gamma -Secretase, Evidence for Multiple Proteolytic Activities and Influence of Membrane Positioning of Substrate on Generation of Amyloid beta  Peptides of Varying Length*

Michael Paul MurphyDagger , Lesley Jill Hickman§, Christopher Benjamin EckmanDagger , Sacha Noelle Uljon, Rong Wang, and Todd Eliot GoldeDagger parallel

From the Dagger  Department of Pharmacology, Mayo Clinic Jacksonville, Jacksonville, Florida 32224, the § Department of Neuroscience, The University of Pennsylvania, Philadelphia, Pennsylvania 19010, and the  Laboratory for Mass Spectrometry, The Rockefeller University, New York, New York 10021

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

gamma -Secretase activity is the final cleavage event that releases the amyloid beta  peptide (Abeta ) from the beta -secretase cleaved carboxyl-terminal fragment of the amyloid beta  protein precursor (APP). No protease responsible for this highly unusual, purportedly intramembranous, cleavage has been definitively identified. We examined the substrate specificity of gamma -secretase by mutating various residues within or adjacent to the transmembrane domain of the APP and then analyzing Abeta production from cells transfected with these mutant APPs by enzyme-linked immunosorbent assay and mass spectrometry. Abeta production was also analyzed from a subset of transmembrane domain APP mutants that showed dramatic shifts in gamma -secretase cleavage in the presence or absence of pepstatin, an inhibitor of gamma -secretase activity. These studies demonstrate that gamma -secretase's cleavage specificity is primarily determined by location of the gamma -secretase cleavage site of APP with respect to the membrane, and that gamma -secretase activity is due to the action of multiple proteases exhibiting both a pepstatin- sensitive activity and a pepstatin-insensitive activity. Given that gamma -secretase is a major therapeutic target in Alzheimer's disease these studies provide important information with respect to the mechanism of Abeta production that will direct efforts to isolate the gamma -secretases and potentially to develop effective therapeutic inhibitors of pathologically relevant gamma -secretase activities.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The 4-kDa amyloid beta  protein (Abeta )1 that is invariably deposited as amyloid in Alzheimer's disease (AD) is a normally secreted proteolytic product of the amyloid beta  protein precursor (APP) (1-3). Generation of Abeta from APP requires two proteolytic events, one at the amino terminus referred to as beta -secretase and one at the carboxyl terminus known as gamma -secretase (Fig. 1). To date, neither of the proteases responsible for these activities has been definitively identified. Comparison of the soluble Abeta secreted by cells, soluble Abeta in cerebrospinal fluid, and insoluble Abeta isolated from the AD brain has revealed that there are numerous Abeta species with extensive amino- and carboxyl-terminal heterogeneity. The major Abeta species in both conditioned cell culture media and human cerebrospinal fluid is Abeta 1-40 (~50-70%) although some Abeta 1-42 (5-20%) is also present along with minor amounts of other peptides (e.g. Abeta 1-28, Abeta 1-33, Abeta 1-34, Abeta 3-34, Abeta 1-37, Abeta 1-38, and Abeta 1-39) (4-6). The importance of the longer forms of Abeta , in particular Abeta 42, has been heightened by the fact that all of the familial Alzheimer's disease (FAD) linked mutations that have been analyzed result in an increase in the concentration of Abeta 42 in a wide variety of model systems (reviewed in Refs. 7 and 8). Biophysical studies have shown that the longer forms of Abeta aggregate at a much faster rate and at lower concentrations than forms ending at Abeta 40 suggesting that alterations in Abeta 42 concentration, as occurs in FAD linked forms of the disease, may account for the observation that the longer forms of Abeta are often the initial species deposited in the parenchyma of the AD and Down's syndrome brain (9-11).


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Fig. 1.   Cleavage of APP to generate Abeta . The APP is cleaved at the amino terminus of the Abeta sequence by an unknown protease referred to as beta -secretase. This cleavage releases a large secreted derivative referred to as sAPPbeta . Subsequent cleavage of the APP COOH-terminal fragment (C99) by gamma -secretase generates Abeta . In the alpha -secretory pathway (not shown) the APP is cleaved within the Abeta region to generate a large secreted derivative referred to as sAPPalpha . Subsequent cleavage of the APP COOH-terminal fragment (C83) by gamma -secretase generates a truncated Abeta species referred to as P3.

In addition to the beta - and gamma -secretase activities that generate Abeta , a third proteolytic activity referred to as alpha -secretase cleaves within the Abeta sequence at Lys16 to release a large secreted derivative (12-14), thus precluding formation of full-length Abeta . Following cleavage of APP at the extracellular/lumenal domain of the APP by either alpha - or beta -secretases, gamma -secretase cleavage of the resulting COOH-terminal fragment results in the release of the p3 or Abeta , respectively.

The findings that all of the FAD-linked mutations in APP and the presenilin (PS) genes alter the concentration of Abeta ending at position 42 and, with the exception of the APPK670N,M671L (NL) mutation, appear to act by altering gamma -secretase activity makes understanding this activity pivotal to our knowledge of the disease process. While previous studies have shown that beta -secretase cleavage, like most proteolytic cleavages, exhibits fairly rigid primary amino acid sequence requirements (15), similar studies of the more complex gamma -secretase activity demonstrates fairly loose specificity (16, 17). These studies, however, which either examine effects on total Abeta production (16) or effects of mutations at a single residue, Abeta 43 (17), provide no compelling mechanisms for the observed alterations in cleavage associated with FAD-linked mutations.

One of the more unusual aspects of gamma -secretase cleavage is that based on hydropathy plots the gamma -cleavage sites lie within the putative transmembrane domain (TMD) of the APP (18). Several other proteins, SREPB (19), Notch (20), and mitochondrial inner membrane proteins (21), have been postulated to undergo such intramembranous cleavage. While recent data for SREBP site two protease cleavage (22) and Notch (20), indicate that the cleavage of these proteins occurs at hydrophobic residues near the transmembrane junction, in no instance has the site of cleavage and the location of the residues with respect to the membrane at the time of cleavage been elucidated. Thus, the concept of intramembranous proteolysis remains controversial. To date, there is no definitive evidence showing that a protease can cleave bonds buried within a membrane.

As a first step toward defining the mechanism of gamma -secretase cleavage, we undertook a mutagenesis study in an attempt to define the specificity and structural requirements that produce Abeta species of varying lengths by using a large number of APP TMD mutants. The TMD mutant APPs are shown in Fig. 2. These mutations can be divided into several different categories. 1) Point mutations at positions Abeta 41 (I637X based on the APP695 sequence) and Abeta 43 (T639X) which correspond to the P1' positions for a gamma -secretase cleavage producing Abeta 1-40 and Abeta 1-42, respectively. 2) Deletion and insertion mutations designed to alter the localization of the gamma -secretase cleavage site within the membrane (del and ins). 3) Point mutations that alter the putative membrane stop anchor signal or increase the number of charged residues on the lumenal side of the membrane and 4) replacement of residues carboxyl to the normal gamma -cleavage sites with alanine. All mutations were made in a background of the APP695NL mutation in order to increase absolute amounts of Abeta peptides generated without affecting gamma -cleavage as this mutation increases activity at the beta -secretase site without altering the relative amounts of either Abeta 40 or Abeta 42 (5, 23-25).


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Fig. 2.   Transmembrane domain mutations of APP695NL. Mutated residues are shown in red. Numbering is based on the APP695 sequence. The ends of Abeta 1-40 and Abeta 1-42 are illustrated.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Generation of Mutant APPs-- A two-step PCR based mutagenesis strategy was employed to generate the various TMD domain mutants. In the first step, the EcoRI/NotI fragment of pcDNA3APP695NL was replaced with a PCR product generated from wild-type APP695 using the forward primer APP+1803 and the reverse primers Delta 26 (5'-CATGCGGCCGCTCGTCTCTTGAACCCACATCTTCTGCA-3'), Delta 39 (5'-CATGCGGCCGCTCGTCTCCAACACCGCCCACCATGAGT-3'), and Delta 52 (5'-CATGCGGCCGCTCGTCTCTCAGCATCACCAAGGTGATGA-3'), respectively, to generate the base constructs pcDNA3APP695NLDelta 26, pcDNA3APP695NLDelta 39, and pcDNA3APP695NLDelta 52. These mutant APPs incorporated a class IIa restriction site, BsmBI, 3' to Abeta 26, Abeta 39, and Abeta 52. When cut with BsmBI and NotI the BsmBI site is lost, leaving a 5' 4-bp overhang at the 3' end, effectively truncating the APP. To produce the various TMD mutants in this study, oligonucleotides incorporating the various mutations were generated containing a BsmBI site at their 5' end. PCR products were amplified using various mutant oligonucleotides and a common reverse primer using wild type APP as template. Subsequent cleavage of these products with BsmBI and NotI followed by cloning into the appropriate base vectors (pcDNA3APP695NLDelta 26, pcDNA3APP695NLDelta 39, and pcDNA3APP695NLDelta 52) generated the desired mutants with no additional base changes. All PCR reactions were carried out using the High Fidelity PCR Kit from Boehringer Mannheim. All mutant cDNAs were sequenced to ensure that no additional mutations were incorporated. Sequences for all of the PCR primers used are available upon request.

Transient Transfection of 293 T Cells and Media Collection-- Cells were plated onto 6-well culture dishes (Corning) and grown to 70-80% confluence in Dulbecco's modified Eagle's medium (HyClone) supplemented with 10% fetal calf serum and 1% penicillin/streptomycin solution (Life Technologies, Inc.). On the day of transfection, culture media was changed to Opti-MEM (Life Technologies, Inc.). After a short period of equilibration, the media was again changed to fresh Opti-MEM (0.5 ml) and 1.5 µg of DNA and 4.0 µg of DOSPER liposomal transfection reagent (Boehringer Mannheim) were added to each well (premixed) in a volume of 0.5 ml of additional media. After overnight incubation, the media were discarded and replaced with Dulbecco's modified Eagle's medium, 10% fetal calf serum (1.0 ml/well). Twenty-four hours later, the media were collected and replaced with serum-free Dulbecco's modified Eagle's medium; complete protease inhibitor mixture (Boehringer Mannheim) was added to each sample. The serum-free media were collected 24 h later, and phenylmethylsulfonyl fluoride (to 1 mM) and EDTA (to 5 mM) added to each sample. In both cases, samples were immediately centrifuged at high speed to pellet cellular debris, transferred to clean Microfuge tubes, and frozen in aliquots at -80 °C until analyzed.

Pepstatin treatment was conducted by adding to the media (post-transfection) pepstatin A to a concentration of 100 µg/ml and Me2SO to 2%. Control cultures were treated with 2% Me2SO alone. Transfections and media collections were otherwise identical to untreated 293T cells.

sAPP Competitive ELISA-- To generate and purify recombinant human sAPP, a 6-histidine tag was placed in-frame near the amino terminus of human APP695 by cleavage with KpnI and in-frame insertion of a double-stranded DNA formed by annealing two oligonucleotides. After construction in the expression vector pAG3, the APP695nterm his was stably transfected into CHO cells. For purification, after 48 h serum-free media was collected and purified using Ni2+ affinity resin (Qiagen). To perform sAPP ELISAs, MaxiSorp 96-well immunoplates (Nunc) were coated overnight at 4 °C with 400 ng/ml purified recombinant sAPP in 100 µl of 0.1 M sodium carbonate buffer per well (pH 9.6). Plates were then blocked overnight at 4 °C with 1% Block Ace, 0.05% NaN3 in PBS (300 µl/well). Blocking solution was discarded, the plates washed twice with PBS, and the wells loaded with 40 µl of 1% bovine serum albumin in PBS (PBSB) and 10 µl of each sample, in duplicate. Standard samples, containing from 31.5 to 8000 ng/ml sAPP in PBSB, were also loaded in duplicate. The NH2-terminal APP antibody 207 was added (100 µl) to each well, diluted 1:32,000 in PBSB. Following overnight incubation at 4 °C, wells were washed twice with PBS, 0.05% Tween 20, twice with PBS, and then loaded with 100 µl of horseradish peroxidase-conjugated anti-goat IgG (1:5000 in PBSB; Rockland Immunochemicals). After a 4-h incubation at room temperature, plates were washed as above, then developed with 100 µl of TMB solution (Kirkegaard & Perry). The reaction was stopped after approximately 5 min with the addition of 100 µl of 6% phosphoric acid and the plates read at 450 nm. These conditions were determined empirically to optimize detection of sAPP in transfected cell lines in our laboratory, with typical expression levels placing sAPP concentrations in a linear region of the standard curve.

Abeta Sandwich ELISAs and Normalization of Abeta Measurements-- For determination of Abeta concentrations we used 3 well characterized sandwich ELISA systems. Total Abeta was determined by 3160 capture and detection with either 4G8 or BNT77 (9, 26), and Abeta 40 and 42 were determined by BAN50 capture and detection with either BA27 or BC05, respectively (5). To control for variance in transfection efficiency and the amount of APP that is appropriately inserted in the membrane and trafficked through the secretory pathway (thus APP available for gamma -secretase cleavage) Abeta levels were normalized to sAPP expression. This was accomplished by dividing the Abeta values (fmol/ml) by the sAPP values (ng/ml) resulting in a normalized Abeta value (fmol/ng). The normalized Abeta value for each mutant was then divided by the normalized Abeta value for APP695NL to give the %NL Abeta .

Metabolic Labeling and Immunoprecipitation of sAPP and Full-length APP-- 48 h after transfection, 293T cells were labeled for 2 h with 100 µCi/ml [35S]methionine. Immunoprecipitation of full-length APP (flAPP) with Ab369W (27) from Triton X-100 cell lysates and sAPP in the 2-h conditioned media with antibody 207 were carried out as described previously (28, 29). Each analysis was performed in duplicate. Immunoprecipitated proteins were separated on 10% Tris-Tricine gels. PhosphorImaging analysis of the dried gels was performed. After subtracting the number of pixels of flAPP or sAPP present in the mock transfected (vector alone) sample from each of the experimental samples, the ratio of sAPP/flAPP was calculated by dividing the pixels of sAPP by the pixels of flAPP. sAPP/flAPP ratios were then compared with the ratio of sAPP/flAPP calculated for APP695NL. TMD mutants showing sAPP/flAPP ratios greater than 50% of APP695NL ratio were assumed to have preserved processing in the secretory pathway.

Mass Spectrometric Abeta Analysis-- Serum-free media (Dulbecco's modified Eagle's medium) collected for a 24-h period beginning 24 h post-transfection was used for mass spectrometric analysis. Abeta peptides were analyzed by immunoprecipitation/mass spectrometric Abeta assay as described previously (4). The Abeta peptides were immunoprecipitated from 1.0 ml of conditioned media using monoclonal anti-Abeta antibody, 4G8 (Senetek, Maryland Heights, MO), and protein G Plus/Protein A-agarose beads (Oncogene Science, Inc., Cambridge, MA) and analyzed using a matrix-assisted laser desorption/ionization time of flight mass spectrometer (Voyager-DE STR BioSpectrometry Workstation, PerSeptive Biosystem). Each mass spectrum was averaged from 500 measurements and calibrated using bovine insulin as the internal mass calibrant. For comparing the peptide levels secreted by cells expressing different mutations and between the treatments of Me2SO and pepstatin, synthetic Abeta (12-28) peptide (10 nM) was used as internal standard.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Transmembrane Domain Mutations: Effects on Total Abeta Levels-- APP appears to be a substrate for gamma -secretase only after it has been cleaved by alpha - or beta -secretase (30). Because cleavage of APP by these activities requires normal membrane insertion and trafficking through the secretory pathway, we have only performed in depth analyses of APP TMD mutants that showed preserved processing by these activities and normalized Abeta production to sAPP levels. Processing in the secretory pathway was assessed by metabolic labeling experiments and determination of the sAPP/flAPP ratio in comparison to APP695NL as described under "Experimental Procedures" (data not shown). To examine Abeta production in the mutants that showed preserved processing in the secretory pathway, sAPP, total Abeta , Abeta 1-40, and Abeta 1-42 were measured in the conditioned media by ELISA. Comparison of normalized total Abeta shows that most mutants resulted in modest to moderate decrements in Abeta production (Table I). Even strikingly non-conservative mutations at residues 637 (Abeta 41) or 639 (Abeta 43) had relatively modest effects on total Abeta production. Basic (Lys) or acidic (Glu) amino acid substitutions at both sites, and substitutions of amino acids with a large hydrophobic side chain (Phe and Trp) or even a proline at position 637, did not prevent gamma -secretase cleavage. In addition, production of Abeta was only modestly decreased by most mutations at or near the lumenal transmembrane domain junction or by deletion or insertion mutants. The notable exceptions being the large decrements in Abeta production by del640-43, 624-626E, and ins625-631 mutants.

                              
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Table I
Summary of ELISA results for TMD mutants
Results of total Abeta ELISAs, standardized to sAPP levels (assayed in duplicate, mock transfection levels subtracted). Values shown are the average of three to five independent transfections for each mutation; control levels for APP695NL are determined from 17 transfections to ensure the most accurate estimate possible. %Abeta 1-40 and %Abeta 1-42 refer to the amount of Abeta 1-40/total Abeta *100 or Abeta 1-42/total Abeta *100, respectively. For APP695NL the values for %Abeta 1-40, and %Abeta 1-42 are expressed as mean ± SD. Statistical significance (*, p < 0.05; **, p < 0.01; ***, p < 0.001) was assessed by computing values from Student's t distribution by comparing each mutation to the control population of APP695NL transfections.

Six mutants (I637R, T639P, 649-651E, 649-651D, del625-631, and del640-647) showed dramatic decreases in Abeta levels (<5% of APP695NL Abeta , data not shown). Pulse-chase analyses showed that these mutations produced equivalent levels of full-length APP, but that the sAPP/flAPP ratio was markedly diminished (<1% compared with APP695NL, data not shown). Although the precise mechanism for the impaired processing of these mutations is not definitively established by these studies, based on previous studies (16) and our metabolic labeling data it is likely that these mutants either alter membrane insertion or prevent proper trafficking through the secretory pathway. In agreement with this, one of these mutations (649-651E) has previously been shown to result in a non-transmembrane, membrane-associated cell surface, full-length APP (31).

Effects on Cleavage Site-- Comparison of the relative amounts of Abeta 1-40 to Abeta 1-42, measured, respectively, by BAN50/BA27 or BAN50/BC05 ELISA, shows that many of these mutants dramatically alter the relative amounts of the major Abeta species produced. These data are expressed as the percent of total Abeta that Abeta 1-40 and Abeta 1-42 species represent (Table I). For the I637X mutants, detection of Abeta 1-42(43) species could be impaired as the 42(43) end specific BC05 detection antibody recognizes an epitope partially determined by the residue at Abeta 41; thus, altered Abeta 42 peptides could be produced but not detected (5). To illustrate how these data indicate shifts in cleavage it is useful to look at the effects on %Abeta 1-40 and %Abeta 1-42 in the ins625-628 mutant. Comparison of the %Abeta 1-40 and %Abeta 1-42 illustrates that the major shift in cleavage is a reduction in processing at the Abeta 40 cleavage site (2% of total versus 51% of total in the APP695NL construct) while Abeta 42 cleavage is preserved (4.6% of total versus 3.3% of total in the APP695NL construct). These analyses also show that only 7% of total Abeta is accounted for by Abeta 1-40 and 1-42 versus 54% in the APP695NL construct, indicating that the vast majority of Abeta peptides generated from this mutant are cleaved at a different site or sites than the ones normally utilized. By these criteria, almost all of the mutants show shifts in gamma -secretase cleavage. In the last column of Table I, the statistically significant shifts in cleavage for each of these mutations are summarized.

Mass Spectral Analysis-- In order to determine exactly how cleavage is shifted by the TMD mutations, Abeta produced from 293T cells expressing APP695NL or select TMD mutant APPs was analyzed by immunoprecipitation/mass spectrometric analysis (4). The monoclonal antibody, 4G8, was used to immunoprecipitate Abeta from serum-free conditioned media. This antibody recognizes Abeta 17-24 (32), a region of the Abeta not altered by the mutants used in this study, making it highly unlikely that this analysis would be biased by selective immunoprecipitation of different Abeta species. The molecular masses of various Abeta peptides were measured in these analyses by using internal mass calibrants, bovine insulin and Abeta 12-28 peptide. These masses were then used to identify Abeta peptides produced by the TMD mutant APPs and infer the gamma -secretase cleavage sites as illustrated in Fig. 3. The relative peak intensity was used to determine the relative abundance of Abeta peptides within each spectra resulting from each TMD mutant APP. For clarity, all Abeta peptides are numbered according to the cleavage sites in wild type Abeta species; thus Abeta 1-43 in del625-628 is a 39-amino acid Abeta peptide derivative. Representative mass spectra for APP695NL, I637F, I637P, and ins644-647 are shown in Fig. 3A.


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Fig. 3.   Mass spectral analysis of Abeta secreted by TMD mutants. A, representative spectra of Abeta produced by 293T cells expressing APP695NL (A), I637F (B), I637P (C), and ins644-647 (D) are shown. Spectra are normalized to the most abundant Abeta peptide species and peaks in the spectra are labeled with Abeta peptide sequence numbers based on the wild type Abeta sequence. Background peaks are labeled with an asterisk (*). B, mass spectrometric analysis of Abeta peptide secreted by the TMD mutants are schematically depicted. Abeta peptides detected in each mutant are indicated by arrows above the cleavage sites, while the height of the arrows indicates relative peak intensity. The large arrows indicate peaks with relative intensity greater than 60%, the medium arrows represent peaks with relative intensity between 20 and 60%, and the small arrows stand for peaks with relative intensity less than 20%. These results are averaged from multiple measurements.

The mass spectrum of Abeta produced from APP695NL shows that Abeta 1-40 was the major Abeta species and that the minor Abeta species were Abeta 1-42, Abeta 1-39, Abeta 1-38, and Abeta 1-37. Based on comparison to APP695NL, several of the different TMD mutations analyzed dramatically shift the gamma -secretase cleavage site. Mass spectrometric Abeta analyses of APP695NL and TMD mutants are schematically summarized in Fig. 3B. The largest shifts in cleavage site utilization are seen in the ins625-628 and del625-628 mutants, while less dramatic effects are seen with the del644-647 and ins644-647 mutations. G625K also increases long Abeta production, while G625P increases production of shorter Abeta peptides. Introduction of Lys at position 637 or 639 has only minor effects on cleavage, while substitution of Ala at 639 increases both long Abeta (Abeta 1-42) and short Abeta (Abeta 1-38) production. Pro or Phe substitutions at Ile637 dramatically shift cleavage away from Abeta 40. Finally, Abeta production is only minimally affected by the 640-648A mutant. Notably, the mass spectral data and the ELISA data are remarkably consistent with the exception being that small amounts of Abeta 1-42 detected by ELISA are not always detected by mass spectrometric analysis (for example, in 640-648A) due to the slightly lower detection efficiency of Abeta 1-42 (4).

Pepstatin Treatment of Selected Mutants-- Given the loose sequence specificity exhibited by gamma -secretase, we postulated that the gamma -secretase activity responsible for generating the various Abeta species was unlikely to be due to a single protease. Therefore, we treated cells transfected with several of the TMD mutants that shift cleavage with pepstatin, an inhibitor of normal gamma -secretase activity. Although it is not known whether pepstatin directly inhibits gamma -secretase, pepstatin treatment of cells transfected with APP COOH-terminal fragments LC99, APP695wt, or APP695NL results both in accumulation of COOH-terminal fragments and a decrease in Abeta production2 without significantly altering sAPP secretion in either APP695wt or APP695NL (Table II), consistent with it being an inhibitor of gamma -secretase activity. Because cells are rather impermeable to pepstatin, it is necessary to treat them with high concentrations (100 µg/ml) in the presence of 2% Me2SO in order to obtain effective intracellular concentrations (33). Again sAPP, total Abeta , and Abeta 1-40 and 1-42 were measured by ELISA from conditioned media. Treatment of cells with vehicle alone and vehicle with pepstatin had no overt toxic effect in the time course studied, and although changes in sAPP levels were observed in individual experiments in some mutants, none of these changes reached statistical significance (Table II). Pepstatin treatment of cells transfected with the APP695NL mutant decreased total Abeta and Abeta 1-40 equally while Abeta 1-42 was less affected. The effects on three of the TMD domain mutants: del625-628, G625P, and I637F were similar to the effect on APP695NL. In contrast, pepstatin treatment did not alter Abeta production from the I637P mutant to any significant extent. The effects of pepstatin on T639K, a mutant that had only subtle effects on cleavage site utilization, were also distinct from APP695NL. Abeta 1-40 was only modestly inhibited and Abeta 1-42 was not inhibited. Although Abeta 1-40 produced from ins625-628 represents only a minor fraction of the total Abeta produced, pepstatin actually increased the amount of Abeta 1-40 produced from 15 to 50 fmol/ml.

                              
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Table II
ELISA analysis of pepstatin treatment on select TMD mutants
Results for sAPP, total Abeta , Abeta 1-40, and Abeta 1-42 are expressed as % of control treatment (+ pepstatin treatment/Me2SO alone*100). Values shown are the average of three independent transfections treated either with Me2SO alone or Me2SO/pepstatin and are representative of several independent experiments. Statistical significance (*, p < 0.05; **, p < 0.01) was assessed by computing values from Student's t distribution by comparing the treated group versus the group treated with vehicle alone.

To definitively identify how pepstatin altered cleavage of these TMD domain mutants, Abeta secreted from treated and untreated cells was analyzed by mass spectrometry (Fig. 4). For APP695NL, G625P, I637F, and T639K, the relative peak heights of each of the major Abeta species was similar before and after pepstatin treatment, indicating that cleavage was inhibited equally at each site (Fig. 4, data shown only for APP695NL). Consistent with the ELISA analysis, the relative peak height of Abeta 1-42 was not reduced as much by pepstatin treatment in APP695NL and G625P. The effect of pepstatin treatment on del625-628 also revealed little difference in relative peak heights of the major Abeta species except that peaks corresponding to Abeta 1-45 and Abeta 1-46 were increased suggesting enhanced utilization of minor cleavage sites (Fig. 4, G and H). In the ins625-628 and I637P TMD mutants, there were some marked shifts in the relative peak intensities of some Abeta species after pepstatin treatment. In ins625-628 the Abeta 1-33 peak was decreased more than the Abeta 1-37 peak (Fig. 4, E and F), while in I637P the relative peak intensity of Abeta 1-37 decreased and the relative peak intensity of Abeta 1-43 increased (Fig. 4, C and D). Thus, there appears to be differential sensitivity of certain gamma -secretase cleavages to pepstatin and increased utilization of alternative sites by pepstatin-insensitive proteases in certain mutants when pepstatin-sensitive cleavage is inhibited.


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Fig. 4.   Mass spectral analysis of Abeta secreted by TMD mutants in the presence or absence of pepstatin. Immunoprecipitation/MS spectra of Abeta peptides secreted by 293T cells expressing APP695NL (A and B), I637P (C and D), ins625-628 (e and f), and del625-628 (G and H) proteins after treatments with either Me2SO (vehicle) (A, C, E, and G) or pepstatin (B, D, F, and H). For each pair of spectra, Me2SO versus pepstatin, the peak heights of Abeta peptides are first normalized to the internal standard, Abeta (12-28) (labeled as 12-28 (Std)). The spectra are then plotted using the relative peak intensities; e.g. the highest peak within the pair of spectra is plotted as 100%. In this way, the relative intensity of peaks between the two spectra can be compared. Peaks in the spectra are labeled with Abeta peptide sequence numbers based on the wild type Abeta sequence. Thus, Abeta (1-37) in ins625-628 is a 41-amino acid peptide whereas Abeta (1-43) in del625-628 is a 39-amino acid peptide, respectively. Background peaks and unidentified peaks are labeled with asterisk (*) and question marks (?), respectively.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

APP Transmembrane Domain Mutants Shift gamma -Secretase Cleavage-- An unanticipated result of this analysis is that many TMD mutations alter the major sites of gamma -secretase cleavage. The most striking finding resulting from the analyses of the mutations, ins625-628 and del625-628, is that the major gamma -secretase cleavage site appears to be determined by the length of the TMD lumenal to the normal gamma -secretase sites. In wild type APP, the major gamma -secretase site carboxyl to Abeta 40 lies 12 amino acids from the lysine at Abeta 28 (KAbeta 28) that is predicted to delineate the lumenal TMD boundary. In these mutations, the normal gamma -site is shifted either 4 amino acids (del625-628) closer or farther away (ins625-628) relative to KAbeta 28. Although the primary sequence surrounding the normal gamma -site is unaltered, the major cleavage shifts to the 13th amino acid (Abeta 37) from KAbeta 28 in ins625-628 or the 11th amino acid (Abeta 43) from KAbeta 28 in del625-628. Only a minor portion of Abeta produced from either mutant is cleaved at the V-I bond at Abeta 40 (14% in del625-628 and 2% in ins625-628). Mutations altering charge at the presumptive lumenal border of the transmembrane domain (625-626K, 624-626E, and 624-626D) had a similar effect as the del625-628 mutant increasing production of longer Abeta peptides. These mutants might be predicted to decrease the length of the TMD proximal to the gamma -secretase site by several amino acids resulting in increased cleavage at sites distal to Abeta 40.

The insertion and deletion mutants designed to alter the length of the TMD distal to the normal gamma -cleavage sites had much subtler effects. The major effect of the ins644-647 mutation was to decrease cleavage at Abeta 40 and increase cleavage at Abeta 38. The major effect of the del644-647 mutant was to modestly decrease Abeta 1-42 production. This is similar to the effect seen in the I637K and T639K mutants, where Abeta 1-42 production is decreased. In all three cases, increased positive charge has been placed closer to the normal Abeta 42 cleavage site. Replacement of residues 640-648 with alanine (640-648A) had only a minor effect on Abeta production shifting cleavage away from the Abeta 40 site (28% Abeta 1-40) indicating that this region is not as crucial in determining the membrane positioning of the gamma -secretase sites in comparison to the region amino to the gamma -secretase cleavage site. This finding is similar to a previous report showing that substitution of APP residues 635-642 or 636-653 with the corresponding residues of the epidermal growth factor receptor TMD did not significantly impair Abeta production (16). Nevertheless, alterations in this region do result in subtle, but important, shifts of gamma -cleavage. All FAD-linked mutations in this region, V641I, V641F, V641G, and I640V (based on the APP695 sequence), replace hydrophobic residues in the transmembrane domain with other hydrophobic residues and each results in increases in Abeta 42 (5, 17, 34; reviewed in Refs. 7 and 8). In this study, a similar substitution, T639A, increased Abeta 42 cleavage. This result is similar to a recent report demonstrating that hydrophobic substitutions at Abeta 43 increased Abeta 42 production relative to Abeta 40 (17). However, that study only looked at ratios of Abeta 42:Abeta 40; thus, alternative cleavages would have been missed, and it is unclear from the data presented whether the increase in the ratio is due to a decrease in Abeta 40 production or an increase in Abeta 42 production. Of the mutations on the carboxyl side of the gamma -site, only del640-643 had dramatic effects on Abeta , significantly decreasing total Abeta production and markedly increasing %Abeta 1-42 production. This mutant's more dramatic effect compared with deletions or insertions downstream could be explained by the fact that this region may represent the end of the APP TMD (16). Taken together, these data indicate that mutations in the lumenal portion of the TMD have effects on cleavage that are much more profound than mutations which alter charge or TMD length on the cytoplasmic side of the normal gamma -secretase site.

gamma -Secretase Is Not a Single Proteolytic Activity-- Our data shed some light on the proteolytic mechanism responsible for generation of Abeta peptides of various lengths. The finding that the del625-628 preferentially secretes an Abeta species of 39 amino acids ending at Abeta 43 is evidence against a mechanism in which a single endoprotease cleavage occurs carboxyl to the major gamma -sites followed by trimming by carboxypeptidase. However, for any given mutation, we cannot rule out the possibility that shorter Abeta species are generated through carboxypeptidase degradation of longer Abeta peptides. Furthermore, the differential effect of pepstatin on select TMD mutants is most consistent with the action of at least two and possibly more proteolytic activities. This differential inhibition is most clearly demonstrated in the I637P mutant. This mutant is readily processed into Abeta but is almost completely pepstatin insensitive. Mass spectral analysis indicates that the gamma -secretase that generates Abeta 1-43 in the I637P mutant is pepstatin-resistant, while a different gamma -secretase that cleaves this mutant at Abeta 1-37 is pepstatin-sensitive. When cleavage is inhibited at Abeta 37 by pepstatin a corresponding increase is seen in the cleavage at Abeta 43, resulting in almost identical amounts of total Abeta secreted. Similarly, other TMD domain mutants as well as APP695NL exhibit both a pepstatin-sensitive and a pepstatin-resistant gamma -secretase activity.

Previous reports based on differential inhibition of Abeta 40 and Abeta 42 cleavage have indicated that these Abeta species may be generated by distinct proteases. Unfortunately, interpretation of these previous studies has been difficult. The compound, MDL 21760, used in several studies could have altered trafficking of APP as it markedly increased sAPP (35, 36). In other studies, reporting an Abeta 1-40 inhibitory effect of calpain 1 inhibitor, no controls for either APP synthesis or sAPP production were reported (37, 38). In fact, only one potential gamma -secretase inhibitor, a substrate-based difluoroketone compound, altered gamma -secretase activity without apparent alteration of other secretase activity (39). In this study, pepstatin had no toxic effects and no significant effect on sAPP production from APP695NL; yet, it was a less effective inhibitor of Abeta 1-42 (~35% of control) production than Abeta 1-40 (~20% control).

The differential inhibition observed at Abeta 40 and Abeta 42 sites could be due to a number of mechanisms. Since Abeta 1-42 has previously been shown to be specifically generated in the endoplasmic reticulum (although endoplasmic reticulum-derived Abeta 1-42 is not secreted), it is possible that this difference simply reflects a varying degree of organelle penetrance (a similar argument could be made for any of the other published inhibitors) (40-42). Alternatively, this differential inhibition could be viewed as additional evidence that multiple proteases generate both Abeta 40 and Abeta 42, but that pepstatin-sensitive cleavage is responsible for a higher percentage of activity at the Abeta 40 site. The finding that differential inhibition of Abeta 1-40 and Abeta 1-42 was constant among several of the TMD mutants which alter cleavage site preference (del625-628, G625P, I637F), but not the T639K mutant supports this notion of multiple proteases as it is most consistent with enhancing the Abeta 40 and Abeta 42 cleavages by a pepstatin-insensitive protease. Based on our results, we would predict that for wild type substrate a pepstatin-sensitive protease cleaves both Abeta 40 and Abeta 42, with preferential specificity for cleavage at Abeta 40 and that additional pepstatin-insensitive proteases account for ~20% of the cleavage events at Abeta 40 and ~35% of the cleavage events at Abeta 42. Thus, when the major Abeta 40 cleavage site is "protected" (see below) from cleavage in the del625-628 mutant (14% Abeta 1-40 versus 51% in APP695NL, Table II) and shifted to Abeta 1-42(43) (48% versus 3.3% in APP695NL, Table II), a remarkably similar degree of inhibition is seen on both species following pepstatin treatment. A final mechanism in which a single pepstatin-sensitive protease cleaves both sites but is differentially inhibited could also be considered. This mechanism, however, is not consistent with known models of proteolytic inhibition by pepstatin and would be an unprecedented mechanism of action for a protease inhibitor that acts in a competitive fashion.

Implications for Models of gamma -Secretase Cleavage: the Length of the Lumenal Portion of the TMD Is the Prime Determinant of Cleavage-- Until the gamma -secretases are definitively identified and the location of the residues at the time of cleavage defined, the issue of intramembranous proteolysis is likely to remain controversial. Unless gamma -secretases are completely novel proteases that can cleave proteins in the hydrophobic environment of the membrane, mechanisms must exist that permit membrane-embedded regions of polypeptides to transfer from the lipid bilayer into a hydrophilic proteinaceous environment that supports proteolysis. This could occur in one of two fashions. First, as has been generally proposed, gamma -secretase activity may be due to the action of proteases or proteolytic complexes that are capable of creating a pore or hydrophilic pocket within the membrane. Based on our data, in this model the interaction of the protease with the TMD domain of the APP lumenal to the gamma -secretase site would appear to be a critical factor in determining cleavage (see Fig. 5A). A second possibility is that these cleavages require translocation of the cleavage sites out of the membrane (Fig. 5B). One of the similarities between the gamma -secretase-like cleavages in APP, Notch and SREBP, is that the potentially intramembranous cleavage occurs only after an initial cleavage or cleavages that remove significant portions of the protein amino-terminal to the intramembranous cleavage site. Thus, it is possible that the sites are normally protected by the membrane prior to the primary lumenal cleavage of the holoprotein. Once the lumenal domain is cleaved, the protein either assumes a different conformation or is actively translocated resulting in exposure of the intramembranous site to the cytoplasm where it can be cleaved. Such a cut-expose-cut model for the APP is illustrated in Fig. 5B. In this model one could envision slippage or translocation of the intramembranous site in either direction; for APP we propose that the gamma -site is exposed to proteases that are associated directly or indirectly with the membrane and have active sites facing the cytoplasm. This model, while subject to some potential thermodynamic penalties associated with translocation of the APP TMD, is consistent with our data showing that length of the TMD lumenal to the gamma -cleavage site has more profound effects on gamma -secretase cleavage than mutations carboxyl to the normal gamma -sites, offers a very simple explanation as to why these sites are resistant to proteolysis in the intact holoprotein, is consistent with another model of intramembranous cleavage (21), and does not implicate an unprecedented type of proteases in this cleavage.


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Fig. 5.   Models of gamma -secretase cleavage. This figure illustrates two potential models for gamma -secretase cleavage. A, intramembranous cleavage. In this model, the APP COOH-terminal fragment generated after beta -secretase cleavage associates with a protease whose active site lies within the membrane. Based on our data, the interaction of the intramembranous protease with the TMD domain of the APP lumenal to the gamma -secretase site would appear to be a critical factor in determining cleavage. Thus, del625-628 and ins625-628 mutants would alter the residues in contact with the active site resulting in different cleavages. B, a cut-expose-cut model. In this model, prior to cleavage by alpha - or beta -secretase, the gamma -site is buried within the membrane, inaccessible to proteolysis. After cleavage to release sAPP, the APP COOH-terminal fragment location within the membrane is altered to expose the gamma -site to the cytoplasm where it could be cleaved by either cytoplasmic proteases or membrane-associated proteases with active sites facing the cytoplasm. This translocation could occur because this is the preferential conformation of the APP COOH-terminal fragment within the membrane or it could be facilitated by interaction with PS, gamma -secretase itself, or other transmembrane proteins. Such a model provides a simple explanation for the cleavages observed in the del625-628 and ins625-628 mutant APPs. Based on this model, thickness of the membrane could influence cleavage site or alternatively interaction with other proteins in the membrane, such as PS, could alter the exposure of the gamma -site. Because the transmembrane region of a protein is not static within the membrane, it is likely that in either model there is some resonance of the COOH-terminal fragment with respect to the membrane. Such resonance could result in either different residues presented to the active site of an intramembranous protease or exposure of different residues to the cytoplasm, which could account for the invariable production of "ragged" ends. Scissors indicate proteolytic events. beta  and gamma  indicate beta - and gamma -secretase cleavage, respectively.

In either model proposed above, it is apparent that the prime determinant of gamma -secretase cleavage is the length of the transmembrane domain proximal to the gamma -secretase cleavage site. In an intramembranous cleavage model, we would predict that the position of the active site of the protease is relatively fixed within the membrane and that the protease recognizes determinants in the APP TMD lumenal to the normal cleavage site. Thus, varying the length of the lumenal TMD alters the residues that contact the active site. In contrast, the membrane serves as the delineating factor in the cut-expose-cut model, residues buried within the membrane are protected from cleavage while exposed residues are cleaved.

Because at least two, and possibly more, proteolytic activities appear to contribute to gamma -secretase cleavage, defining the substrate specificity is problematic until the secretases are definitively identified. Nevertheless, it is almost certain that any individual gamma -secretase would exhibit at least some sequence specificity as even nonspecific proteases such as proteinase K preferentially cleave certain substrates (43, 44). Consistent with this, altering sequence at the normal gamma -cleavage site has dramatic effects on recognition of the normal cleavage site by the major pepstatin-sensitive gamma -secretase activity which results in either increased cleavage by a pepstatin-sensitive protease at sites other than Abeta 40 and Abeta 42, increased cleavage by a pepstatin-insensitive protease at normally utilized sites, increased cleavage by a pepstatin insensitive protease at Abeta 40 and Abeta 42, or a combination of these alterations.

Multiple Cellular Factors Could Influence gamma -Secretase Cleavage-- If gamma -secretase cleavage is primarily dependent upon the location of the gamma -secretase cleavage site with respect to the membrane or active site of a protease within the membrane, then it is likely that a number of cellular factors could influence this cleavage including membrane thickness or composition and interaction with other proteins. PSs, which are important regulators of gamma -secretase activity (reviewed in Refs. 7 and 8), could influence gamma -secretase cleavage of APP by altering trafficking of APP to different cellular microdomains where cleavage would be influenced by membrane thickness, by directly interacting with APP carboxyl-terminal fragments and positioning them within the membrane, by translocating the gamma -site out of the membrane, or by altering the position of gamma -secretase with respect to the APP transmembrane domain. Alternatively, the possibility that PSs, which do not resemble any known protease, could in fact be gamma -secretase has not been excluded, in which case altered interaction between PS and APP could provide a simple explanation for shifts in cleavage induced by FAD-linked PS mutants and would be entirely consistent with decreased production of Abeta from PS 1 knockout mice (45).

Conclusions-- gamma -Secretase appears to represent a membrane protein secretase defined as a proteolytic activity that cleaves a proteolytically sensitive region of a transmembrane protein resulting in secretion of the proximal portion of the cleaved protein (46). Compared with other secretase activities, gamma -secretase activity appears to be distinct in that it does not cleave its substrate within an extracellular/lumenal "stalk" like region proximal to the membrane, but rather within a hydrophobic region purportedly within the TMD of the APP COOH-terminal fragment. The demonstration in this study that the gamma -secretase is not a single proteolytic activity and that the membrane plays a critical role in determining gamma -secretase cleavage of APP will be important in developing strategies for isolation of the various gamma -secretase activities, which remain a major therapeutic target in AD. Additional studies will be needed to determine whether the pathological shifts in Abeta cleavage are caused by the alterations in the major pepstatin-sensitive gamma -secretase activities or by additional proteases, which might play a role in pathogenic processing. Given that the common effect of all early onset FAD-linked mutations is to modestly increase long Abeta production, this study offers important insight into how various Abeta peptides are generated and suggests possible mechanisms whereby various FAD-linked mutations might shift gamma -secretase cleavage. Because other biologically important cleavage events in SREBP and Notch may be intramembranous, it will be important to perform similar studies on such proteins to determine if similar, atypical, proteolytic mechanisms are responsible for such cleavages. Together, these studies should also offer additional insights into the important question of how transmembrane domains, in general, are degraded.

    ACKNOWLEDGEMENTS

We thank T. Dechert, J. Shockely, D. Yager, and T. Smith for excellent technical assistance. Antibodies 207 and 369W were kindly provided by B. Greenberg and S. Gandy, respectively. BC05, BAN50, and BA27 were gifts of Takeda industries.

    FOOTNOTES

* This work was supported by a Beeson Award from American Federation for Aging Research (to T. G.) and National Institutes of Health/National Institute of Aging Grant AG-16065 (to R. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel To whom correspondence should be addressed. Tel.: 904-953-2538; Fax: 904-953-7370; E-mail: tgolde{at}mayo.edu.

2 C. Eckman, unpublished results.

    ABBREVIATIONS

The abbreviations used are: Abeta , amyloid beta  protein; APP, amyloid beta  protein precursor; AD, Alzheimer's disease; FAD, familial Alzheimer's disease; NL, APPK670N,M671L mutation (numbering based on APP770 isoform); TMD, transmembrane domain; PS, presenilin; Me2SO, dimethyl sulfoxide; KAbeta 28, lysine at Abeta 28; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; PBSB, phosphate-buffered saline with bovine serum albumin; flAPP, full-length APP; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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