Characterization of
2,6-Sialyltransferase Cleavage by
Alzheimer's
-Secretase (BACE1)*
Shinobu
Kitazume
,
Yuriko
Tachida
,
Ritsuko
Oka
,
Norihiro
Kotani
,
Kazuko
Ogawa
,
Minoru
Suzuki
,
Naoshi
Dohmae§,
Koji
Takio§,
Takaomi C.
Saido¶, and
Yasuhiro
Hashimoto
From the
Glyco-chain Functions Laboratory,
Supra-biomolecular System Group, Frontier Research System and
¶ Proteolytic Neuroscience Laboratory, Brain Science Institute,
Institute of Physical and Chemical Research, RIKEN and the
§ Department of Biomolecular Characterization, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 51-0198, Japan
Received for publication, June 24, 2002, and in revised form, December 4, 2002
 |
ABSTRACT |
BACE1 is a membrane-bound aspartic protease that
cleaves the amyloid precursor protein (APP) at the
-secretase site,
a critical step in the Alzheimer disease pathogenesis. We previously
found that BACE1 also cleaved a membrane-bound sialyltransferase,
ST6Gal I. By BACE1 overexpression in COS cells, the secretion of ST6Gal I markedly increased, and the amino terminus of the secreted ST6Gal I
started at Glu41. Here we report that BACE1-Fc
chimera protein cleaved the A-ST6Gal I fusion protein, or ST6Gal
I-derived peptide, between Leu37 and Gln38,
suggesting that an initial cleavage product by BACE1 was three amino
acids longer than the secreted ST6Gal I. The three amino acids,
Gln38-Ala39-Lys40, were found to be
truncated by exopeptidase activity, which was detected in detergent
extracts of Golgi-derived membrane fraction. These results suggest that
ST6Gal I is cleaved initially between Leu37 and
Gln38 by BACE1, and then the three-amino acid sequence at
the NH2 terminus is removed by exopeptidase(s) before
secretion from the cells.
 |
INTRODUCTION |
The deposition of amyloid
-peptide
(A
)1 in the brain is a
hallmark of the pathogenesis of Alzheimer's disease (1). A
, a 39-43-amino acid peptide, is a proteolytic product derived from the
amyloid precursor protein (APP). The
-secretase initially generates
the NH2 terminus of A
, cleaving APP to produce a soluble NH2-terminal fragment (APPs
) and a 12-kDa COOH-terminal
fragment (C99) that remains membrane bound. C99 is further cleaved by
-secretase, resulting in the production of pathogenic A
peptide
(2, 3). As an alternative processing pathway,
-secretase cleaves
within the A
sequence to produce a soluble NH2-terminal
fragment (APPs
) and a 10-kDa membrane-bound COOH-terminal fragment
(C83) (4, 5). C83 is also cleaved by
-secretase to produce the
nonpathogenic p3 peptide. BACE1 (
-amyloid-converting enzyme 1), a
pepsin-like membrane-bound aspartic protease, has recently been
identified as
-secretase (6-10). In the case of
-secretase,
functional presenilin is required (2, 3), and recent reports have shown that Notch and ErbB4 are also its substrates (11-14). Nevertheless, inhibitors toward both
- and
-secretases are still promising therapeutics for Alzheimer's disease (15-18).
We previously found that BACE1 is involved in the cleavage and
secretion of a membrane-bound sialyltransferase, ST6Gal I (19), and the
secreted ST6Gal I starts at Glu41. Several reports show
that BACE1 prefers Leu at position P1 (20-22). Indeed, APP
substitution of Leu at the P1 position for Met together with
substitution of Asn at the P2 for Lys, a Swedish mutation of familial
Alzheimer's disease, clearly facilitates BACE1-dependent cleavage and induces rapid progression of pathological symptoms. In
this article we describe in vitro cleavage of ST6Gal
I-derived peptide or protein by BACE1.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Eight-week-old male Wistar rats,
maintained in specific-pathogen free conditions, were purchased from
the Shizuoka Agricultural Cooperative Association for Laboratory
Animals (Shizuoka, Japan). Tissue culture media and reagents, including
Dulbecco's modified Eagle's medium and Lipofectin, were
purchased from Invitrogen. Protein A-Sepharose Fast Flow was purchased
from Amersham Biosciences. CDP-hexanolamine-Sepharose was a gift
from Dr. K. J. Colley (University of Illinois at Chicago). Columns
for DNA purification were obtained from Qiagen Inc. (Chatsworth, CA).
PCR was performed using LA Taq polymerase (Sigma). Protein
molecular weight standards were purchased from Bio-Rad. BACE inhibitor,
KTEEISEVN(Sta)VAEF (in which Leu was substituted with statine
(Sta) for the P1 position of an APP analogue peptide), was purchased
from Bachem (Bubendorf, Switzerland). A polyclonal antibody, Q38, which
specifically recognizes the NH2 terminus of ST6Gal I-Q38
form, was prepared by immunizing a synthetic peptide, QAKEFQC,
conjugated with keyhole limpet hemocyanin (23).
Expression Plasmid--
For the transient transfection
experiment, ST6Gal I FLAG-pSVL, protein A ST6Gal I-pcDSA, and BACE
Fc-pEF were constructed as described previously (19, 24, 25). To
generate ST6Gal I FLAG-pcDSA, polymerase chain reaction was performed
using ST6Gal I FLAG-pSVL templates with primer 1 (5'-CGCGAATTCAAGAAAGGGAGCGACTATGA-3') and primer 2 (5'-GCGCTCGAGGCTCACTTGTCATCGTCGTCC-3'). PCR product purified with the
QIAEX II Gel extraction system (Qiagen Inc.) was digested with
EcoRI and XhoI and then ligated into the pcDSA EcoRI-XhoI site. STK40A FLAG-pcDSA
was generated from ST6Gal I FLAG-pcDSA using a QuikChange site-directed
mutagenesis kit (Invitrogen) with primer 3 (5'-CTTACACTGCAAGCAGCAGAGTTCCAGATGCCC-3') and primer 4 (5'-GGGCATCTGGAACTCTGCTGCTTGCAGTGTAAG-3') (24).
Detection of ST6Gal I Protein--
COS-7 or rat hepatoma FTO2B
cells maintained in Dulbecco's modified Eagle's medium, 10% fetal
bovine serum were plated on 100- or 150-mm tissue culture dishes and
grown in a 37 °C, 5% CO2 incubator until 50-70%
confluent. Cells were transfected using Lipofectin and Opti-MEM I. Expression of proteins was typically allowed to continue for 24 to
48 h.
To analyze the soluble secreted form of ST6Gal I-FLAG, COS
cells were transfected with rat ST6Gal I FLAG-pSVL. At 48 h after transfection, soluble ST6Gal I-FLAG secreted in the media was pulled down with M2-agarose (Sigma) and analyzed by immunoblotting using either the E41 (1:500), Q38 (1:500), or anti-ST6Gal I polyclonal antibody (1:1000). Pre-absorption of the E41 antibody was performed using peptide EFQMPKC (10 µg/ml) or FQMPKC (10 µg/ml). Horseradish peroxidase-goat anti-rabbit IgG (Cappel, 1:1000) was used as a secondary antibody, and chemiluminescent substrate (Pierce) was used
for detection (19).
Rat hepatoma FTO2B cells that endogenously express ST6Gal I
at a high level were transfected with human BACE1-myc-pcDNA3.1 or
the control vector. At 48 h after transfection, soluble ST6Gal I
secreted in the media was immunoprecipitated with anti-ST6Gal I rabbit
polyclonal antibody. One-third of the immunoprecipitant was used for
detection with the anti-ST6Gal I antibody, and the rest of sample was
used for detection with the E41 antibody. Each sample was treated with
Laemmli sample buffer (26), subjected to 4-20% gradient SDS-PAGE, and
then transferred to a nitrocellulose membrane. The membrane was
incubated with either anti-ST6Gal I or the E41 polyclonal antibody.
Rat plasma (200 µl) was diluted with the buffer containing 10 mM sodium cacodylate, pH 6.5, 0.1% Triton CF-54, and 0.15 M NaCl. Protein A-Sepharose (30 µl) was added to the
mixture and rotated for 30 min to remove adhesive proteins.
After the beads were removed by centrifugation, 20 µl of
CDP-hexanolamine-agarose was then added to pull down sialyltransferase
proteins. After rotation for 16 h, beads were washed with
phosphate-buffered saline. Sialyltransferase proteins
immobilized to the beads were analyzed by immunoblotting with either
anti-ST6Gal I or the E41 polyclonal antibody.
In Vitro BACE1 Assay Using Protein A-ST6Gal I Fusion
Protein--
When protein A-ST6Gal I-FLAG or protein
A-STK40A FLAG was used as a substrate, both BACE1-Fc and
protein A-ST6Gal I fusion proteins were purified from 20 ml of culture
media of COS cells that transiently expressed these proteins by
absorbing them in 20 µl of protein A-Sepharose and IgG-Sepharose
(50% suspension in phosphate-buffered saline), respectively. These
preparations of BACE1-Fc and protein-A-ST6Gal I were mixed, resulting
in a final volume of 20 µl, which comprised 50 mM sodium
acetate buffer (pH 4.5) and protease inhibitors for possible
contaminating proteases associated with Sepharose beads (Complete
(Roche), 10 µM pepstatin, 1 µM leupeptin, 1 mg/ml pepstatin, and 2 µM amastatin). The mixture was
incubated at 37 °C for 2 h with rotation. The reaction was then
terminated, and the product was analyzed by immunoblotting with
anti-FLAG (M2) antibody.
In Vitro BACE1 Assay Using Synthetic Peptides--
When peptides
were used as substrates for assay, BACE1-Fc was purified from 50 ml of
culture medium of COS cells. Each peptide (0.1 mM) was
incubated for 16 h in 50 µl of the reaction mixture as described
above. KTEEISEVN(Sta)VAEF (0.1 µM, Bachem) was used as a
-secretase inhibitor. After the incubation the reaction mixture was
centrifuged to remove immobilized BACE1-Fc. The products were
separated on a reversed-phase HPLC C30-UG-5 column (4.6-mm i.d. × 250 mm, Nomura Chemical Co., Japan) using a Waters model 600E HPLC
system, equipped with a Senshu model SSC-5200 (Tokyo, Japan) UV
detector. The sample, applied to the column equilibrated in
10% acetonitrile, was then eluted with a gradient of 10-50% acetonitrile for 40 min. The elution rate was 1 ml/min. For MALDI-TOF MS analysis, peptide DYEALTLQAKEFQMPKSQE was incubated with BACE1-Fc in
a 5-fold scale, and each peak, separated by HPLC, was collected manually.
NH2-terminal Amino Acid Sequence
Analysis--
Protein A-ST6Gal I-FLAG, prepared from 120 ml of culture
medium, was cleaved by BACE1-Fc, and the soluble proteins yielded were
precipitated with 75% ice-cold ethanol at
20 °C for 16 h. An
aliquot of the precipitant was analyzed by SDS-PAGE and stained with
silver nitrate (Daiichikagaku, Tokyo, Japan). The rest of the
sample was subjected to SDS-PAGE followed by electrical blotting to an
Immobilon membrane (Millipore). After staining with Coomassie Blue, the
band of soluble ST6Gal I-FLAG (~49 K) was excised, and its
amino-terminal amino acid sequence was determined with a Procise 492 cLC protein sequencer (Applied Biosystems).
Exopeptidase Assay Trims NH2-terminal QAK
Sequence of Q38 Form of ST6Gal I--
ST6Gal I- or ST6Gal
IK40A-Q38 form that starts at Gln38 was
prepared by cleaving protein A-ST6Gal I-FLAG or protein A-ST6Gal IK40A-FLAG by BACE1 in vitro as described above.
The Q38 form protein as a substrate was mixed with a microsomal
fraction (10 µg of protein) or rat Golgi membrane (10 µg of
protein) pretreated with 1% of Triton X-100. The mixture was incubated
at 37 °C for 2 h in 20 µl of 50 mM sodium acetate
buffer (pH4.5) containing 0.1 mg/ml bovine serum albumin. Incubation
was terminated by the addition of a Laemmli sample buffer (25). An
aliquot of the incubation mixture was subjected to 4-20% gradient
SDS-PAGE, and then the separated proteins were transferred onto a
nitrocellulose membrane. The ST6Gal I-FLAG protein was detected by
anti-FLAG antibody, and E41 form was detected by the E41 antibody.
Several protease inhibitors such as amastatin, bestatin,
1,10-phenanthrolin, and EDTA were added to the reaction mixture to
for the purpose of examining their inhibitory potency.
 |
RESULTS |
Secreted ST6Gal I from the Cells Starts at
Glu41--
Previously, we used ST6Gal I as a model
molecule for studying how glycosyltransferases are cleaved and secreted
from the cells (24). We found that BACE1 was responsible for the
cleavage and secretion of ST6Gal I, i.e. overexpression of
BACE1 together with rat ST6Gal I in COS cells increased the secretion
of a soluble ST6Gal I, which had a
Glu41-Phe42-Gln43-Met44-Pro45-Lys46
sequence at the NH2 terminus of soluble ST6Gal I
(19). We prepared a polyclonal antibody, E41, that
recognized the NH2-terminal sequence of soluble ST6Gal I. In the present study, the specificity of the E41 antibody was further
characterized by a pre-absorption experiment showing that the antibody
was absorbed by the peptide EFQMPK but not by FQMPK (Fig.
1A). Using the E41 antibody,
we also examined the secretion of endogenous ST6Gal I in rat hepatoma FTO2B cells, which express high levels of the ST6Gal I protein. Soluble
ST6Gal I, immunoprecipitated with anti-ST6Gal I antibody from the media
of FTO2B cells, was detected with the E41 antibody (Fig.
1B). With BACE1 overexpression in FTO2B cells, the ST6Gal I
secretion markedly increased, and the increased soluble enzyme was also
recognized by the E41 antibody. A similar form of soluble ST6Gal I was
also present in rat plasma. The plasma enzyme was partially purified
using CDP-hexanolamine-agarose resin and subjected to immunoblotting
analysis. The plasma enzyme reacted with the E41 antibody as well as
anti-ST6Gal I antibody (Fig. 1C). By pre-absorption of E41
antibody with peptide EFQMPK, E41 staining of the plasma enzyme was
reduced (data not shown). These data suggest that in vivo
cleavage and secretion of endogenous ST6Gal I are also mediated by
BACE1.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 1.
Soluble ST6Gal I secreted from rat hepatoma
cells started at Glu41. A, COS
cells were transfected with ST6Gal I FLAG-pSVL. After 48 h,
soluble ST6Gal I-FLAG in the media were pulled down with
M2-agarose and then detected with either the E41 or anti-ST6Gal I
antibody (Ab.). The E41 antibody was pre-absorbed with
peptide EFQMPKC (10 µg/ml), FQMPKC (10 µg/ml), or control buffer.
B, FTO2B cells were transiently transfected with either
human BACE1 in the pcDNA3.1 expression vector or control pcDNA
vector. At 48 h after transfection, soluble ST6Gal I secreted in
the media was immunoprecipitated with the anti-ST6Gal I antibody.
Immunoprecipitants were analyzed by immunoblotting with either
anti-ST6Gal I or the E41 polyclonal antibody (19). C, 20 µl of CDP-hexanolamine-agarose was added to 200 µl of rat plasma.
Immobilized ST6Gal I to the beads was detected by
immunoblotting with either anti-ST6Gal I or the E41 polyclonal
antibody.
|
|
BACE1 Cleaved ST6Gal I-derived Peptide between Leu37
and Gln38--
We confirmed that the secreted ST6Gal I
starts at Glu41 in vivo as well as in
cultured cells (19), but in vitro studies by others (21, 22)
on BACE1 cleavage site preference showed that Lys40 residue
at the P1 position is not a preferable amino acid for BACE1 cleavage.
We therefore analyzed BACE1-dependent cleavage of a peptide
substrate, DYEALTLQAKEFQMPKSQE, which corresponds to
Asp31~Glu49 of ST6Gal I sequence. The
peptide substrate was incubated with purified BACE1-Fc chimera, and the
products yielded were analyzed by reversed-phase HPLC. We detected two
peptide peaks as products, the retention times of which corresponded to
those of authentic peptides, DYEALTL and QAKEFQMPKSQE (Fig.
2, A and
B). These products were subjected to MALDI-TOF MS analysis,
and their protonated molecular ions, [M+H]+, were
observed at m/z 824 and 1451 (Table
I and Fig. 2, C and D). Several other peaks (marked with an asterisk)
in the HPLC chromatogram were also observed in the control reaction
mixture without peptide substrate and shown by MALDI-TOF MS analysis to be non-peptide components. We did not detect peptide peaks
corresponding to DYEALTLQAK and EFQMPKSQE on HPLC and MALTI-TOFMS
analyses. This result indicates that BACE1-Fc cleaves the peptide
substrate exclusively between Leu37 and
Gln38.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 2.
MALDI-TOF MS of the reaction products.
The BACE1-Fc was incubated with the synthetic peptide
DYEALTLQAKEFQMPKSQE (ST-WT). The reaction products were
then analyzed by reversed-phase HPLC. Both product and substrate were
monitored by absorbance at 215 nm. Each separated peak was then
analyzed by MALDI-TOF MS. The peptide substrate (ST-WT) and
its expected peptide fragments were then successfully separated by
reversed-phase HPLC. A, cleaved peptides and substrate
peptide in the reaction products were separated by reversed-phase HPLC. The retention times
of peaks X, Y, and Z corresponded to
those of authentic peptides, QAKEFQMPKSQE, DYEALTLQAKEFQMPKSQE, and
DYEALTL, respectively. Several other peaks, each marked with an
asterisk, were also observed in the control reaction mixture
without peptide substrate and were demonstrated by MALDI-TOF MS
analysis to be non-peptide components. B, protonated
molecular ions [M+H]+ of two major products (peaks
Z and X) were observed at
m/z 824 (C) and 1451 (D) by
MALDI-TOF MS.
|
|
We further characterized the cleavage of the ST6Gal I peptide, by
comparison with those of APPwt (KTEEISEVKMDAEFRHDSG) and APPsw (KTEEISEVNLDAEFRHDSG) peptides, which cover the
-cleavage site of APP. The APPsw peptide was the more
preferable substrate (Fig. 3). BACE1-Fc
cleaved ST6Gal I peptide with higher efficiency than APPwt
peptide. By the addition of a BACE inhibitor at 0.1 µM,
cleavage of the STGal I peptide, as well as that of the
APPsw peptide, was inhibited nearly 50%. When mutant
substrate ST-LA was used, in which Leu at the P1 position was replaced
by Ala, cleavage efficiency was reduced to 40% compared with the
wild-type substrate (ST-WT), suggesting that the Leu residue
in the ST6Gal I peptide is recognized as a preferable amino acid at the
P1 position. This result is consistent with those reported previously
by others (21, 22); Leu at the P1 position in the APP sequence
is recognized preferentially by BACE1.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 3.
BACE1-Fc-dependent cleavage of
APP-derived or ST6Gal I-derived peptides. The BACE1-Fc protein,
expressed in COS cells and then purified with protein A-Sepharose, was
incubated with a series of synthetic peptides (APP wild type
(APP-WT), KTEEISEVKMDAEFRHDSG; APP Swedish mutant
(APP-SW), KTEEISEVNLDAEFRHDSG; ST6Gal I wild type substrate
(ST-WT), DYEALTLQAKEFQMPKSQE; and ST6Gal IL37A
mutant (ST-LA), DYEALTAQAKEFQMPKSQE) for 16 h.
KTEEISEVN(Sta)VAEF (0.1 µM) was used as the BACE1
inhibitor. Cleaved peptides and substrate were separated by
reversed-phase HPLC. The rate of BACE1-dependent cleavage
was expressed as the ratio (%, mean ± S.D., n = 3) of the product to the total peptide signal by measuring their
absorbance at 215 nm.
|
|
BACE1 Cleaved Protein A-ST6Gal I Chimera between Leu37
and Gln38 in Vitro--
We also examined
BACE1-dependent cleavage of protein A-ST6Gal I-FLAG
chimera, which lacked a transmembrane domain, instead containing a
signal peptide plus protein A and COOH terminally tagged with
FLAG. The Protein A-ST6Gal I chimera was reported to have
sialyltransferase activity (25), suggesting that the catalytic domain
was correctly folded as a native enzyme. The chimeric protein was
incubated with BACE1-Fc. The cleaved protein was purified to near
homogeneity (Fig. 4) and then transferred onto a polyvinylidene difluoride membrane for sequencing.
Micro-sequence analysis revealed that the
NH2-terminal sequence of the soluble protein was
exclusively qAKEFQMpks, where lowercase letters indicate ambiguous
identification (the first cycle of amino acid also contained Gly, most
likely derived from the transfer buffer which contained a large amount
of glycine). Thus, protein A-ST6Gal I-FLAG was cleaved exclusively
between Leu37 and Gln38, as was the case with
the peptide substrate.

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 4.
Purity of the protein A-ST6Gal I-FLAG cleaved
by BACE1-Fc. The BACE1-Fc protein was incubated with protein
A-ST6Gal I-FLAG. An aliquot of the products was analyzed by SDS-PAGE.
Proteins were detected with silver nitrate.
|
|
We also examined whether BACE1 recognized Lys40
at P3' in the ST6Gal I sequence. We constructed a mutant
substrate in which Lys40 of protein A-ST6Gal I-FLAG was
replaced with Ala. BACE1-Fc cleaved the protein A-ST6Gal
IK40A-FLAG with efficiency similar to the wild type protein
(Fig. 5). When we took a time course to
compare the cleavage efficiency between these protein A-ST6Gal I
proteins in detail, protein A-ST6Gal IK40A-FLAG was cleaved
at almost the same efficiency as the wild type (data not shown).
These results suggest that Lys40 of ST6Gal I is not
critical for BACE1 recognition.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 5.
BACE1 did not recognize
Lys40 of ST6Gal I. Purified BACE1-Fc was
incubated with protein A-ST6Gal I-FLAG and then analyzed by
immunoblotting with anti-FLAG antibody.
|
|
Exopeptidase Activity Trimmed NH2-terminal QAK Sequence
of the Q38 form of ST6Gal I--
Our results described above suggest
that BACE1 cleaves ST6Gal I between Leu37 and
Gln38; the cleaved product (Q38 form) was three amino acids
longer than the secreted one (E41 form). We speculated that the
three-amino acid QAK sequence of the Q38 form was removed by
endogenous exopeptidase(s) before secretion, and hence we set up an
assay for detecting possible exopeptidase activity. As a substrate for
the assay we used the Q38 form of ST6Gal I, which had been prepared by
cleavage of protein A-ST6Gal I-FLAG with BACE1-Fc. When the substrate
was mixed with detergent extracts of a microsomal fraction of COS
cells, its NH2-terminal QAK sequence was
trimmed to generate the E41 form of ST6Gal I, which was detected by the
E41 antibody (Fig. 6A). This
suggests that the extracts contained protease activity, as we had
expected. The protease activity was not detected by adding the
microsomal fraction without detergent treatment. As shown in Fig.
6B, we also detected such a protease activity in the
detergent-solubilized Golgi fraction prepared from rat livers (27).
These results suggest that the protease was localized mainly in the
Golgi apparatus and its catalytic domain faces the luminal side. To
rule out the possibility that the protease has endoprotease activity,
we added protein A-ST6Gal I-FLAG as substrate to the
detergent-solubilized Golgi fraction as the enzyme fraction to see
whether the ST6Gal I E41 form was produced. Because we did not detect
the production of ST6Gal I E41 form, we surmised that the
protease in the Golgi fraction is a kind of exopeptidase that acts on
ST6Gal I-Q38 form. We also confirmed that BACE1 itself has no trimming
activity, because we did not detect production of the ST6Gal I-Q38 form without detergent-solubilized Golgi fraction (Fig. 6B). Thus
we demonstrate the presence of luminal exopeptidase activity that trims
the NH2-terminal QAK-sequence of the Q38 form. To further characterize the trimming activity, we tested the sensitivity of the
putative peptidase to the various aminopeptidase inhibitors (Table II). As both EDTA and
1,10-pheananthroline significantly inhibited the peptidase activity at
1 mM, the enzyme seems to have metallopeptidase-like
character. Moreover, we found that bestatin, but not amastatin,
significantly inhibits the activity. These results suggest that the
protease that trims NH2-terminal QAK sequence of ST6Gal
I-Q38 form belongs to the bestatin-sensitive metalloaminopeptidase.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 6.
NH2-terminal QAK
sequence of a ST6Gal I-Q38 form was truncated by exopeptidase activity
in detergent-treated Golgi membrane fraction. A,
protein A-ST6Gal I-FLAG was cleaved by BACE1-Fc. The resulting Q38
form, in which the NH2-terminal amino acid sequence starts
at Gln38, was incubated with either a microsomal or
cytosolic fraction prepared from COS cells. The microsomal fraction was
pretreated with or without 1% Triton X-100. The product generated was
analyzed by immunoblotting the anti-FLAG or E41 antibodies.
B, protein A-ST6Gal I-FLAG was incubated with either the
BACE-Fc or Golgi membrane fraction (10 µg of protein) or both. After
incubation for 2 h, the reaction products were analyzed by
immunoblotting with the E41 antibody.
|
|
View this table:
[in this window]
[in a new window]
|
Table II
Effect of various protease inhibitors on the exopeptidase activity
ST6Gal I-FLAG Q38 form was incubated with detergent-solubilized Golgi
membrane fraction (10 µg of protein) in the presence or absence of 1 mM protease inhibitors. The reaction products were then
analyzed by immunoblotting with both the anti-FLAG and the E41
antibodies. The amounts of total ST6Gal-FLAG substrate and the product
E41 form were quantitated using an LAS 1000 chemiluminescence analyzer
(Fuji). Values are the average of relative residual activity obtained
from the three independent experiments when E41 form/total substrate
was taken as 100% in the control sample.
|
|
ST6Gal I Q38-form Was Not Secreted from the
Cell--
We show here that BACE1 cleaves ST6Gal I between
Leu37 and Gln38. Although amino-terminal
sequencing analysis showed that most of soluble secreted ST6Gal I
started at Glu41, there might be small amount of soluble
ST6Gal I starting at Gln38. We prepared an antibody that
specifically recognizes the NH2 terminus of ST6Gal I-Q38
form. This Q38 antibody failed to detect soluble secreted ST6Gal I from
COS cells, even though E41 antibody did detect the soluble ST6Gal I
(Fig. 7). The results indicate that
secreted ST6GalI start mostly at Glu41.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 7.
Soluble secreted ST6Gal I was not detected
with Q38 antibody, which recognizes the ST6Gal I-Q38 form. Protein
A-ST6Gal I-FLAG was cleaved by BACE1-Fc. The resulting Q38 form, in
which the NH2-terminal amino acid sequence starts at
Gln38, was analyzed by immunoblotting with either the Q38
or E41 polyclonal antibodies (Ab.). As a negative control,
Q38 antibody pre-absorbed with peptide QAKEFQ (10 µg/ml) was also
used for staining. ST6Gal I-FLAG was overexpressed in COS cells, and
soluble ST6Gal I-FLAG proteins secreted in the media were pulled down
with M2-agarose and then detected with either the Q38 or E41 polyclonal
antibodies as well.
|
|
K40A Mutation Reduced the Efficiency of Exopeptidase
Activity--
A simple explanation for the efficient conversion from
ST6Gal I-Q38 form to E41 form is that the amount of exopeptidase
activity exceeds that of the substrate. An alternative idea is that
exopeptidase-dependent trimming may be a prerequisite for
secretion. The latter speculation is supported by our previous cellular
experiment (19) in which a ST6Gal IK40A mutant expressed in
COS cells was poorly secreted (40% of wild type level), although this
mutation did not affect in vitro BACE1-dependent
cleavage (Fig. 5). The mutation may affect exopeptidase-dependent
trimming and then the secretory process. We therefore compared the
exopeptidase activity toward ST6Gal I-Q38 form with that for its K40A
mutant. Exopeptidase cleavage rate of the K40A mutant was half of wild
type protein over a particular time course; i.e.
when we set the cleavage rate of wild type after a 2-h incubation as
100%, the cleavage rate of K40A was 46%. After a 1-h
incubation, the cleavage rate of the wild type was 40% and that of
K40A was 17% (Fig. 8). These results
suggest that K40A mutation reduces the efficiency of the exopeptidase
activity.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 8.
Exopeptidase activity, which trims the
NH2-terminal QAK sequence of ST6Gal I-Q38 form
and its K40A mutant. The ST6Gal I-FLAG-Q38 form and its K40A
mutant were prepared from their respective protein A-ST6Gal I
proteins by BACE1 cleavage. The resulting substrates were then
incubated with 10 µg of Golgi fraction solubilized with 1% Triton
X-100 for 0, 0.5, 1, and 2 h. Each product was divided in half;
one-half of the sample was analyzed by immunoblotting with anti-FLAG
antibody to quantitate total ST6Gal I-FLAG and the other half was
analyzed with E41 antibody to quantitate the E41 form by Luminoimage
Analyzer LAS-1000 PLUS (Fujifilm). The percentage of cleavage was an
average of two independent experiments, with the cleavage rate of wild
type ST6Gal I (E41 form/total ST6Gal I-FLAG) after 2 h of
incubation taken as 100%.
|
|
 |
DISCUSSION |
In the present experiment, we used ST6Gal I peptide and protein
A-ST6Gal I-FLAG as substrates for BACE1, both of which were cleaved
between Leu37 and Gln38. The observation fits
other previous reports (20-22) describing residue preferences for
subsites of BACE1; i.e. P1 site is most stringently
recognized by BACE1 and only large hydrophobic residues such as Leu,
Phe, Met, and Tyr are accepted at this position. Previous reports (22,
28) showing that BACE1 prefers bulky hydrophobic residues at the P3
position also correspond well with the presence of Leu at the P3 of
ST6Gal I, suggesting that BACE1-Fc preferably recognizes the
Leu35-Thr36-Leu37 sequence of
ST6Gal I and cleaves exclusively at this site. This cleavage is also
supported by our own previous cellular experiment, in which replacement
of Leu37 with Ala (ST6Gal IL37A), an
unfavorable substitution for BACE1 cleavage in vitro,
significantly reduced the secretion from the cells (61 ± 20% of
the control level, p < 0.05). Taken together, we
speculate that BACE1 cleaves ST6Gal I between Leu37 and
Gln38 inside the cells and generates the Q38 form of
soluble ST6Gal I as an initial product.
Our data suggest that luminal exopeptidase activity is involved in the
trimming of the NH2-terminal flanking sequence of Q38 form
(Fig. 6). A proposed model for ST6Gal I processing and secretion is
summarized in Fig. 9. In some cases,
exopeptidase activities are critical for the processing of luminal
proteins as well as physiologically important peptides (29-31). A
recent report by Komlosh et al. (32) has identified
a novel luminal endoplasmic reticulum exopeptidase that is involved in
the trimming of major histocompatibility complex class I antigenic
peptides. It is notable that the main portion of soluble ST6Gal I
secreted from the cells is the E41 form (24); we did not detect
the Q38 form in the culture medium (Fig. 7), suggesting
that the Q38 form generated by BACE1 is efficiently converted to the
E41 form before secretion. Moreover, our previous observation,
in which a ST6Gal IK40A mutant expressed in COS cells was
poorly secreted (24), supports the idea that the mutation may affect
exopeptidase-dependent trimming and then the secretory process.
Indeed, the present data indicate that the K40A mutation somewhat
reduces exopeptidase trimming efficiency. At present, however, we
cannot exclude the possibility that an additional unveiled mechanism
other than the ST6Gal I cleavage process exists to regulate the ST6Gal
I secretion. The data presented here suggest that the exopeptidase has
a bestatin-sensitive metalloprotease-like character. Because we
used a crude Golgi membrane fraction as an enzyme source in this study,
purification of this exopeptidase will be required for further
characterization in the future. Identification and characterization of
the exopeptidase will be important for a better understanding of the
molecular mechanisms underlying the cleavage and secretion of ST6Gal
I.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 9.
Proposed mechanism of ST6Gal I processing and
secretion in the cell. Membrane-bound ST6Gal I protein is cleaved
initially by BACE1 to produce the soluble Q38 form, which starts at
Gln38. Luminal aminopeptidase(s) activity then trims its
NH2-terminal
Gln38-Ala39-Lys40 sequence, with
the resultant E41 form secreted from the cell (21).
|
|
 |
ACKNOWLEDGEMENTS |
We thank Dr. Tae-Wan Kim (Harvard Medical
School, Boston, MA) for providing human BACE1 myc-pcDNA and Dr.
Carolyn Bruzdzinski (University of Illinois, Chicago) for FTO2B rat
hepatoma cells. We also thank Dr. Marcos Milla (University of
Pennsylvania School of Medicine, Philadelphia), Dr. Shoichi Ishiura
(University of Tokyo, Japan), and Drs. Masafumi Tsujimoto and Akira
Hattori (RIKEN) for helpful discussions on the assay for exopeptidase
activity and Drs. Akemi Suzuki and Tamio Yamakawa (RIKEN Frontier
Research System) for encouragement throughout the study. Kazuko
Hashimoto is also acknowledged for invaluable secretarial assistance.
 |
FOOTNOTES |
*
This work was supported in part by Grants-in-aid 13780512 (to S. K.), 14030089 (to S. K.), and 14016003 (to Y. H.) for
Scientific Research from the Ministry of Education, Science, Sports,
and Culture of Japan, by a Sasagawa scientific research grant from the
Japan Science Society (to S. K.), and by funding from the Yamanouchi
Foundation for Research on Metabolic Disorders (to Y. H.) and RIKEN,
the Intra-RIKEN Collaboration Fund (to Y. H.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Glyco-chain
Functions Laboratory, Supra-biomolecular System Group, Frontier
Research System, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan. Tel.: 81-48-467-9613; Fax: 81-48-462-4690; E-mail:
yasua@postman.riken.go.jp.
Published, JBC Papers in Press, December 7, 2002, DOI 10.1074/jbc.M206262200
 |
ABBREVIATIONS |
The abbreviations used are:
A
, amyloid
-peptide;
APP, amyloid precursor protein;
BACE,
-site
APP-cleaving enzyme;
Fc, the hinge and constant region of IgG;
HPLC, high performance liquid chromatography;
MALDI-TOF MS, matrix-assisted
laser desorption/ionization time-of-flight mass spectrometry.
 |
REFERENCES |
1.
|
Selkoe, D. J.
(2001)
Physiol. Rev.
81,
741-766[Abstract/Free Full Text]
|
2.
|
De Strooper, B.,
Saftig, P.,
Craessaerts, K.,
Vanderstichele, H.,
Guhde, G.,
Annaert, W.,
Von Figura, K.,
and Van Leuven, F.
(1998)
Nature
391,
387-390[CrossRef][Medline]
[Order article via Infotrieve]
|
3.
|
Wolfe, M. S.,
Xia, W.,
Ostaszewski, B. L.,
Diehl, T. S.,
Kimberly, W. T.,
and Selkoe, D. J.
(1999)
Nature
398,
513-517[CrossRef][Medline]
[Order article via Infotrieve]
|
4.
|
Lammich, S.,
Kojro, E.,
Postina, R.,
Gilbert, S.,
Pfeiffer, R.,
Jasionowski, M.,
Haass, C.,
and Fahrenholz, F.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
3922-3927[Abstract/Free Full Text]
|
5.
|
Buxbaum, J. D.,
Liu, K. N.,
Luo, Y.,
Slack, J. L.,
Stocking, K. L.,
Peschon, J. J.,
Johnson, R. S.,
Castner, B. J.,
Cerretti, D. P.,
and Black, R. A.
(1998)
J. Biol. Chem.
273,
27765-27767[Abstract/Free Full Text]
|
6.
|
Vassar, R.,
Bennett, B. D.,
Babu-Khan, S.,
Kahn, S.,
Mendiaz, E. A.,
Denis, P.,
Teplow, D. B.,
Ross, S.,
Amarante, P.,
Loeloff, R.,
Luo, Y.,
Fisher, S.,
Fuller, J.,
Edenson, S.,
Lile, J.,
Jarosinski, M. A.,
Biere, A. L.,
Curran, E.,
Burgess, T.,
Louis, J. C.,
Collins, F.,
Treanor, J.,
Rogers, G.,
and Citron, M.
(1999)
Science
286,
735-741[Abstract/Free Full Text]
|
7.
|
Sinha, S.,
Anderson, J. P.,
Barbour, R.,
Basi, G. S.,
Caccavello, R.,
Davis, D.,
Doan, M.,
Dovey, H. F.,
Frigon, N.,
Hong, J.,
Jacobson-Croak, K.,
Jewett, N.,
Keim, P.,
Knops, J.,
Lieberburg, I.,
Power, M.,
Tan, H.,
Tatsuno, G.,
Tung, J.,
Schenk, D.,
Seubert, P.,
Suomensaari, S. M.,
Wang, S.,
Walker, D.,
John, V.,
et al..
(1999)
Nature
402,
537-540[CrossRef][Medline]
[Order article via Infotrieve]
|
8.
|
Yan, R.,
Bienkowski, M. J.,
Shuck, M. E.,
Miao, H.,
Tory, M. C.,
Pauley, A. M.,
Brashier, J. R.,
Stratman, N. C.,
Mathews, W. R.,
Buhl, A. E.,
Carter, D. B.,
Tomasselli, A. G.,
Parodi, L. A.,
Heinrikson, R. L.,
and Gurney, M. E.
(1999)
Nature
402,
533-537[CrossRef][Medline]
[Order article via Infotrieve]
|
9.
|
Luo, Y.,
Bolon, B.,
Kahn, S.,
Bennett, B. D.,
Babu-Khan, S.,
Denis, P.,
Fan, W.,
Kha, H.,
Zhang, J.,
Gong, Y.,
Martin, L.,
Louis, J. C.,
Yan, Q.,
Richards, W. G.,
Citron, M.,
and Vassar, R.
(2001)
Nat. Neurosci.
4,
231-232[CrossRef][Medline]
[Order article via Infotrieve]
|
10.
|
Cai, H.,
Wang, Y.,
McCarthy, D.,
Wen, H.,
Borchelt, D. R.,
Price, D. L.,
and Wong, P. C.
(2001)
Nat. Neurosci.
4,
233-234[CrossRef][Medline]
[Order article via Infotrieve]
|
11.
|
De Strooper, B.,
Annaert, W.,
Cupers, P.,
Saftig, P.,
Craessaerts, K.,
Mumm, J. S.,
Schroeter, E. H.,
Schrijvers, V.,
Wolfe, M. S.,
Ray, W. J.,
Goate, A.,
and Kopan, R.
(1999)
Nature
398,
518-522[CrossRef][Medline]
[Order article via Infotrieve]
|
12.
|
Struhl, G.,
and Greenwald, I.
(1999)
Nature
398,
522-525[CrossRef][Medline]
[Order article via Infotrieve]
|
13.
|
Ni, C. Y.,
Murphy, M. P.,
Golde, T. E.,
and Carpenter, G.
(2001)
Science
294,
2179-2181[Abstract/Free Full Text]
|
14.
|
Lee, H. J.,
Jung, K. M.,
Huang, Y. Z.,
Bennett, L. B.,
Lee, J. S.,
Mei, L.,
and Kim, T. W.
(2002)
J. Biol. Chem.
277,
6318-6323[Abstract/Free Full Text]
|
15.
|
Li, Y. M.,
Xu, M.,
Lai, M. T.,
Huang, Q.,
Castro, J. L.,
DiMuzio-Mower, J.,
Harrison, T.,
Lellis, C.,
Nadin, A.,
Neduvelil, J. G.,
Register, R. B.,
Sardana, M. K.,
Shearman, M. S.,
Smith, A. L.,
Shi, X. P.,
Yin, K. C.,
Shafer, J. A.,
and Gardell, S. J.
(2000)
Nature
405,
689-694[CrossRef][Medline]
[Order article via Infotrieve]
|
16.
|
Esler, W. P.,
Kimberly, W. T.,
Ostaszewski, B. L.,
Diehl, T. S.,
Moore, C. L.,
Tsai, J. Y.,
Rahmati, T.,
Xia, W.,
Selkoe, D. J.,
and Wolfe, M. S.
(2000)
Nat. Cell Biol.
2,
428-434[CrossRef][Medline]
[Order article via Infotrieve]
|
17.
|
Doerfler, P.,
Shearman, M. S.,
and Perlmutter, R. M.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
9312-9317[Abstract/Free Full Text]
|
18.
|
Marcinkeviciene, J.,
Luo, Y.,
Graciani, N. R.,
Combs, A. P.,
and Copeland, R. A.
(2001)
J. Biol. Chem.
276,
23790-23794[Abstract/Free Full Text]
|
19.
|
Kitazume, S.,
Tachida, Y.,
Oka, R.,
Shirotani, K.,
Saido, T. C.,
and Hashimoto, Y.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
13554-13559[Abstract/Free Full Text]
|
20.
|
Citron, M.,
Teplow, D. B.,
and Selkoe, D. J.
(1995)
Neuron
14,
661-670[Medline]
[Order article via Infotrieve]
|
21.
|
Turner, R. T., III,
Koelsch, G.,
Hong, L.,
Castanheira, P.,
Ermolieff, J.,
Ghosh, A. K.,
Tang, J.,
Castenheira, P.,
and Ghosh, A.
(2001)
Biochemistry
40,
10001-10006[CrossRef][Medline]
[Order article via Infotrieve]
|
22.
|
Gruninger-Leitch, F.,
Schlatter, D.,
Kung, E.,
Nelbock, P.,
and Dobeli, H.
(2002)
J. Biol. Chem.
277,
4687-4693[Abstract/Free Full Text]
|
23.
|
Saido, T. C.,
Iwatsubo, T.,
Mann, D. M.,
Shimada, H.,
Ihara, Y.,
and Kawashima, S.
(1995)
Neuron
14,
457-466[Medline]
[Order article via Infotrieve]
|
24.
|
Kitazume-Kawaguchi, S.,
Dohmae, N.,
Takio, K.,
Tsuji, S.,
and Colley, K. J.
(1999)
Glycobiology
9,
1397-1406[Abstract/Free Full Text]
|
25.
|
Kitazume-Kawaguchi, S.,
Kabata, S.,
and Arita, M.
(2001)
J. Biol. Chem.
276,
15696-15703[Abstract/Free Full Text]
|
26.
|
Laemmli, U. K.
(1970)
Nature
227,
680-685[Medline]
[Order article via Infotrieve]
|
27.
|
Leelavathi, D. E.,
Estes, L. W.,
Feingold, D. S.,
and Lombardi, B.
(1970)
Biochim. Biophys. Acta
211,
124-138
|
28.
|
Hong, L.,
Koelsch, G.,
Lin, X.,
Wu, S.,
Terzyan, S.,
Ghosh, A. K.,
Zhang, X. C.,
and Tang, J.
(2000)
Science
290,
150-153[Abstract/Free Full Text]
|
29.
|
Chesneau, V.,
Pierotti, A. R.,
Barre, N.,
Creminon, C.,
Tougard, C.,
and Cohen, P.
(1994)
J. Biol. Chem.
269,
2056-2061[Abstract/Free Full Text]
|
30.
|
Serwold, T.,
Gaw, S.,
and Shastri, N.
(2001)
Nat. Immunol.
2,
644-651[CrossRef][Medline]
[Order article via Infotrieve]
|
31.
|
Stoltze, L.,
Schirle, M.,
Schwarz, G.,
Schroter, C.,
Thompson, M. W.,
Hersh, L. B.,
Kalbacher, H.,
Stevanovic, S.,
Rammensee, H. G.,
and Schild, H.
(2000)
Nat. Immunol.
1,
413-418[CrossRef][Medline]
[Order article via Infotrieve]
|
32.
|
Komlosh, A.,
Momburg, F.,
Weinschenk, T.,
Emmerich, N.,
Schild, H.,
Nadav, E.,
Shaked, I.,
and Reiss, Y.
(2001)
J. Biol. Chem.
276,
30050-30056[Abstract/Free Full Text]
|
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.