) . {gamma}-Secretase activity cannot be reconstituted in nonmammalian cells without coexpression of presenilin 1 or 2 with Aph-1, Pen-2, and Nicastrin (16 , 19) . SPP activity can be reconstituted in yeast by expression of SPP alone (11) . {gamma}-Secretase inhibitors targeting presenilins are currently under investigation for both Alzheimer therapeutics (21) and possibly as anticancer agents (22 , 23) . SPP inhibitors have been suggested as potential therapeutic agents for hepatitis C infection (24) .

There are four human homologs of SPP, which have been referred to as presenilin homologs (PSH) (25) , intramembrane proteases (IMPAS) (26) , and signal peptide peptidase like (SPPL) (11) . Besides SPP, which was identified based on a search for the proteolytic activity that cleaved the signal peptide of MHC class I molecules and several viral preproproteins, little is known about the function of the various SPPLs. Several recent papers suggest that SPPLs play critical roles in development. In Caenorhabditis elegans, deficiency of ce-imp-2 (a SPP like gene) causes a severe developmental phenotype (27) . Drosophila deficient in one of two SPP genes, CG11840, had defective trachea and died as larvae (28) . In Danio rerio (Zebrafish), when either the SPP or SPPL3 homologs were knocked down, a similar cell death phenotype was achieved (29) . However, knockdown of the SPPL2b homologue resulted in a distinct developmental phenotype with an enlarged caudal vein (29) .

Based on these genetic studies, it is clear that SPP and its homologs have important physiological roles essential for normal development. However, the molecular determinants of these functional effects have not been elucidated. Indeed, only SPP has been shown to cleave a transmembrane substrate directly, and it is unlikely that the SPP cleavage of the endogenous substrates identified to date (HCV core protein, human leukocyte antigen-E eptitopes) mediates the phenotypes associated with SPP deficiency (11 , 12 , 24 , 30) .

Plasmodium falciparum (Malaria) effects ~300 million people a year, with >1 million deaths attributable to plasmodium infection (31) . Treatment of malaria is now complicated by the development of widespread resistance of many Plasmodium species to common affordable drugs such as choloroquine, mefloquine, and sulfadoxanine (32 33 34) . Thus, there is a need to identify novel targets, a process that can be aided by utilizing information generated by the sequencing of the Plasmodium falciparum genome (3D7) (35) . A search of the Plasmodium falciparum genome for SPP homologs identified only one SPP gene that encodes a putative protein (NP_702432) that is ~50% homologous to human SPP. As SPP homologs in C. elegans, Drosophila, and Zebrafish appear essential for development, we hypothesized that given the complex life cycle of malaria, a single malarial SPP may be a novel therapeutic target. Therefore, we synthesized a full-length cDNA encoding this gene and have expressed the malarial SPP (mSPP) in mammalian cells.

In this study we used a cell-based reporter assay for intramembrane cleavage of type II proteins to assess proteolytic activity of two human homologs of SPP, SPPL3 and SPPL2b and mSPP. Using this assay we found that SPPL3 and mSPP cleave the type II membrane reporter construct but that SPPL2b, a more distant homologue, did not. We also show that inhibitors of human SPP and SPPL3 also inhibited the mSPP.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
DNA constructs
SPP and the SPP substrate (SPPSub) constructs were described previously (12 , 20) . Full-length SPPL3 was cloned by amplifying a 1188 bp sequence from a human brain cDNA library. The wild-type SPPL3 cDNA contains the entire coding sequence of SPPL3, and 17 bases of 5' untranslated sequence and 10 bases of 3' untranslated sequence, and was cloned into HindIII (5') and XhoI (3') sites of the pFLAG-cytomeglovirus-2 (Sigma, St. Louis, MO, USA) to generate an amino-terminally FLAG epitope-tagged SPPL3 (SPPL3-NTFLAG). In SPPL3-NTFLAG, full-length SPP was cloned into the HindIII (5') and XhoI(3') sites of pFLAG-cytomeglovirus-2 such that a vector-derived three amino acid spacer (LLA) separates the FLAG tag from the amino terminus of SPPL3. Full-length SPPL2b was cloned by amplifying a 1779 bp sequence from a human brain cDNA library. The wild-type SPPL2b cDNA contains the entire coding sequence of SPPL2b and six bases of 5' untranslated sequence and six bases of 3' untranslated sequence, and was cloned into HindIII (5') and XbaI (3') sites of the pAG3 expression vector (36) . Full-length SPP was also cloned into the pcDNA6-V5-his vector (Invitrogen, San Diego, CA, USA) to generate a carboxyl terminally V5 + 6 X his epitope tagged SPP (SPPL2bCTV5). All constructs were verified by sequencing.

Malaria SPP (mSPP) was identified by searching the Plasmodium falciparum genome with the entire human SPP protein sequence. The malaria amino acid sequence (NP_702432) was then human codon optimized and synthesized by GenScript. The resulting gene was cloned into pCDNA6, which puts a V5 his epitope tag at the COOH terminus (mSPPCTV5).

SPP and mSPP phylogenetic tree analyses and identities were generated using the AlignX@ feature of Vector NTI@.

DNA transfection of 293 cells
The luciferase reporter assays were performed as described previously (12) . In short, HEK 293T cells were plated at 70% confluency and transiently transfected using 100 µl serum free Opti MEM® (Life Technologies, Inc., Grand Island, NY, USA), 8 µl of fugene, 0.02 µg of pRL-Null Renilla expression plasmid (Promega, Madison, WI, USA), 0.25 µg of pGL3 5x ATF6 reporter plasmid, 0.25 µg of pAG3 SPPsub plasmid, and the indicated amount of a SPP construct expression plasmid or control plasmid to total 2 µg of DNA in each well of 12-well plate. Each well of cells were lysed using 100 µl of passive lysis buffer (Promega) firefly and Renilla luciferase activities were measured using the Dual-Luciferase® kit (Promega) and a Veritas microplate Luminometer (Turner Biosystems, Sunnyvale, CA, USA) with Veritas 2.0.40 software package. Transfections were performed in triplicate. Results were normalized to the Renilla luciferase activity control. In some experiments, where substrate was analyzed by Western blot, transfections were modified such that 2 µg of SPPsub and 2 µg of SPPL3NTFLAG plasmids were used.

Antibodies and Western blot
Anti-V5 (Invitrogen, San Diego, CA, USA) and anti-FLAG (Sigma) antibodies were used at 1:1000. Anti-ß-actin antibody (Ab) (Sigma) was used as a loading control at 1:1000. The anti-ATF6 Ab (Imgenex, San Diego, CA, USA) was raised to the first 273 amino acids and recognizes the NH2-terminal, pre-transmembrane portion of our substrate. This Ab was used at 1:150. Antipeptide antisera were raised in rabbits to the carboxyl (anti-SPPL3ct, residues 364–384) terminal domains of human SPPL3 (Covance, Princeton, NJ, USA). Peptides corresponding to these domains of SPP were synthesized and coupled to keyhole limpet hemocyanin prior to immunization.

Cells were lysed in 1% triton X 100 (TX-100) with 1x complete protease inhibitor (Roche, Nutley, NJ, USA) unless otherwise stated. Cell lysates were then spun at 14,000 rpm for 2 min to remove nuclei. Bio-Rad XT loading buffer with reducing solution was added to each sample. SDS-PAGE was performed using Bio-Rad Criterion gel system. 10% Bis-Tris XT gels were used unless otherwise stated with Bio-Rad MES buffer. Gels were transferred to Millipore lowfluor PVDF for 90 min and 160 V. Membranes were blocked in a casein solution and primary antibodies were used at the reported concentration in the blocking solution overnight at 4°C. Odyssey secondary antibodies containing either the 680 or 800 fluorophore were incubated with the membrane for 1 h at room temperature at 1:20,000. Fluorescently labeled protein detection was performed using the Odyssey Scanner.

Gradient fractionation
Sucrose gradients were run (37) and glycerol velocity gradients were run as described previously (38) . Briefly, HEK cells stably expressing the SPPCTV5 or SPPL2bCTV5 constructs (5 confluent 150 mM plates) were lysed in either CHAPSO or TX-100. Lysates were spun at 3220 g to remove nuclei, insoluble material, and cellular debris and 1 ml of supernatant was loaded onto the top of an 11 ml 10–40% linear glycerol gradient with either 0.5% CHAPSO 150 mM NaCl and 25 mM HEPES or 0.1% TX-100 in TBS. The samples were then spun in a SW 41 rotor for 15 h at 110,000 g at 4°C. One ml fractions were collected from the top and analyzed by SDS-PAGE for SPP or SPPL2b proteins. Control gradients were prepared identically but included a combination of commercially available, characterized molecular weight standards (SERVA).

Immunoprecipitation
HEK 293T cells were transiently transfected using a calcium phosphate transfection method (39) . 67 µg of SPPL3NTFLAG plasmid was added to each 15 cm plate. Cells were lysed in 1% TX-100 (1xTBS) and immunoprecipitation (IP) was performed as described (40) .

Data analysis
Data were analyzed using Sigma Stat. For comparison of multiple experimental values relative to controls an ANOVA was performed using a Dunnet’s post hoc t test. Variance is reported as the standard error of the mean.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Sequence analysis and expression of human SPP family members and mSPP
Phylogenetic tree analyses have been used to show that the SPP family members can be placed into two families (13) . SPPL3, also known as PSH1 (25) and IMP2 (26) , is the nearest evolutionary homologue to SPP (13) . SPPL2b, also known as PSH4 (25) and IMP4 (26) , is a more distant relative of SPP and SPPL3 (13) . In fact, SPPL2a, b, and c are more closely related to each other than SPP and SPPL3. We have generated a phylogenetic tree to determine the relative identity between human SPP family members and mSPP. The single SPP gene present in Plasmodium falciparum (NP_702432) is most closely related to SPP and SPPL3 (30% identity) and is more distantly related to SPPL2a, b, and c (Fig. 1 a). To further characterize these SPP homologs, we expressed SPP, SPPL3, SPPL2b, and mSPP in mammalian cells.


Figure 1
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  		Figure 1. mSPP is most closely related to SPP. a) A phylogenetic tree was generated using the Align X feature in Vector NTI@. Percent identity scores were reported relative to mSPP. mSPP is most closely related to SPP and SPPL3 whereas SPPL2a, b, and c are more closely related to each other. b) HEK 293T cells were transiently transfected with SPPNTFLAG, SPPL3NTFLAG, SPPL2bCTV5, or mSPPCTV5. Cells were lysed in 1% triton X 100; lysates were run on SDS-PAGE and Western blots were probed with the antibodies listed.

The SPP glycoprotein has been shown to run on SDS-PAGE as both a monomer (45 kDa) and a dimer (90 kDa) (20) , as seen in Fig. 1b . Overexpression of SPPL3NTFLAG and analysis by SDS-PAGE and Western blot lead to a predominant band detected at ~60 kDa (Fig. 1b ). In experiments where the amount of SPPL3NTFLAG was highly overexpressed, a significant increase in both the ~60 kDa and a smaller ~30 kDa band was observed (13 , 29) . We suspected that, like SPP, SPPL3NTFLAG might exist as both a monomer and homodimer. SPPL3 is predicted to be ~38 kDa, which is slightly larger than observed by SDS-PAGE and Western blot. To show that both the ~30 and the ~60 kDa bands contain full-length SPPL3, we immunoprecipitated SPPL3NTFLAG with an anti-SPPL3 Ab raised against the COOH terminus of SPPL3. The immunoprecipitated material was then run on SDS-PAGE and Western blotted with anti-FLAG Ab (FLAG epitope is at the NH2 terminus of SPPL3). Two anti-FLAG positive bands were observed at ~60 and ~30 kDa, indicating that both contain the SPPL3 holo-protein (data not shown). SPPL2bCTV5 is observed on a Western blot as a group of diffuse bands from 65 to 95 kDa (Fig. 1b ; see Fig. 3b ), which is consistent with the immature and mature forms of SPPL2b observed previously (29) . One predominant and specific band was detected for mSPPCTV5 by Western blot analysis at ~35 kDa (Fig. 1b ). A minor ~70 kDa was also detected, although the ratios of these bands tend to vary from experiment to experiment (Fig. 1b ; see Fig. 4b ). These are consistent with monomer and dimer formation; however, in the case of mSPP we predominantly detect the smaller ~35 kDa band, indicating that the dimer may either be less prevalent or less stable in SDS than human SPP.


Figure 2
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  		Figure 2. SPPL3 cleaves a SPP substrate. a) Luciferase activity increased with increasing SPPL3NTFLAG protein expression. SPPL3NTFLAG expression plasmid was cotransfected into HEK 293T cells at the concentrations reported and as described in Materials and Methods. Normalized reporter activity expressed as % of the activity present in cells expressing endogenous levels of SPP (% endogenous activity) was plotted. SPP inhibitor, ZLL (0.2 µM), and {gamma}-secretase inhibitor, LY411,575 (50 nM) both inhibited SPPL3NTFLAG activity significantly. SPP activity levels increased proportionally with increasing levels of SPPNTFLAG expression plasmid. Western sample were prepared from passive lysis buffer lysates used for luciferase measurements. Two distinct SPPL3NTFLAG band were observed at ~60 kDa and ~30 kDa. The SPPNTFLAG was observed as the dimer (labeled) at ~95 kDa and monomer (labeled) at ~45 kDa as detected with anti-FLAG. *P < 0.05. b) Luciferase activity increases with increasing SPPNTFLAG protein expression. Normalized reporter activity expressed as a % of the activity present in cells expressing endogenous levels of SPP (percent over endogenous activity) were plotted vs. SPPL3NTFLAG protein levels expressed as a fold detected over background (Fold SPPL3 detected over background).


Figure 3
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  		Figure 3. SPPL2b does not cleave the SPP substrate and unfolded protein response does not interfere with the assay. a) Overexpression of either SPPNTFLAG or SPPL3NTFLAG significantly increased luciferase activity (% endogenous activity) observed in the reporter assay. Conversely, overexpression of either SPPL2bCTV5 or SPPL2b (not shown) did not significantly increase SPP luciferase activity. Overexpression of SPPNTFLAG, SPPL3NTFLAG, or SPPL2bCTV5 did not contribute to luciferase activity observed by eliciting an endogenous unfolded protein response. This was determined by overexpressing the SPP construct in the presence of 5x ATF6 reporter but an absence of SPPsub. *P < 0.05. b) Samples that did not contain SPPsub (lysed in passive lysis buffer) were run on SDS-PAGE and probed by Western blot with anti-FLAG (SPPNTFLAG and SPPL3NTFLAG) and anti-V5 (SPPL2bCTV5). The amount of SPP and SPPL3 levels were relatively comparable. In our hands anti-V5 and anti-FLAG recognize the SPP epitope tagged constructs with relatively similar affinities, suggesting that the SPPL2b levels may have been slightly higher than SPP and SPPL3. c, d) Previously we showed that unmasking of the V5 epitope was a surrogate marker for SPP cleavage (12) . The amount anti-V5 positive of SPPsub detectable increased with increasing SPPNTFLAG expression (12) . Though SPPsub levels remained relatively constant (as detected by anti-ATF6) SPPsub detected by anti-V5 (anti-V5 relative light units, RLU; % increase) increased significantly when SPPL3NTFLAG was overexpressed. *P < 0.05. e) SPPsub cleavage was observed directly in cells not transfected with either SPP or SPPL3 by probing the SPPsub with anti-V5. An increase in anti-V5 detected SPPSub was observed when either SPPNTFLAG or SPPL3NTFLAG were transiently overexpressed. The increase in the soluble anti-V5 detectable SPPsub decreased as a result of inhibition by ZLL (an SPP inhibitor).


Figure 4
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  		Figure 4. Sucrose gradient fractionation and glycerol velocity gradient sedimentation of SPP and SPPL2b. a) 10–40% linear, glycerol velocity gradients of HEK cells stably overexpressing SPPCTV5 or SPPL2bCTV5 lysed in CHAPSO or TX-100 were analyzed by SDS-PAGE on 4–15% Tris-HCl gels and probed with anti-V5. SPPCTV5 dimer sediments in fractions consistent with ~160 kDa complex and a monomer that sediments in even smaller fractions (not shown). SPPCTV5 overexpressing cells lysed in either CHAPSO or TX-100 show a similar sedimentation, but SPPL2bCTV5 sedimentation differs significantly when the cells are lysed in CHAPSO instead of TX-100. Similarly, significant differences in the PS1 fragment sedimentation are observed in the two different lysis conditions. Gradient molecular weight kDa at bottom refers to control proteins loaded on a separate but identically poured gradients. Lysate and control gradients have been performed in triplicate. Comparable results were observed for endogenous SPP from HEK cell shown previously (20) . b) HEK cells stably overexpressing SPPCTV5 or SPPL2bCTV5 were lysed in 2% CHAPSO and sucrose gradient fractionated. Fractions were collected from the top making 1–4 buoyant fractions and 9–12 dense fractions. All fractions were analyzed by SDS-PAGE on 4–15% Tris-HCl gels and probed with anti-V5. Much like PS1, SPPCTV5 and SPPL2bCTV5 fractionate to buoyant fractions 4, 5, and 6.

Functional SPP activity analyses of SPPL3, SPPL2b, and mSPP
The SPP reporter assay utilizes a SPP cleavable type II membrane domain fused to the COOH terminus of ATF6 transcription factor (SPPsub) (12) . Upon cleavage, ATF6 is released from the membrane, translocates to the nucleus, and activates a luciferase reporter construct (12 , 41) . To determine whether the SPPsub could be cleaved by SPPL3NTFLAG and activate the 5x ATF6 luciferase reporter construct, we examined whether increased expression of SPPL3NTFLAG would increase the luciferase activity. For these studies, we transiently transfected 293T cells with increasing amounts of SPPL3NTFLAG, as indicated in Fig. 2 a, and performed the luciferase assay as described in Materials and Methods. As increasing amounts of SPPL3NTFLAG expression vector were transfected, a significant increase in luciferase activity was observed (Fig. 2a ). Addition of a SPP inhibitor (ZLL)2-ketone (ZLL) or LY411,575, a {gamma}-secretase inhibitor that has been shown to inhibit SPP, inhibited the increase in luciferase activity due to SPPL3NTFLAG overexpression. When comparable amounts of SPPNTFLAG or SPPL3NTFLAG were detected by anti-FLAG Western blot, a similar increase in luciferase activity was observed (Fig. 2a ). The increase in activity observed, as an increasing amount of SPPL3NTFLAG expression plasmid was transfected, corresponded to an increase in the amount of SPPL3NTFLAG detected by Western blot (Fig. 2b ).

The reporter construct for the SPP cell-based assay is a 5x repeat of the ATF6 binding region coupled to the luciferase promoter. In the event of a significant endogenous unfolded protein response, as a result of overexpressing a foreign protein there is the potential for endogenous ATF6 NH2-terminal signal peptide binding to the 5x ATF6 reporter construct contributing to an artificially high luciferase signal. Assays without SPPsub were performed to confirm that the increase in activity seen as a result of overexpression of SPP constructs was due to SPPSub cleavage and not the result of an endogenous ATF6 unfolded protein response. As seen in Fig. 3 a, the significant increase in activity observed was due to the inclusion of the SPPSub. When an empty vector was substituted for the SPPsub, no significant luciferase activity was observed as a result of SPPNTFLAG, SPPL3NTFLAG, or SPPL2bCTV5 overexpression, indicating that these constructs did not elicit a significant endogenous ATF6 unfolded protein response.

Unlike SPPL3 and SPP, overexpression of SPPL2bCTV5 did not activate the SPPsub to generate significant activity above endogenous activity (Fig. 3a ). In our hands anti-V5 and anti-FLAG recognize the SPP epitope tagged constructs with relatively similar affinities, suggesting that the SPPL2b levels may have been slightly higher than SPP and SPPL3 (Fig. 3b ).

The increase in luciferase activity as a result of SPPL3NTFLAG overexpression was further examined by Western blot, the SPPSub with anti-V5. Previously we demonstrated that the anti-V5 signal increased as a result of SPPSub cleavage, making the V5 epitope a good indicator for both cleavage and solubilization of SPPSub (12) . An increase in the anti-V5 detectable SPPSub was observed as a result of SPPNTFLAG or SPPL3NTFLAG overexpression (Fig. 3c, d ). As shown before, the anti-ATF6 detectable substrate remained constant but the anti-V5 detectable SPPsub increased when conditions favor substrate cleavage (Fig. 3c ). The cleavage detected by anti-V5 Western blot was inhibited by SPP inhibitor ZLL (Fig. 3e ). A doublet of the SPPsub (presumably phosphorylated and unphosphorylated ATF6 NH terminus; ref 42 ) was resolved and detected on the Western blot when a 10% Bis-Tris gel was used (Fig. 3c ) as apposed to the single band observed for the substrate resolved on a 4–15% Tris-HCl gel (Fig. 3e ).

Glycerol velocity gradient fractionation and sucrose gradient fractionation of SPPL2b and SPP
Glycerol velocity gradient fractionation can be used to provide estimates of the molecular weight of proteins and protein complexes. This technique provided initial evidence that PS1 existed in a high molecular weight complex (38) . Previously we showed that both weight and overexpressed SPP with an epitope tag ran comparably on glycerol velocity gradients (20) . We found that the SPP dimer distributed primarily in the 100–160 kDa range, with a small portion of the SPP dimer sedimenting at higher molecular weight.

To explore whether SPP or SPPL2b exists as high molecular weight complexes, we analyzed the distribution of SPP and SPPL2b in glycerol velocity gradients in the presence of TX-100 or CHAPSO detergent and compared both to exogenous molecular weight marker proteins and the distribution of endogenous PS1. A similar glycerol velocity sedimentation of SPP was observed when cells overexpressing SPPCTV5 were lysed in either CHAPSO or TX-100 (Fig. 4 a). These results contrast with the distribution of PS1 under varying detergent lysis conditions. Under conditions that maintain {gamma}-secretase activity (CHAPSO lysis), the NTF and CTF of PS1 sediment to high molecular weight fraction (>200 kDa, Fig. 4a ). Using detergents that disrupt and inactivate the {gamma}-secretase complex (TX-100), PS1 is found in lower molecular weight fractions. Like PS1, SPPL2b appears to exist as a high molecular weight complex that is detergent sensitive. In the presence of TX-100, SPPL2bCTV5 is observed from 25 to 160 kDa with a small amount of SPPL2b in the higher molecular weight range. In contrast, SPPL2bCTV5 fractionated in the presence of CHAPSO migrates from 160 to 450 kDa. These results are reminiscent of PS1 and suggest that SPPL2b may exist as a higher molecular weight complex.

It has been demonstrated that {gamma}-secretase activity and PS1 are enriched by sucrose gradient fractionation to buoyant cholesterol rich membranes, also known as lipid rafts (37 , 43) . These buoyant membrane are also enriched in GS27, Flotilin, and other raft markers (37 , 43) . We found that the majority of SPPCTV5 and SPPL2bCTV5 are enriched in the these same buoyant membrane fractions (Fig. 4b ).

Comparable experiments with SPPL3 could not be performed due to our inability to generate a SPPL3 overexpressing stable cell line. A variety of different SPPL3 plasmids encoding SPPL3 with various epitope tags at either the NH2 terminus or the COOH terminus have been generated. In all cases these plasmids appear to express SPPL3 transiently in a variety of different cell lines (CHO, HEK, and H4). Though SPPL3 can be overexpressed transiently, stable cell lines are devoid of detectable SPPL3. Fractionation studies were attempted by transiently overexpressing SPPL3 and SPP. However, transient overexpression of SPP gave fractionation results that were completely inconsistent with the stable cell lines and endogenous results previously generated and essentially uninterruptible (not shown).

mSPP cleaves the SPPsub and is inhibited by SPP inhibitors
Only one SPP homologue is encoded by the Plasmodium Falciparum genome. We synthesized a cDNA encoding the entire predicted open reading frame (NP_702432) that was codon optimized for expression in mammalian cells. The cDNA was then cloned into a mammalian expression vector and either transiently or stably transfected into mammalian cells. Overexpression of mSPPCTV5 caused a significant increase in the SPPsub luciferase activity (Fig. 5 a). The increase in activity observed due to mSPPCTV5 overexpression was inhibitable with either LY411,575 or ZLL, both of which have been shown to effectively inhibit SPP (12) and SPPL3 activity (Figs. 2 , 3) . Overexpression of mSPPCTV5 did not generate an endogenous unfolded protein response that falsely signals the 5x ATF6 reporter as observed by expression of the reporter construct and mSPPCTV5 but no SPPsub in Fig. 5a . In addition, a significant increase in the V5 epitope of the SPPsub was observed as a result of mSPPCTV5 overexpression (Fig. 5b, c ). Two specific bands were observed for mSPPCTV5 by Western blot analysis at ~35 kDa and ~70 kDa (Fig. 5b ).


Figure 5
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  		Figure 5. mSPP cleaves the SPPsub. a) Overexpression of mSPPCTV5 provided a significant increase in luciferase SPPsub activity relative to endogenous activity (expressed and % control). The activity was inhibitable by two SPP inhibitors (ZLL and LY411,575). *P < 0.05. b, c) Unmasking of the V5 epitope was shown to be a surrogate marker for SPPsub cleavage (12) . The amount anti-V5 positive of SPPsub detectable increased as a result of mSPPCTV5 overexpression (12) (anti-V5 relative light units, RLU; % increase). The SPPsub substrate was detectable at ~53 kDa. The mSPPCTV5 was detected at ~35 kDa and ~70 kDa. *P < 0.05.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Using a cell-based reporter assay for SPP, we provide strong evidence that the nearest evolutionary homologue of SPP, SPPL3, has proteolytic activity. Reporter activity and substrate cleavage (detected by anti-V5 analysis) both increased with increasing SPPL3NTFLAG expression. Moreover, inhibitors of SPP activity inhibited SPPL3 activity.

Though all members of the SPP family have been speculated to be I-CLiPs, overexpression of SPPL2b did not increase SPPsub luciferase activity. Opposite active site topology between presenilin and SPP has been thought to be the key factor responsible for their differences in cleaving type II membrane proteins and type I membrane proteins, respectively (5 , 6 , 11) . All SPP family members appear to have similar topological orientation (11 12 13) . Hence, SPPL2b contains the apparent active site orientation necessary for SPPsub cleavage to occur, but no cleavage was detected. These data appear to be consistent with genetic data from Zebrafish, demonstrating that when the SPPL2b gene was knocked down a unique phenotype was observed relative to the SPP and SPPL3 knockdown phenotype. The absence of detectable substrate cleavage by SPPL2b may be attributable to its inability to cleave the substrate or a lack of colocalization of the SPPsub and SPPL2b. SPP and SPPL3 were both shown to localize to the ER (29) , similar to ATF6 (44) . However, SPPL2b was found predominantly in vesicular structures, which were positive for LAMP-2, a marker for late endosomes/lysosomes (29) , implying that even if SPPL2b could theoretically cleave the SPPsub, substrate and active protease may not localize to the same cellular compartment.

Much like PS, SPPL2b may exist as a high molecular weight complex that is sensitive to various detergents. As seen in the glycerol velocity gradient fractionation, SPPL2b fractionated to a much higher molecular weight when the lysate and sedimentation were performed in the presence of CHAPSO. It is possible that, like PS1, SPPL2b has additional cofactors. In contrast to this, SPP did not appear to be sensitive to either detergent used in these experiments and runs on a glycerol velocity gradient from 100 to 160 kDa. These data do not exclude the possibility that SPP exists as a higher molecular weight complex and has cofactors, but indicate that if cofactors of SPP exist they have different solubilization properties from PS and SPPL2b.

Having established a method for assessing whether SPP candidate enzymes exhibit SPP activity in vivo, we cloned the only SPP gene identifiable in the malaria genome and tested for activity. mSPP contains the hallmark features of a SPP protein in that it has multiple transmembrane domains, active site YD/GXGD aspartates in adjacent transmembrane domains, and a PAL motif near the COOH terminus. Each of these features has been shown to be critical for both presenilin and SPP function (45) . The mSPP gene is most closely related to SPP and SPPL3. We found that luciferase activity increased due to cleavage of the SPPsub by mSPPCTV5 overexpression. The increase in activity was inhibitable by both a SPP inhibitor, ZLL, and potent {gamma}-secretase inhibitor, LY411,575, demonstrating the potential to target mSPP as an antimalaria therapy.

As the Malaria parasite becomes increasingly resistant to traditional drugs, the need for novel targets is increasing. The potential to target novel malarial aspartyl proteases has been established in the plasmepsin protein family, and inhibitors in the low nanomolar range have shown potential in cell culture and animal models (46 47 48) . In addition, recent studies have shown that clinically utilized HIV protease inhibitors can inhibit the in vitro growth of Plasmodium falciparum at or below concentrations found in human plasma after oral drug administration (49) . We hypothesize that mSPP may be a novel drug target because elimination or inhibition of SPP activity causes embryonic death in C. elgans, Drosophila, and Zebrafish even though each contains at least two SPP homologs (27 28 29) . Thus, it is possible that mSPP is a critical gene for malaria development. If this is the case, then the pharmacologically inhibition of mSPP may be lethal to the parasite. As shown here, mSPP is targetable by drug-like compounds. Moreover, numerous inhibitors of {gamma}-secretase and SPP have been reported in the literature, and the pharmaceutical industry has generated thousands of compounds that target {gamma}-secretase. Some of these compounds have moved into clinical trials, and those that target {gamma}-secretase do so with incredible potency (subnanomolar IC50s) and excellent tissue distribution, including the central nervous system (21 , 50 , 51) .

Due to potential toxicity associated with complete {gamma}-secretase inhibition, it would be ideal to develop compounds that selectively target mSPP. SPP and presenilin activities are differentially inhibitable as demonstrated by two structurally similar {gamma}-secretase inhibitors, DAPT and LY411,575, having very different effects on SPP inhibition. LY411,575 is an effective inhibitor of SPP but DAPT is not (12 , 52) . As the overall homology of mSPP with human SPP or SPPL3 is low, it should be possible to identify compounds that selectively inhibit mSPP and not human SPP. Finally, a similar analysis of the genomes of other human parasites that cause significant morbidity and mortality reveals that each contains a single SPP homologue (Leishmaniasis XP_847754, Trypanosomiasis EAN96276, and Giardia EAA41466). Like malaria, these genes all have greater sequence homology to SPP than other SPPs. Thus, if it can be shown that mSPP is a good drug target, similar techniques and reagents could be developed to target multiple human parasitic pathogens. The SPP assay used to generate the data in this manuscript, or a close variant, should be useful in developing testing potential inhibitors.


   ACKNOWLEDGMENTS
 
This work was supported by the Mayo Foundation and an NIH/NINDS grant NS39072 to T.E.G., NS44734 to A.C.N. pCGNATF6 1–373 plasmid, and the pGL3 5x ATF6 luciferase reporter plasmids were generously provided by Ron Prywes.

The authors have declared that no competing interests exist.

Received for publication January 11, 2006. Accepted for publication March 31, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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