Báo cáo Y học: Enzymic properties of recombinant BACE2 pdf

Enzymic properties of recombinant BACE2 Yong-Tae Kim 1 , Deborah Downs 1,2 , Shili Wu 1,2 , Azar Dashti 1,2 , Yujun Pan 1 , Peng Zhai 1,2 , Xinjuan Wang 1,2,3 , Xuejun C. Zhang 1 and Xinli Lin 1,2,4 1 Functional Proteomics Laboratory and Crystallography Program, Oklahoma Medical Research Foundation, Oklahoma City, USA; 2 ProteomTech, Inc., Oklahoma City, USA; 3 Department of Biochemistry and Molecular Biology, Peking University Health Science Center, Beijing, China; 4 Department of Pathology, University of Oklahoma Medical Center, Oklahoma City, USA BACE2 (Memapsin 1) is a membrane-bound aspartic pro- tease that is highly homologous with BACE1 (Memapsin 2). While BACE1 processes the amyloid precursor protein (APP) at a key step in generating the b-amyloid peptide and presumably causes Alzheimer’s disease (AD), BACE2 has not been demonstrated to be directly involved in APP pro- cessing, and its physiological functions remain to be deter- mined. In vivo, BACE2 is expressed as a precursor protein containing pre-, pro-, protease, transmembrane, and cyto- solic domains/peptides. To determine the enzymatic prop- erties of BACE2, two variants of its pro-protease domain, pro-BACE2-T1 (PB2-T1) and pro-BACE2-T2 (PB2-T2), were constructed. They have been expressed in Escherichia coli as inclusion bodies, refolded and purified. These two recombinant proteins have the same N terminus but differ at their C-terminal ends: PB2-T1 ends at Pro466, on the boundary of the postulated transmembrane domain, and PB2-T2 ends at Ser431, close to the homologous ends of other aspartic proteases such as pepsin. While PB2-T1 shares similar substrate specificities with BACE1 and other ÔgeneralÕ aspartic proteases, the specificity of PB2-T2 is more con- strained, apparently preferring to cleave at the NH 2 -terminal side of paired basic residues. Unlike other ÔtypicalÕ aspartic proteases, which are active only under acidic conditions, the recombinant BACE2, PB2-T1, was active at a broad pH range. In addition, pro-BACE2 can be processed at its in vivo maturation site by BACE1. Keywords: Alzheimer’s disease; b-amyloid precursor protein; BACE2; propeptide processing enzyme; b-secretase. Most genetic and pathological evidence indicates that the formation of b-amyloid plaques in the brain is a major pathological event in Alzheimer’s disease (AD) [1,2]. The plaques are formed by aggregated b-amyloid peptides (Ab), which are produced from proteolytic cleavages of the b-amyloid precursor protein (APP) by two proteases known as b-andc-secretases. The activity of c-secretase is believed to be either a protease regulated by presenilin-1 (PS1) or PS1 itself [3,4]. APP cleavage by b-secretase is believed to be the rate-limiting step in Ab production and therefore one of the most promising pharmaceutical targets for treating AD [5,6]. Recently, b-secretase has been positively identified as a new transmembrane aspartic protease, BACE1 (Memap- sin 2), by several laboratories [6–10]. Its three-dimensional structure complexed with an inhibitor has also been determined [11]. These findings provide new opportunities to design inhibitor drugs against this enzyme for the prevention and treatment of AD. Newly published results on BACE1-deficient mice [12,13] demonstrate two facts: first, no detectable Ab peptide has been produced in the brain of the BACE1 –/– mice, and second, the BACE1 –/– mice appear normal in the observation period of more than 1 year [12]. These results further support the contention that BACE1 is a strong candidate as a therapeutic target for AD treatments. Successful development of inhibitory drugs against a given target usually requires a good understanding of the physiological and pathological functions of the target and related enzymes. BACE2 (Memapsin 1), another human aspartic protease (AP), was simultaneously identified with BACE1 [8,10,14–16] because of the high sequence homo- logy between them and the characteristic sequences around the two catalytic aspartic acid residues. Currently, there are five human APs of well-characterized physiological func- tions: pepsin and gastricsin (food digestion), cathepsin D and cathepsin E (intracellular protein catabolism), and renin (blood pressure regulation) [17]. Eukaryotic APs are homologous at both the gene and protein levels. A typical AP is usually synthesized as a single-chain zymogen and is directed to intracellular compartments. It is generally activated by a self-catalyzed process, by which an N-terminal pro-segment of  45 residues is cleaved off, resulting in a mature enzyme [17]. However, few pro-APs, including pro-renin and pro-BACE1, are activated by other proteases in vivo [18–21]. The catalytic domains of APs share the same overall folding in their three-dimensional struc- tures [17]. A typical structure contains two subdomains with a substrate-binding cleft located between them, which can accommodate six to eight residues from the substrate. Four new human APs have been identified in recent years, namely BACE1, BACE2, Napsin1, and Napsin2 [6–10,22,23]. Correspondence to Y T. Kim, Oklahoma Medical Research Foundation, 825 NE 13th St., Oklahoma City, OK73104, USA. Fax: + 1 405 271 1795, Tel.: + 1 405 271 7641, E-mail: kimy@omrf.ouhsc.edu, and X. Lin, Oklahoma Medical Research Foundation, 825 NE 13th St, Oklahoma City, OK73104, USA. Fax: + 1 405 271 7544, Tel.: + 1 405 271 1368, E-mail: lin@proteomtech-inc.com Abbreviations: AD, Alzheimer’s disease; Ab, b-amyloid peptides; APP, b-amyloid precursor protein; AP, aspartic protease; BACE, beta-site APP cleaving enzyme; NCH-c,Notchc-secretase cleavage site; PB1-T1, pro-BACE1-T1; PB2-T1, pro-BACE2-T1. (Received 11 July 2002, revised 12 September 2002, accepted 23 September 2002) Eur. J. Biochem. 269, 5668–5677 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03277.x Although the pathological function of BACE1 in AD has been clearly demonstrated, the physiological functions of these newly identified APs remain unknown. There is widespread interest in these human APs because of their possible important physiological and pathological roles in general. The BACE2 gene was mapped to human chromosome 21, where the Down’s Syndrome-associated genes are located [14–16], suggesting that the corresponding enzyme may function as a second b-secretase involved in the pathology of Down’s Syndrome as well as AD. Such a gene location is consistent with an early prediction that BACE2 may not only be structurally but also functionally homo- logous to BACE1. Furthermore, both BACE1 and BACE2 are expressed in all parts of the brain [24]. Like BACE1, BACE2 can cleave the b-secretase site of APP both in vivo and in vitro [24,25], thus it is thought to provide b-secretase activity. Contradictory to this point of view, however, it has been found that unlike BACE1, BACE2 is not coexpressed with APP and ADAM-10 (a putative a-secretase), the latter of which is involved in alternative APP processing [26]. Due to the expression patterns in different tissues, it was also proposed that BACE2 is more likely to function as a pro- hormone processing enzyme [27]. Moreover, the fact that BACE1-deficient cells could not produce detectable levels of Ab [12,13] suggests that BACE2 has little ability to complement BACE1 activity in neurons. Detailed bio- chemical studies on BACE2 are therefore desirable for better understanding of its functions and clarification of the contradictory data. While working towards this goal, two different forms of recombinant pro-BACE2 have been purified and characterized. The results show that BACE2 possesses some unique enzymatic properties when com- pared to BACE1 and other known aspartic proteases. EXPERIMENTAL PROCEDURES Cloning, Escherichia coli expression and purification of pro-BACE2 A schematic presentation of the two human pro-BACE2 variants, pro-BACE2-T1 (PB2-T1) and pro-BACE2-T2 (PB2-T2), is shown in Fig. 1, as compared to pro-BACE1-T1 (PB1-T1) [10,11]. The cDNA of PB2-T1 and PB2-T2 was amplified from a human placenta cDNA library (Clontech) using oligonucleotide primers: 5¢ primer, 5¢-GGATCCGCCGCCCCGGAGCTGGCCCCCGCGC 3¢;3¢ primer for T1, 5¢-GGATCCTCAGGGCTCGCTCAA AGACTGAGCGGG-3¢;and3¢ primer for T2, 5¢-GGAT CCTCAGCTCGCTGCGAAGCCCACCCTC-3¢. These primers contain a BamHI site at the 5¢ end(shownin italics). In addition, a stop codon was inserted prior to the BamHI site in the 3¢ primers (shown in boldface). The PCR products were cloned into the BamHI site of pET11a (Novagen), resulting in pET11-PB2-T1 and pET11-PB2-T2. A schematic presentation of the resulting expressed proteins is shown in Fig. 1. Expression, inclusion body isolation, refolding, and purification of BACE2 are described below. E. coli BL21 (DE3) cells transformed with the expression vector (pET11-PB2-T1 or pET11-PB2-T2) were grown in Luria–Bertani broth and induced by the addition of isopropyl-b- D -thiogalactopyranoside (final concentration, 1m M ) for inclusion body production. The inclusion body was dissolved in 50 mL of a denaturation buffer (8 M urea, 1m M glycine, 0.1 m M EDTA, 10 m M b-mercaptoethanol, 10 m M dithiothreitol, 1 m M reduced glutathione, 0.1 m M oxidized glutathione, 20 m M Tris/HCl, pH 10.5) to a protein concentration of  1.2 mgÆmL )1 . The denatured proteins were refolded in 10 vols 20 m M Tris base using a rapid dilution method [10,28], followed by adjusting the pH to 8.0. The refolded protein was concentrated by ultrafiltration, and further purified by two steps of chromatography on columns of Sephacryl S-300 (5 · 100 cm, Amersham Phar- macia Biotech) and Resource-Q (1.6 · 3 cm, prepacked, Amersham Pharmacia Biotech). The enzyme fractions obtained from the last column were pooled, concentrated by ultrafiltration, and used for further experiments. Activity assay and kinetics measurement of pro-BACE2 To rudimentarily identify the substrate specificity of the purified PB2-T1 and PB2-T2, each enzyme sample was incubated separately with different polypeptide substrates (40 lg) in 40 lL of a reaction mixture containing 50 m M sodium phosphate buffer (pH 6.5) at 37 °Cfor2or20h. Some of the peptide substrates were custom synthesized by a commercial source (Research Genetics; Huntsville, AL, USA), and the remainder were purchased (Sigma). The 11 Fig. 1. Schematic diagram of the primary structures of pro-BACE1-T1 (PB1-T1), pro- BACE2-T1 (PB2-T1), and pro-BACE2-T2 (PB2-T2). The primary structure of each of these enzymes consists sequentially of a T7 tag sequence, a pro, and a mature protease domain (with or without the C-terminal extension). Two active-site aspartic acids in D(T/S)G motifs (D-93/289 for BACE1 and D-110/303 for BACE2) are marked. The cysteine residues and possible disulfide bonds are labeled. Open circles indicate possible free cysteine residues in PB2-T2. Ó FEBS 2002 Enzymatic properties of BACE2 (Eur. J. Biochem. 269) 5669 polypeptides are as follows (sequences shown in Table 1): NCH-c, c-secretase cleavage site of notch [29]; APP-a, a-secretase cleavage site of APP; APP-b, b-secretase clea- vage site of APP; swAPP-b, b-secretase cleavage site of Swedish APP; APP-c, c-secretase cleavage site of APP; ENK-1, preproenkephalin fragment 129–138 peptide; insu- lin B chain (Sigma, I6383); kinetensin (Sigma, K1879); mastoparan (Sigma, M3545); neuropeptide (Sigma, M0421); and preproenkephalin fragment 128–140 (Sigma, P7162). The peptide fragments produced from the enzy- matic reaction were separated by HPLC using a Magic 2002 system (Michrom BioResources, Inc., Aubum, CA, USA) and a Magic C18 reverse-phase column (1.0 · 150 mm). Elution was performed with a gradient from 5% acetonitrile in 0.06% trifluoroacetic acid to 95% acetonitrile in 0.08% trifluoroacetic acid and monitored at 215 nm. The incuba- ted samples were also subjected to HPLC/MS (LC/MS, Molecular Biology Resource Facility, University of Okla- homa Medical Center) to identify the hydrolytic products (average error in mass determination was 0.02%). For LC/ MS analysis, the HPLC effluent was fed into the electro- spray ion source of the mass spectrometer at 40 lLÆmin )1 .A Sciex QSTAR hybrid quadruple time-of-flight mass spec- trometer (Applied Biosystems-Sciex, Inc.) was used to produce positive ions from a pneumatically assisted elec- trospray interface. Sample ions were analyzed over the mass range of 300–3000 amu. The two BACE2 variants were also incubated with different proteins (40 lg) in 40 lLofa reaction mixture containing 50 m M sodium phosphate buffer (pH 6.5) at 37 °C for 4 h. The proteins (Sigma) used were as follows: human serum albumin, cytochrome C, lysozyme, alcohol dehydrogenase, b-amylase, and carbonic anhydrase. The reaction mixtures were run in 20% SDS/ PAGE under reducing conditions for identification of the possible hydrolytic products. Kinetic parameters (K m and V max )ofPB2-T1were routinely determined using the NCH-c peptide as a substrate. In a typical assay, the reaction was carried out at 37 °C for 5–30 min in a 40-lL reaction mixture containing 50 m M sodium phosphate buffer (pH 6.5), and 0.8 m M substrate with an enzyme concentration of 6.26 l M . The reaction was initiated by the addition of substrate at concentrations varying in the range of 0.1–2 m M ,andwas terminated with 40 lL 2% trifluoroacetic acid. The reaction 5670 Y T. Kim et al. (Eur. J. Biochem. 269) Ó FEBS 2002 mixture was analyzed by HPLC as described above. The kinetic parameters were obtained from the fitting of the data using nonlinear regression analysis software GraFit [30]. The protein concentration was estimated colorimetrically with a protein assay kit (Bio-Rad) using BSA as standard. Activation of pro-BACE2 by BACE1 To identify the interaction between BACE1 and BACE2, PB2-T1 was incubated with PB1-T1. The reaction was carried out at 37 °Cfor60minin50m M Tris/BisTris/ sodium acetate/Caps buffer pH 4.5–12 and the aliquots were applied to a 10% tricine/SDS gel (Novex). The gel bands produced from the reaction were transferred to a PVDF membrane and the N-terminal sequence was analyzed by using automated Edman degradation. Determination of enzymatic properties The pH dependencies of PB2-T1 activity toward two synthetic peptide substrates (NCH-c and ENK-1) were determined in 50 m M sodium acetate (pH 3.0–5.0), 50 m M sodium phosphate (pH 5.5–6.5), 50 m M Tris/HCl (pH 7.0– 9.0), 50 m M Caps/NaOH (pH 9.5–10.5), and 50 m M Na 2 HPO 4 /NaOH (pH 11.0–13.0). To investigate the pH stability, the enzymes were preincubated for 2 h at 25 °Cin the buffers listed above. The pH of the mixture was adjusted to 10.0 by the addition of 0.6 vol 0.5 M Caps/NaOH (pH 10.0) or 0.1 M NaOH, and then the enzymatic activity with NCH-c was determined as described above. To test the effects of different protease inhibitors, the enzyme solution containing each inhibitor was preincubated in 50 m M sodium phosphate (pH 6.5) and 50 m M Caps/NaOH (pH 10.0) at 37 °C for 10 min, respectively, then assayed using NCH-c as a substrate. The following inhibitors were tested: 0.1 m M antipain, 0.1 m M chymostatin, 0.1 m M E-64, 0.1 m M leu- peptin, 0.5 m M pepstatin, 0.2 m M phosphoramidon, 1.0 m M pefabloc SC, 10 m M EDTA, and 0.01 m M aprotinin. CD spectroscopic study on the thermal stability of pro-BACE2 CD measurements of PB2-T1 and PB2-T2 at different temperatures were performed using a JASCO 715 spectro- polarimeter equipped with a Peltier temperature control accessory PTC348WI. The temperature scans of the molar ellipticity were recorded using an optical cell with a 0.1-cm pathlength for the far-UV region and performed at a rate of 30 °CÆh )1 . The protein concentrations of PB2-T1 and PB2- T2 were 23.1 l M and 29.7 l M , respectively. RESULTS Cloning, expression, purification, and activity of pro-BACE2 variants Two designed E. coli expression constructs of pro-BACE2, named pro-BACE2-T1 (PB2-T1) and pro-BACE2-T2 (PB2-T2) are shown in Fig. 1, as compared with pro- BACE1-T1 (PB1-T1) [10,11]. PB2-T1 was constructed based on the sequence homology between BACE2 and BACE1 (PB1-T1) of which a crystal structure has been recently solved [11]. PB2-T2 was constructed based on the sequence homology with the pepsin catalytic domain. Both variant forms of the enzyme were expressed in E. coli BL21 (DE3), then refolded in vitro as described in ÔExperimental proceduresÕ. The enzymes were purified by consecutive column chromatographic procedures using Sephacryl S-300 and Resource-Q (data not shown), and gave a single band on SDS/PAGE (Fig. 2A). Although two free cysteines, Cys233 and Cys292, exist in PB2-T2 based on sequence homology (Fig. 1), no intermolecular disulfide bond was found, as demonstrated by the nonreducing SDS/PAGE (Fig. 2A). The molecular masses of recombinant PB2-T1 and PB2-T2 were estimated to be 49 183 and 45 747 Da, respectively, by MALDI-TOF MS (data not shown). These molecular masses are consistent with the molecular mass calculated from the deduced amino acid sequences for PB2- T1 (49 173) and PB2-T2 (45 756), with the standard error of the MS at  0.02%. The N-terminal sequences of the recombinant proteins were determined to be Ala-Ser-Met- Thr-Gly, consistent with the designed sequence. The enzymatic activities of PB2-T1 and PB2-T2 were determined using a synthetic peptide substrate, NCH-c (Fig. 2B). The specific activity of PB2-T1 enzyme was 15 120 (pmolÆ min )1 Æmg )1 ). In contrast, the PB2-T2 enzyme exhibited activity that was only 17% of that of PB2-T1. These results show that the refolded and purified pro-BACE2 enzymes (PB2-T1 and PB2-T2) are active in hydrolyzing a synthetic peptide, NCH-c. Fig. 2. SDS/PAGE and activities of the puri- fied PB2-T1 and PB2-T2. (A) SDS/PAGE of the purified PB2-T1 and PB2-T2. SDS/PAGE (12.5%) was run under nonreducing condi- tions followed by Coomassie brilliant blue staining. Protein standards are shown on the left. (B) Specific activities of PB2-T1 and PB2- T2. The enzyme activity was determined in 50 m M sodium phosphate buffer (pH 6.5) with 0.8 m M NCH-c at 37 °Cfor30minas described in ÔExperimental proceduresÕ. Ó FEBS 2002 Enzymatic properties of BACE2 (Eur. J. Biochem. 269) 5671 Processing of BACE2 propeptide by BACE1 To test whether PB2-T1 can auto-activate either intra or intermolecularly, the zymogen was incubated under various conditions, including different pH, buffers, and tempera- tures. The pH range used was from 4.5 to 12.0, the incubation time used was 2 or 20 h, and the temperature was 25 and 37 °C. Auto-activation was not observed under any of the conditions tested (Fig. 3A). To clarify the relationship between BACE1 and BACE2 and to study their possible interactions, PB2-T1 was incubated with PB1- T1 [10]. Under experimental conditions, pro-BACE2 (PB2- T1) could be ÔactivatedÕ by BACE1 (PB1-T1), while BACE2 did not activate pro-BACE1 (Fig. 3B and C). The N-terminal sequence of the lower band in the gel shown in Fig. 3B (left lane, pH 4.5 and 6.0) contained the sequence Ala-Leu-Glu-Pro-Ala as the first five amino acid residues, which is the N-terminal sequence of mature BACE2 observed in vivo [24]. Therefore, these results indicate that BACE1 is capable of activating pro-BACE2 by removing its pro-peptide. pH Dependency and stability The pH dependence of the PB2-T1 activity toward a synthetic substrate (NCH-c) is shown in Fig. 4A. PB2-T1 was active over a broad pH range, from 6.0 to 11.0, with maximum activity at pH 9.5. PB2-T2 was also active in the same range with maximum activity at pH 9.0–10.0 (data not shown). To confirm whether the pH dependence of PB2-T1 activity could be changed depending on the substrate used, the pH dependence of PB2-T1 was also determined using a different substrate (ENK-1). The optimum pH of the enzyme using ENK-1 substrate was 6.0 (Fig. 4B), closer to a ÔnormalÕ aspartic protease. These results show that the pH dependence of PB2-T1 activity varied depending on the substrate. To investigate the stability of BACE2 at different pH levels, PB2-T1 and PB2-T2 were preincubated at various pHs before the activity was measured. As shown in Fig. 4C, PB2-T1 retained > 80% of the maximum activity after preincu- bation in the buffers at pH between 4 and 12. The pH stability of PB2-T2 is similar to that of PB2-T1 (data not shown). These data show that BACE2 is a new type of aspartic protease in spite of the conservation of two active-site aspartic acid residues in D(T/S)G motifs and the high degree of homology to BACE1 [10]. Thermostability of the secondary structure of BACE2 In PB2-T2, the C-terminal ÔextensionÕ of the protease domain of BACE2 was deleted, resulting in potential disruption of two disulfide bonds (Fig. 1). Therefore, the structure of PB2-T2 may be less stable than that of PB2-T1. To assay the structural stability, a CD spectropolarimeter was used to monitor the secondary structure of the proteins at increasing temperatures. The thermal unfolding of PB2- T1 and PB2-T2, measured by the changes in ellipticity at 215 nm, is shown in Fig. 5. The figure shows that the major transition of the secondary structure of PB2-T1 occurs between 90 and 120 °C, while that of PB2-T2 occurs between 50 and 80 °C. The secondary structure of PB2-T2 was completely denaturated at temperatures over 80 °C. However, even at 120 °C, PB2-T1 exhibits  50% of the far-UV ellipticity of the native enzyme. These results indicate that the secondary structure of PB2-T1 is unusually stable, while that of PB2-T2 is considerably less stable. Possible inhibition of BACE2 by different protease inhibitors and metal ions Using NCH-c as a substrate, the possible inhibitory effects of different protease inhibitors and metal ions were tested on PB2-T1. The potential inhibitors are listed in Experi- mental procedures. None of the protease inhibitors tested, including a high concentration of pepstatin, had any significant inhibitory effect toward BACE2 (data not shown). These results are consistent with similar experi- ments on BACE1 [10]. Two metal ions (Cu 2+ and Zn 2+ ), however, did inhibit PB2-T1 significantly (> 70% inhibi- tion) at 1 m M concentration. It was previously shown that the inhibition of proteolytic activity by metal ions could be nonspecific. For example, E. coli leader peptidase is inhib- ited nonspecifically by Hg 2+ and Cu 2+ ions (60% inhibition Fig. 3. Processing of pro-BACE2 (PB2-T1) by BACE1 (PB1-T1). PB2-T1, PB2-T1/PB1-T1, and PB1-T1 were incubated in 50 m M Tris/ BisTris/sodium acetate/Caps buffer (pH 4.5, 6.0, 8.0, 10.0, and 12.0) at 37 °C for 60 min, respectively. The reaction mixtures were separated by SDS/PAGE (12.5%) under reducing conditions. The arrowheads indicate pro-BACE2-T1 (PB2-T1), pro-BACE1-T1 (PB1-T1), and the mature form of BACE2-T1 (B2-T1). (A) SDS/PAGE of PB2-T1. (B) SDS/ PAGE of PB2-T1/PB1-T1. (C) SDS/PAGE of PB1-T1. 5672 Y T. Kim et al. (Eur. J. Biochem. 269) Ó FEBS 2002 [31]); an endoprotease from porcine antral mucosal mem- branes is inhibited by Fe 2+ ,Cu 2+ ,Zn 2+ ,andHg 2+ ions (100% inhibition [32]), among others [33,34]. Therefore, it is speculated that the inhibition of BACE2 by the metal ions is also nonspecific. Activity and specificity of PB2-T1 and PB2-T2 toward NCH-c The specificities of PB2-T1 and PB2-T2 towards NCH-c were measured. The two variants of pro-BACE2 clearly had different substrate specificities. In this case, PB2-T1 pre- ferred to cleave between Leu and Ser with a minor cleavage site between Ser and Arg, while PB2-T2 preferred to cleave between Ser and Arg with a minor cleavage site between Leu and Ser (Table 1). These results suggest that the BACE2 variants have at least two different substrate specificities. The steady-state enzyme kinetics of PB2-T1 toward substrate NCH-c was also determined (data not shown). Under the experimental conditions, the processing site of the substrate was mainly VGSGVLL/SRK, and the Ser–Arg processing site was insignificant. Therefore, only a single processing site was measured in the kinetic experi- ments. The kinetic parameters of PB2-T1 toward the NCH- c substrates are: K m ¼ 0.2 m M ,andV max ¼ 0.054 l M Æs )1 . Activity of PB2-T1 and PB2-T2 toward various peptide and protein substrates Because BACE2 is highly homologous to BACE1, the enzymatic activity of PB2-T1 and PB2-T2 toward various peptide substrates designed according to the a-, b-, and c-secretase cleavage site of APP was investigated. The substrate cleavage was assayed and quantified by HPLC and HPLC/MS. In addition, due to the initial discovery that PB2-T2 cut at the N-terminal site of the paired basic residues in NCH-c, some specific peptides derived from enzyme processing sites of pro-hormones were also tested. Table 1 summarizes the results of the specificity of PB2-T1 and PB2-T2 toward some of the peptides used. The table shows that recombinant pro-BACE2 cleaves at b-secretase recognition site (M/D and L/D, b-secretase recognition site of APP and Swedish mutation APP, respectively) of both APP-b and swAPP-b. However, APP-c substrate is not cleaved by the pro-BACE2 variants under the experimental conditions used. These results indicate that recombinant BACE2 exhibits the same activity as that of b-secretase (BACE1), although the cleavage rate of the b- secretase recognition site by the enzyme is low. PB2-T1 and PB2-T2 cleaved several positions of kinetensin, mastoparan, neuropeptide, and preproenkephalin frag- ment 128–140 at a significant rate. The APP-a,ENK-1 and oxidized insulin B chain were also hydrolyzed at several sites with poor cleavage rate. These results show that PB2-T1 demonstrates broad substrate specificities, preferring bulky residues at the P1 site, and various residues at the P1¢ site. The substrate specificity of PB2- T2, in contrast, seems more constrained, apparently preferring small residues at the P1 site, and basic residues Fig. 5. Thermostability of the secondary structure of PB2-T1 and PB2- T2. CD spectropolarimeter was used to measure the thermo- unfolding of the secondary structures. The ellipticities of PB2-T1 (solid line) and PB2-T2 (dotted line) were monitored at 215 nm in 20 m M Tris/HCl, pH 8.0, 0.4 M urea. Fig. 4. pH dependence and pH stability of the activity of PB2-T1. (A) pH dependence of PB2-T1 toward NCH-c. Assay of the enzyme activity was carried out as described in ÔExperimental proceduresÕ, using a synthetic peptide substrate (NCH-c), except for the use of the following buffers: 50 m M sodium acetate (pH 3.0–5.0); 50 m M sodium phosphate (pH 5.5–6.5); 50 m M Tris/HCl (pH 7.0–9.0); 50 m M Caps/NaOH (pH 9.5–10.5); and Na 2 HPO 4 /NaOH (pH 11.0–13.0). (B) pH dependence of PB2-T1 toward ENK-1. The enzyme assay was carried out as described in ÔExperimental proceduresÕ with the exception of the above buffers. (C) pH stability of PB2-T1. The enzyme was preincubated for 2 h at 25 °Cinthe same buffers used for the pH dependence study. Then, the pH of each preincubation mixture was adjusted to 10.0 by the addition of 0.6 vol. 0.5 M Caps/NaOH (pH 10.0) or 0.1 M NaOH, and the enzyme activity was determined. Ó FEBS 2002 Enzymatic properties of BACE2 (Eur. J. Biochem. 269) 5673 at P1¢ and P2¢ sites. These results show that the substrate specificity of PB2-T1 is different from that of PB2-T2 (Table 1). Thus, the C-terminal extension domain of BACE2 (Pro432–Pro466) may affect the substrate speci- ficity of the enzyme. To explore further the substrate specificity of PB2-T1 and PB2-T2 toward intact protein substrates, some commercially available proteins, which include human serum albumin, cytochrome C, lysozyme, alcohol dehy- drogenase, b-amylase and carbonate anhydrase, were used in the activity assays. The substrate proteins were incubated with PB2-T1 in a 1 : 10 enzyme/substrate weight ratio and various reaction conditions were as follows: the pH range used was 4.5–12.0, the temperature was 25 and 37 °C, and the incubation time was 2 or 20 h. None of the above proteins were processed by PB2-T1 (data not shown). These results suggest that BACE2 is different from general purpose aspartic proteases, such as pepsin, but similar to BACE1, which has also been shown to lack the ability to process native protein substrates in vitro [10]. DISCUSSION BACE2 is a newly identified human aspartic protease. To study its biochemical properties and possible biological functions, two variants of pro-BACE2, PB2-T1 and PB2- T2, have been constructed, expressed in E. coli,and purified. PB2-T1 consists of the pro and protease domains, similar to a pro-BACE1 variant, PB1-T1, for which a high- resolution crystal structure has been determined [11]. The other variant, PB2-T2, is a truncated version of PB2-T1 as illustrated in Fig. 1. Its protease domain is terminated at the C-terminal position of homologous pepsin, and is 34- residues shorter at the C terminus than PB2-T1. Although the primary structures of the enzymes are in pro-forms, both PB2-T1 and PB2-T2 have apparent enzymatic activity consistent with enzymatically active pro-BACE1 (PB1-T1) [10], indicating that the conformation of the pro-domain of BACE2 is flexible and that an equilibrium exists under the reaction conditions between an ÔopenÕ, or active conforma- tion, and a ÔclosedÕ, or inactive conformation [35]. The activation of most mammalian aspartic proteases is brought about by removal of the pro-peptide by either auto- activation or other proteolytic enzymes [17,36]. We showed here that PB2-T1 does not auto-activate in the wide ranges of pH, temperature and buffer conditions tested. We started the experiment with the following intriguing facts in mind. First, it has been shown that pro-BACE1, which is highly homologous to pro-BACE2, can be Ôauto-activatedÕ in acidic conditions [10], although the cleavage site in such activation is different from that of the in vivo activation site. In fact, the in vivo pro-BACE1 processing is catalyzed by furin or related enzymes that recognize basic residues at the cleavage site [19–21]. Since BACE2 often cleaves at basic or paired basic residues (Table 1), it was interesting to test whether BACE2 is able to activate BACE1. Second, cell culture experiments [24] showed that a mature BACE2 protein starts from residue Ala63, suggesting its in vivo activation site is the peptide bond between Leu62 and Ala63. As there is no basic amino acid residue at, or near, this activation site, it is unlikely that pro-BACE2 is also activated by furin or related enzymes. Third, we found in previous experiments that one cleavage site preferred by BACE1 is between Leu and Ala (data not shown). The results presented here demonstrate that under the experi- mental conditions used, BACE2 cannot activate pro- BACE1 (Fig. 3B), while pro-BACE2 can be activated by BACE1 at the in vivo maturation position. These results raise an interesting possibility that BACE1 may be one of the physiological enzymes activating BACE2. Although we have shown that both BACE1 [10] and BACE2 (this paper) cleaves various peptide substrates in vitro, it remains to be demonstrated that protein substrates can be processed under similar conditions. To date, the only confirmed cleavage site of protein substrate for BACE1 is the b-secretase site of APP or related mutants. Thus PB2-T1 becomes the second protein substrate in this list. It has been suggested [21] that the pro-peptide of BACE1 is not evolutionarily developed for the regulation of enzyme activity, as some other zymogens are [36], but to facilitate protein folding. Whether the in vivo activity of pro-BACE2 requires preactivation remains the subject of further inves- tigation. Nevertheless, both BACE1 and BACE2 are activated in vivo, leaving a defined N terminus of the mature enzyme [7,8,24]. Thus, the possibility still exists that zymogen activation of BACE1 and BACE2 may be a means of regulating their enzymatic activities under an in vivo condition. Our results apparently contradict recent reports from other laboratories [37,38], which show that mamma- lian and insect cell expressed fusion protein BACE2 can self- activate under acidic conditions. This contradiction may be due to the different expression systems used. In the case of BACE1, the rate of substrate turnover (k cat /K m )ofBACE1 expressed in insect cells is  10-fold higher than that of the enzyme expressed in E. coli [39]. Furthermore, it has been shown that glycosylation of BACE1 influences the proteo- lytic activity and ensures optimum interaction between BACE1 and a substrate [40]. Therefore, unglycosylated BACE2 expressed by E. coli may exhibit different activity from those expressed in mammalian or insect cell lines. A ÔtypicalÕ aspartic protease is active at acidic pH between 2 and 5 [17]. For example, pepsin has an optimum pH of near 2.0 [17], gastricsin at pH 3.0 [41], cathepsin D at pH 3.5–5.0 [42], yapsin at pH 4.5 [43], and BACE1 at pH 4.0 (recombinant BACE1 from E. coli) [10], or 4.5 (recombinant BACE1 from mammalian or insect cells) [7,39]. Thus it is surprising to find that the activity of BACE2 continuously rose with increasing pH up to pH 9.5 when NCH-c was used as a substrate (Fig. 4A). In this work, a synthetic substrate, NCH-c (Val-Gly-Ser-Gly-Val- Leu-Leu-Ser-Arg-Lys), was mainly used for the activity assay. The substrate has a Lys residue (P2¢/P3¢ site)attheC terminus, which may influence the pH-dependent activity for this particular substrate. The pK of the e-amino group in the Lys side chain is close to the pH optimum of the enzyme activity. Thus it is probable that the enzyme prefers the deprotonated-Lys form (free base) of the substrate. Com- pared with the BACE1 substrate binding pockets, S4–S4¢ [11], BACE2 contains the following nonconservative muta- tions in its substrate binding cleft: Arg307 fi GlninS4, Gln12 fi Arg in S3, Pro70 fi Lys in both S2¢ and S3¢,and Glu125 fi Thr in P4¢. The + 2 net charge increase in S2¢– S4¢ pockets in neutral conditions may provide an explan- ation for the observation that the optimum enzymatic activity towards substrate NCH-c shifts to a more basic pH region relative to other substrates. To demonstrate this 5674 Y T. Kim et al. (Eur. J. Biochem. 269) Ó FEBS 2002 point, a different peptide substrate was used for measuring the pH dependent activity. The result showed that the optimum pH of PB2–T1 using ENK-1 (Fig. 4B) was at pH 6.0. Some results differ from those using purified BACE2 from different expression systems or using different substrates [37,38]. It seems that the precise optimum pH of BACE2 varies depending on substrates, buffers, expression systems (E. coli, insect cell line, and mammalian cell line), and expression vector construction (full-length form, trun- cated form, and full-length/T7 or His tag form). Further- more, there exist several other examples of aspartic proteases that are enzymatically active at neutral and weakly alkaline pH as follows: renin has an optimum pH of 5.5–7.5 [44]; mouse submandibular renin at pH 6.5–8.3 [45]; and signal peptidase II at pH 7 [46]. BACE2 has a high degree of homology with BACE1, with more than 50% amino acid sequence identity. All six cysteine residues are conserved between BACE1 and BACE2. Based on the crystal structure of BACE1 [11], one can predict, with reasonable confidence, the three- dimensional positions of most residues of BACE2, including three disulfide bonds formed by the six cysteine residues (Figs 1 and 6). Thus in BACE2, the three disulfide bonds are assumed to be Cys233–Cys433, Cys292–Cys457, and Cys344–Cys393. Such a disulfide bond pattern of BACE1 and BACE2 is distinctively different from, for example, that observed in pepsin and cathepsin D [47]. Particularly, the two disulfide bonds in the C-terminal subdomain, Cys233– Cys433 and Cys292–Cys457, fasten the C-terminal peptide to the main body of the catalytic unit (Fig. 6). Both disulfide bonds as well as the C-terminal peptide are absent in pepsin and other eukaryotic aspartic proteases. It suggests that the catalytic domain of BACE2 may be tolerable to a trunca- tion from the C terminus up to Ser432 without interfering with the overall folding. The corresponding construct, PB2- T2, is likely to result in two free cysteine residues, Cys233 and Cys292. Spatial positions of these two cysteine residues in the homologous model (30 A ˚ for the C 233 a –C 292 a distance) prohibit them from forming an intramolecular disulfide bond, if the same overall folding of BACE1 is assumed for BACE2. In addition, the fact that PB2-T2 shows a monomeric molecular weight in nonreduced SDS/PAGE (Fig. 2A) indicates that the refolding and purification protocol used is sufficient to produce protein samples without introducing intermolecular disulfide bonds, in spite of the fact that both the free cysteine residues are probably exposed to solvent. The high primary sequence homology between BACE2 and BACE1 suggests that their soluble domains share essentially the same three-dimensional structure. There are only three deletions in the soluble domain of BACE2 relative to that of BACE1: a three-residue deletion around residue 240, and two one-residue deletions around residues 390 and 455, respectively. All are located in the corres- ponding variable loop regions in BACE1 as compared to pepsin. These deletions in BACE2 change the loop length only slightly, thus presumably perturbing the overall structure very little. The C-terminal tail, which is unique to BACE1 and BACE2, is located on the backside of the catalytic domain from the active site, connecting the catalytic domain to the transmembrane domain. The one- residue deletion in the C-terminal loop region (around residue 455) in BACE2 relative to BACE1 is unlikely to affect the formation of the last putative disulfide bond Fig. 6. Ribbon diagram of the BACE2 cata- lytic domain. This BACE2 molecular model is built based on the crystal structure of BACE1 and the primary sequence homology between them. The view is of the opposite side from the active site with the substrate binding cleft roughly horizontal. The C-terminal tail is shownindarkblue.Catalyticasparticresidues are shown as yellow stick models. The three disulfide bonds are shown as red stick models. Regions containing insertion/deletion as compared to BACE1 are colored orange. This figure was produced with the program MOLSCRIPT [48]. Ó FEBS 2002 Enzymatic properties of BACE2 (Eur. J. Biochem. 269) 5675 (Cys292–Cys457). In addition to connecting the soluble domain to the trans-membrane domain, the C-tail also provides structural enforcement to the soluble domain through the two disulfide bonds, and an extended b-sheet and hydrophobic side chain interactions. Together, they are believed to contribute significantly to the overall stability in BACE1 [11]. The dramatic thermal stability difference between PB2-T1 and PB2-T2 observed using CD spectro- scopic method provides direct evidence supporting the same notion in BACE2 (Fig. 5). On the other hand, our data indicate that these structural enforcements are not essential for the enzymatic activity of BACE2. Deletion of the C-tail is tolerable for the enzyme activity, although some subtle structural changes may occur that are associated with the substrate specificity changes. Such structural integrity of the soluble domain in the absence of the C-tail is consistent with the high degree of homology in three-dimensional structures between BACE1, BACE2 and pepsin, the latter of which does not contain the C-tail. In addition to the overall structural stability, the presence/absence of the C-tail apparently affects the substrate specificity of the enzyme. Indirectly, the rigidity associated with the C-tail, particularly the two disulfide bonds, may keep the dynamic structure of BACE2 in a more open form, thus making it more accessible to different substrates. In a more direct way, the loss of the disulfide bond Cys233–Cys433 may affect the substrate binding at P4 position mediated through a b-turn around residue 88. Similarly, the free C-terminal end of the longer version of our BACE2 constructs may wrap around the soluble domain and reach the putative S4¢ substrate binding pocket in BACE2. The corresponding terminus in BACE1 is mobile in the crystal structure [11] and likely to become more fixed if it attaches to the trans-membrane domain. ACKNOWLEDGEMENTS The authors thank K. Takahashi, School of Life Science, Tokyo University of Pharmacy and Life Science, for helpful discussion of this work; K. K. Rodgers, Department of Biochemistry and Molecular Biology, University of Oklahoma Medical Center, for advice on CD experiments; and K. Jackson and C. Batson, Molecular Biology Resource Facility, Warren Medical Research Institute, University of Oklahoma Medical Center for assistance with MS, amino acid analysis, and N-terminal sequencing. This work is supported by the National Institute of Health Grant RO1-AI46298 (to X. Lin). REFERENCES 1. Selkoe, D.J. 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