HEK293 cells were transfected with the β-secretase candidates ASP-1 or ASP-2 pcDNA3.1mycHis clones and selected for stable transfection with Geneticin (800 μg/ml). HEK293 cell lines have been used by others to overexpress these enzymes 3435 and have also been used to study the effects of β-secretase cleavage on recombinant APP 3336. Cell lysates from positive clones were analysed by SDS-PAGE and western blotting with an anti-myc antibody to detect the recombinant ASP-1 and ASP-2 proteins. The myc epitope is encoded in frame downstream of the C-terminal of the proteins followed by a 6-Histidine tag for affinity purification. Both recombinant ASP-1 and ASP-2 were expressed and detected by western blotting as 55–70-k and 65–80-k bands, respectively, higher than the estimated molecular weights of the pro-apoproteins (53.4 and 53.7-kDa including the pro-peptide domains), mainly due to glycosylation as predicted by their amino acid sequences and shown in recent reports by Charlwood et al., 2001. Recombinant ASP-2 was purified by Ni²⁺-affinity chromatography. Silver-stained SDS-PAGE confirmed the purity of ASP-2 from other contaminants as seen in figure 1A; lower bands (dotted arrows) seen in the purified preparation are due to artefactual materials also present in the negative control sample (note the presence of higher molecular weight forms of ASP-2). Immunoblotting of the same preparation of ASP-2 detected with the anti-myc antibody, (figure 1, 1B) confirms purification of ASP-2. The most prevalent of these ASP-2 proteins was a 75-k band. We used an affinity-purified antibody raised against the C-terminal peptide of ASP-2 (anti-ASP-2) to characterise the recombinant proteins. This antibody was specific for ASP-2 and did not cross-react with ASP-1 or other cellular proteins as characterised by ELISA and western blotting.

Removal of N-linked oligosacharides increased the relative mobility of ASP-2 from 65–80-k to 50–60-k, a size resembling the estimated molecular weights of the core protein confirming glycosylation as reported by others either by suppression of N-glycosylation or deglycosylation 1834. When increased amounts of protein were loaded (as in figure 2, 2A) the anti-ASP-2 IgG detected ASP-2 bands of higher Mw, larger than 200-k or at Mr~140-k, shown with triple and double arrows, respectively; the 140 k band was also visible by silver staining as indicated by the double arrow in figure 1A. These correspond in size to three or two ASP-2 molecules, respectively, implying ASP-2 oligomerisation into a trimer and dimer. The sensitivity of the putative dimer (140-k) to PNGase resulted in a reduction of size by ~30-k, to yield a band migrating as 100-k, equivalent to the behaviour of 2 linked monomers. Reduction with DTT and β-mercaptoethanol (figure 2, 2B) almost completely removed the ~200-k protein and increased the intensity of the 75-k band, but failed to affect the appearance of the putative dimer (140-k) suggesting strong intramolecular interactions. The oligomers were visible in freshly produced and purified ASP-2, though the 200-k protein was more prominent (data not shown) upon longer storage and at higher protein concentrations indicating that this was partly due to non-specific aggregation. An additional observation was the delayed mobility of ASP-2 in reducing conditions (figure 2, 2B) which is possibly due to loss of globular structure during reduction of intramolecular disulphide links described previously by Haniu et al., 2000.

We investigated the processing of ASP-2 at different pH values, since self-activation of other aspartic proteases such as pepsinogen 23 has been shown to be sensitive to pH. Pure ASP-2 was dialysed into a buffer of ph 5 or pH 8.5, immediately following purification. Incubation of the protein at room temperature was carried out prior to PAGE analysis. A sample before dialysis was also analysed alongside the ASP-2 at pH 8.5 and ph 5. A western blot analysis of these samples, depicted in figure 3, 3A, reveals a small shift in molecular weight of 3 k for the monomeric band and ~8 k for the dimer at ph 5. This was the first indication that cleavage of the pro-peptide (2.56 k) was occurring, which was also mirrored, by increased mobility in the putative dimer. PNGase treatment of the ph 5 and pH 8.5 ASP-2 samples revealed the deglycosylated proteins to be resolved in 3 and 2 bands (figure 3, 3B), respectively. The bands of the ph 5 sample were smaller (~3-k) than the ASP-2 bands in the pH 8.5 sample. The fact that PNGase did not reduce the ASP-2 protein to a single band of the unmodified protein is probably due to other modifications mentioned earlier, such as palmitoylation 20 and phosphorylation 1819. The triplet at ph 5 is consistent with deglycosylated proteins with the pro-segments removed (processed), with and without additional modifications. The highest band is possibly non-processed deglycosylated protein, because its intensity decreases upon incubation at 37°C.

Pure ASP-2 that had been incubated at ph 5 after adjustment of the pH with acetic acid was analysed by mass spectrometry to further detect the cleaved pro-peptide (figure 4). The sample dialysed at ph 5 could not be used since the small, cleaved fragments would have been dialysed out of the 4 k-exclusion membrane. Compared to pH 8.5 (profile B), the sample at ph 5 (profile A) displays two distinct peaks of size 1094 and 1352 Da. The peak at 1094 corresponds closely to the estimated m/z of the N-terminal fragment of the pro-sequence namely, QHGIRLPLR (1089Da) (the N-terminal fragment from the Pro-sequence from which the N-terminal T had been removed-as found by N-terminal sequencing-see below, P1) and the peak at 1352 corresponds to a dehydrated form (-18 mass) of the penultimate N-terminal fragment of the Pro-sequence, P2 SGLGGAPLGLRLPR (1364 Da) respectively.

The mass spectra indicated firstly that pro-peptide cleavage occurs readily at ph 5 at two sites within the pro-domain. N-terminal sequencing of these protein preparations revealed the presence of two sequences in the pH 8.5 preparation, namely ETDEEPEE (mature protein starting at E46), and QHGIRL (immature protein with an intact pro-domain starting at Q23). The presence of protein without its pro-domain, within the pH 8.5 sample, is not surprising as this protein was purified from the cells which themselves are expected to process nascent ASP-2. In contrast, Edman degradation of the preparation at pH 5 gave the sequence ETDEEPEE, predominantly matured protein, which was produced by processing in vitro following purification and dialysis -at low pH, suggesting that pro-peptide cleavage of ASP-2 can occur at two sites by autocatalysis at ph 5 in vitro. To further ascertain autocatalytic cleavage we added EDTA to inhibit endogenous cleavage by furin-like proteases as well as the BACE inhibitor (H-Lys-Thr-Glu-Glu-Ile-Ser-Glu-Val-Asn-Sta-Val-Ala-Glu-Phe-OH, Bachem, UK) to the incubations at ph 5. Mass spectrometric analysis of those samples (figure 5) showed that EDTA had no effect on the pro-peptide cleavage of ASP-2 as the previously identified peaks of 1094 and 1352 Da are still present in this sample as they are in the control which did not contain any inhibitor. Most importantly incubation of ASP-2 with the BACE inhibitor resulted in the disappearance of these peaks suggesting that pro-peptide cleavage is a result of autocatalysis.

The activity of recombinant ASP-2 was assessed using the Swedish mutation APP (APPswe) purified using the anti-CT15 IgG from stably transfected HEK293 cells. Initially [³⁵S]-labelled C-terminal APPswe polypeptides were isolated by immunoprecipitation with anti-CT15 IgG and protein-A-agarose and resuspended in reaction buffer (pH 8.5) prior to the addition of ASP-2. Figure 6, 6A, depicts an autoradiogram of a 16.5% Tris-Tricine gel where APPswe was incubated with or without ASP-2. In the absence of ASP-2 we noted the presence of a small band of ~9.25 (± 0.6, n = 3)-k corresponding to the C-83 APPswe fragment produced by endogenous α-secretase cleavage. The addition of ASP-2 resulted in an additional band at ~12.4-k consistent with the estimated size of the C-terminal stub after cleavage of β-secretase at the Aβ (1–40/42) site. To confirm the identity of this band the same reactions were carried out with non-radiolabelled substrate and the proteolytic fragments were detected by western blotting using either the anti-CT15 antibody (figure 6, 6B top panels) or the 6E10 (figure 6, 6B bottom panels) antibody, specific for the N-terminal of Aβ. These reactions were carried out both at optimum pH for aspartic protease activity, pH 5, as well as at pH 8.5 for comparison and the whole sample was analysed. ASP-2 was also pre-incubated at the respective pH. The arrows indicate the α-secretase cleaved fraction to be present in both pH 8.5 and ph 5. Two fragments (12.39 (± 0.24, n = 3) and 10.43 (± 0.45, n = 3)-k, designated β₁-C100 and β₂-C90, respectively) appear from ASP-2 cleavage at ph 5, consistent with fragments occurring by β-secretase cleavage at the Aβ (1–40/42)-producing β₁ site starting with D1, and the E11, β₂-site resulting in Aβ (11–40/42). Incubation of the purified recombinant ASP-2 with the APPswe at ph 8.5 also produced cleavage at the β₁-site as only the 12.39 k band is visible. The 12.4-k band was also labelled by the 6E10 antibody further confirming that it is the result of APPswe cleavage at the β₁-site. These results reveal that the activity of ASP-2 is influenced by pH which apparently governs the site at which ASP-2 cleaves the APPswe protein.

Culture media and antibiotics were from Life Technologies, Paisley, UK. All culture plasticware, were from Nalge Nunc International, Loughborough, UK. Amino Acids for peptide synthesis were from Nova Biochem, Nottingham, UK. Freunds Adjuvant, standard chemicals, 9E10 clone ascites fluid (anti-myc), Triton X-100, Nonidet P40, and Kodak X-Omat film were purchased from Sigma-Aldrich Company Ltd, Dorset, UK, 6E10 (anti-Aβ) from ID labs, P.O. Box 3556, Glasgow, Scotland, United Kingdom. C-18 ZIP tips, Centricon-30 concentrators, PVDF membrane were purchased from Millipore. Automated N-terminal peptide sequencing was carried out by AltaBiocscience, University of Birmingham, Edgbaston, UK. All secondary horseradish peroxidase-linked anti-species IgG, Enhanced chemiluminescence reagents, ECL Hyper film, Superdex 200HR, Sephadex G-10, were from Amersham Pharmacia Biotech, UK Ltd, Buckinghamshire, UK, whereas the Ni²⁺-affinity resin NiNTA was from Novagen, Nottingham, UK. Pfx2 Lipids were initially purchased from Invitrogen Life Technologies, Paisley, UK. Ready-made Tris-Tricine gels and low molecular weight Kaleidoscope Polypeptide Standards were from BIORAD, Hemel Hempstead, UK. Prestained protein molecular weight markers and Acrylamide mixture (30% acrylamide: 0.8% (wt/vol) bisacrylamide) from National Diagnostics, East Riding of Yorkshire, UK. Protease Inhibitors and N-glycosidase-F were purchased from Roche Molecular Biochemicals, East Sussex, UK.

pCEP4APP695 Hygromycin resistant, APPswe Neomycin resistant. pcDNA3.1MycHis (A) ASP-1 and 2 were kindly donated by the GlaxoSmithKline laboratories, Harlow, UK. The clones were engineered in frame with the Histidine tag and the Myc epitope, which in that order are downstream of the coding sequence of the Aspartic proteases (ASP).

For the production of stable colonies HEK293 cells were transfected using pFX2 lipids as described by the manufacturers for 2.5 × 10⁴ cells per transfection reaction. Positive transfectants were identified and selected using Geneticin. Colonies were screened by SDS-PAGE and Western Blotting analysis of cell lysates prepared by direct suspension of the cells in SDS, 0.1% (wt/vol) followed by sonication and suspension in gel loading buffer. HEK293 cells were cultured and propagated as described in Frears et al., 1999. Cell lysates for affinity- or immuno-purification were prepared by lysis of the cells with frequent agitation for 2 hrs at 4°C in phosphate buffered saline (PBS: 150 mM NaCl 2.7 mM KCl, 10 mM Na2HPO4 and 1.75 mM KH₂PO₄, at pH7) supplemented with protease inhibitors, 0.5 mM EDTA, 1 μM Leupeptin, 1 μM Pepstatin and 1 mM PMSF, and Triton X-100, 1% (wt/vol) and NP40, 1% (wt/vol). For affinity purification of ASP-2 EDTA and pepstatin were omitted. Prior to purification the lysate was diluted to 0.5% (wt/vol) detergent with the above buffer/inhibitor solution. Ni²⁺ – affinity purification was carried out as recommended by the manufacturers using a pre-charged Ni²⁺ column, 1 ml of settled bed resin for 50 × 10⁶ cells with minor alterations; all binding and washes (with increasing imidazole concentrations, pH 7.4) were carried out in PBS buffer supplemented with 0.2% (wt/vol) NP40 to reduce non-specific binding. Elution was achieved with 300 mM imidazole/0.2% (wt/vol/) detergent.

Freshly purified ASP-2 was dialysed immediately into 50 mM CH₃COONa, 20 mM NaCl, 0.2% (wt/vol) NP-40, at either pH 8.5 or 5, at 4°C overnight, in the presence of 1 μM Leupeptin and 1 mM PMSF. The protein solution was then incubated at room temperature for two hours before SDS-PAGE, in vitro cleavage reactions, or N-terminal sequencing. For Mass Spectrometry the proteins were first dialysed in the above buffer at pH 8.5, and then the pH was adjusted with dilute acetic acid to ph 5. For N-terminal peptide sequencing the samples were immobilised on a PVDF membrane.

Anti-CT15 (2 μg/ml) was used to immunoprecipitate (Stephens and Austen 1996) APPswe from HEK293 cell lysates/extracts prepared as above. The pellet was resuspended in reaction buffer (20 mM CH₃COONa, ph 5, 50 mM NaCl, 0.2% NP40, 1 mM PMSF and 1 μM Leupeptin) prior to the addition of ASP-2 (purified and pre-incubated in reaction buffer at designated pH). Reactions were carried out for 2 hours at 37°C. Samples were analysed by SDS-PAGE on a 16.5% Tris-Tricine gel as described by the manufacturers followed by western blotting and detection with anti-CT15 IgG (0.5 μg/ml) or 6E10 (4 μg/ml).

Purified ASP-2 which had been dialysed at pH 8.5 as described above was pre-incubated in the reaction buffer titrated at the designated pH without detergent prior to absorption to a C-18 ZIP tip (Millipore) equilibrated with 0.1% (vol/vol) trifluorocetic acid, (TFA). Peptides were eluted with 50% (vol/vol) acetonitrile and analysed on an Axima-CFR KRATOS Mass Spectrometer after addition of an equal volume of 10 mg/ml α-cyano-4-hydroxycinnamic acid (Sigma) in 50% (vol/vol) acetonitrile. Angiotensin 1, the dimeric form of α-cyano-4-hydroxycinnamic acid, and neuropeptide Y were used as external calibrants spotted on an adjacent spot on the chip.

The C-terminal peptide of ASP-2 (CLRQQHDDFADDISLLK residues 482–501) was synthesised on a Milligen 9050 synthesizer using Fmoc N-terminal protection and, after deprotection and release from resin was purified by HPLC on a column of Vydac C4 with gradients of acetonitrile in 0.1% TFA. In brief, bovine thyroglobulin was activated with succinimidyl 4-(N-maleimido-methyl) cyclohexane-1-carboxylate, desalted on a Sephadex G-10 column and coupled to the HPLC-purified synthetic peptide utilising its free-cysteine as described in 45. The coupled peptide was used to immunise rabbits and its immunoreactivity was screened by ELISA using immobilised antigenic peptide. Reactive serum was purified on a peptide-conjugated Sepharose column, aliquoted and stored at -20°C. Pure anti-ASP-2 IgG was tested for its specificity towards ASP-2 by western blotting of extracts of recombinant clones as well as mock-transfected cells using purified IgG pre-incubated or not with excess antigenic peptide.

80% Confluent flasks of HEK293 APPswe-trasfected cells (~5 × 10⁶ cells) were depleted of Methionine by incubation in Met-free media supplemented with FCS and Glutamax and pyruvate for 1 hr. The same media was then supplemented with [³⁵S]-Met at 50 μCi/ml in 5 mls, and incubated for 3 hrs. Cells were first rinsed with PBS and then lysed and extracted as described earlier. The [³⁵S]-labelled protein was immunoprecipitated as described earlier and reactions with ASP-2 were carried out. These were analysed by 7% SDS-PAGE, soaked in Amplify^TR for 15 minutes and dried before exposure to Kodak X-Omat film.

Proteins were first denatured by boiling in SDS sample buffer (0.25 M Tris-HCl, pH 8.8, 2.2% (wt/vol) SDS, 10% (v /v) Glycerol, 0.05% (wt/vol) bromophenol blue) with or without reducing agent (1% β-mercaptoethanol and/or 10 mM DTT) and electrophoresed on SDS-PAGE gels prepared as described by the manufacturers of the acrylamide solution (National Diagnostics) and run using a BioRad Mini-Gel system at 20 mA per gel. The gels were subsequently transferred onto PVDF membrane using a Biorad semi-dry blotter in 25 mM Tris, 192 mM Glycine, and 20% (vol/vol) methanol. Antibody detection was carried out by immunoblotting as described in Stephens and Austen (1996). For silver staining, gels were fixed with methanol, 40% (vol/vol) and acetic acid 10% (vol/vol), for one hour, before they were soaked for 30 min in DTT (0.5 μg/ml). The gels were rinsed twice with water and soaked for one hour in AgNO₃, 0.1% (wt/vol) prior to development with a solution of Na₂CO₃, 3% (wt/vol) and formaldehyde, 0.0185% (vol/vol).

Cellular extracts or purified ASP-2 were first heat-denatured in the presence of 0.5% (wt/vol) SDS in PBS, pH8 and the rest of the procedure was carried out as described by the manufacturers.