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 va
Trang 1Enzymic properties of recombinant BACE2
Yong-Tae Kim1, Deborah Downs1,2, Shili Wu1,2, Azar Dashti1,2, Yujun Pan1, Peng Zhai1,2,
Xinjuan Wang1,2,3, Xuejun C Zhang1and Xinli Lin1,2,4
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
colias 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 general aspartic proteases, the specificity of PB2-T2 is more con-strained, apparently preferring to cleave at the NH2-terminal side of paired basic residues.Unlike other typical 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- and c-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, Notch c-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)
Trang 2Although 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
E X P E R I M E N T A L P R O C E D U R E S
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
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¢; and 3¢ primer for T2, 5¢-GGAT CCTCAGCTCGCTGCGAAGCCCACCCTC-3¢.These primers contain a BamHI site at the 5¢ end (shown in 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,
1 mM) for inclusion body production.The inclusion body was dissolved in 50 mL of a denaturation buffer (8Murea,
1 mMglycine, 0.1 mM EDTA, 10 mMb-mercaptoethanol,
10 mM dithiothreitol, 1 mM reduced glutathione, 0.1 mM oxidized glutathione, 20 mMTris/HCl, pH 10.5) to a protein concentration of 1.2 mgÆmL)1.The denatured proteins were refolded in 10 vols 20 mM 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 mM sodium phosphate buffer (pH 6.5) at 37C for 2 or 20 h 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 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.
Trang 3polypeptides 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 cleaclea-vage 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 subjecincuba-ted 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 lL of a reaction mixture containing 50 mM sodium phosphate buffer (pH 6.5) at 37C 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 (Km and Vmax) of PB2-T1 were routinely determined using the NCH-c peptide as a substrate.In a typical assay, the reaction was carried out
at 37C for 5–30 min in a 40-lL reaction mixture containing 50 mMsodium phosphate buffer (pH 6.5), and 0.8 mMsubstrate with an enzyme concentration of 6.26 lM The reaction was initiated by the addition of substrate at concentrations varying in the range of 0.1–2 mM, and was terminated with 40 lL 2% trifluoroacetic acid.The reaction
Trang 4mixture 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 37C for 60 min in 50 mM 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 mMsodium acetate (pH 3.0–5.0), 50 mM
sodium phosphate (pH 5.5–6.5), 50 mMTris/HCl (pH 7.0–
9.0), 50 mM Caps/NaOH (pH 9.5–10.5), and 50 mM
Na2HPO4/NaOH (pH 11.0–13.0) To investigate the pH
stability, the enzymes were preincubated for 2 h at 25C in
the buffers listed above.The pH of the mixture was adjusted
to 10.0 by the addition of 0.6 vol 0.5M Caps/NaOH
(pH 10.0) or 0.1MNaOH, 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 mMsodium
phosphate (pH 6.5) and 50 mMCaps/NaOH (pH 10.0) at
37C for 10 min, respectively, then assayed using NCH-c as
a substrate.The following inhibitors were tested: 0.1 mM
antipain, 0.1 mM chymostatin, 0.1 mM E-64, 0.1 mM
leu-peptin, 0.5 mMpepstatin, 0.2 mMphosphoramidon, 1.0 mM
pefabloc SC, 10 mMEDTA, and 0.01 mMaprotinin
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
30CÆh)1.The protein concentrations of T1 and PB2-T2 were 23.1 lMand 29.7 lM, respectively
R E S U L T S
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 Experimental procedures.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)1Æmg)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 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 C for 30 min as
described in Experimental procedures.
Trang 5Processing 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 37C.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 activated by BACE1 (PB1-(PB2-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 normal 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 extension 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 120C, while that of PB2-T2 occurs between 50 and 80C.The secondary structure of PB2-T2 was completely denaturated at temperatures over 80C However, even at 120C, 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 (Cu2+and Zn2+), however, did inhibit PB2-T1 significantly (> 70% inhibi-tion) at 1 mMconcentration.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 Hg2+and Cu2+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.
Trang 6[31]); an endoprotease from porcine antral mucosal
mem-branes is inhibited by Fe2+, Cu2+, Zn2+, and Hg2+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: Km¼ 0.2 mM, and Vmax¼ 0.054 lMÆs)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 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 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 Experimental procedures, 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
Experimental procedures with the exception of the above buffers.(C) pH stability of PB2-T1.The enzyme was preincubated for 2 h at 25 C in the 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.
Trang 7at 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 37C, 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]
D I S C U S S I O N
BACE2 is a newly identified human aspartic protease.To
study its biochemical properties and possible biological
functions, two variants of pro-BACE2, 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 open, or active
conforma-tion, and a closed, 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 auto-activated 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 (kcat/Km) of BACE1 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 typical 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) at the C 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: Arg307fi Gln in S4, Gln12fi Arg in S3, Pro70 fi Lys in both S2¢ and S3¢, and Glu125fi Thr in P4¢.The + 2 net charge increase in S2¢– S4¢ pockets in neutral conditions may provide an explan-ation for the observexplan-ation that the optimum enzymatic activity towards substrate NCH-c shifts to a more basic pH region relative to other substrates.To demonstrate this
Trang 8point, 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 C233
a –C292
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
shown in dark blue Catalytic aspartic residues
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
[48].
Trang 9(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
A C K N O W L E D G E M E N T S
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).
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