From the Department of Analytical Sciences,
SmithKline Beecham Pharmaceuticals, Harlow, Essex CM19 5AW, United
Kingdom, the § Department of Neurociences, SmithKline
Beecham Pharmaceuticals, Harlow, Essex CM19 5AW, United Kingdom, the
¶ Department of Protein Biochemistry, SmithKline Beecham
Pharmaceuticals, King of Prussia, Pennsylvania 19406, the
Department of Molecular Screening Technologies, SmithKline
Beecham Pharmaceuticals, Harlow, Essex CM19 5AW, United Kingdom, the
** Department of Computational and Structural Sciences, SmithKline
Beecham Pharmaceuticals, Harlow, Essex CM19 5AW, United Kingdom, and
the
Department of Gene Expression Sciences,
SmithKline Beecham Pharmaceuticals, King of Prussia,
Pennsylvania 19406
Received for publication, October 13, 2000, and in revised form, January 29, 2001
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ABSTRACT |
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Amyloid 39-42 One of the key pathological features of Alzheimer's disease is
the formation of brain plaques primarily due to the fibrilization of
amyloid 39-42 Asp-2 contains four potential N-linked glycosylation sites
at the following asparagine residues: Asn153,
Asn172, Asn223, and Asn354. The
close proximity of some of these glycosylated sites to the three
intramolecular disulfide linkages and the catalytic site of Asp-2 has
been the subject of more recent interest (10), especially because the
type and extent glycosylation of a protein can have a profound effect
on its physico-chemical properties (11). For instance, it is well known
that N-linked oligosaccharides can influence
glycoprotein folding in the endoplasmic reticulum (12) and can protect
a protein from protease attack (13).
The N-linked oligosaccharide structures on Asp-2 have not
yet been characterized in detail, although it has been reported that
all four asparagine sites have a heterogeneous mixture of carbohydrate
attached (10). Another report has indicated that the glycans in mature
Asp-2 are of the complex type, as removal of this carbohydrate is not
possible by endoglycosidase H treatment (14).
In this study we have released and analyzed the N-linked
glycans from Asp-2 expressed as an Fc fusion in CHO and COS cells, expressed in baculovirus-infected insect cells and expressed in CHO
cells after subsequent proteolytic removal of the Fc moiety. We have
identified the different oligosaccharides attached to Asp-2 and
demonstrate that the heterogeneity in glycan distribution is very
similar when Asp-2 is expressed in mammalian cell systems; under these
conditions, only complex-type glycans are attached to the asparagine
residues of the four glycosylation motifs found in the primary sequence
of this protease. We have also used site-directed mutagenesis to
ascertain that glycosylation makes a significant contribution to the
activity of Asp-2.
Materials--
Gradient Tris-glycine (4-20%) slab gels were
purchased from NOVEX (San Diego, CA). PNGase F was obtained from Roche
Molecular Biochemicals (Lewes, United Kingdom (UK)). Dithiothreitol was obtained from Calbiochem (La Jolla, CA). Arthrobacter
ureafaciens sialidase was obtained from Glyko (Novata, CA). The
following samples of Asp-2 (numbering from the initiator methionine)
and the corresponding Fc fusion proteins were used for glycan release: Asp-(1-501), wild-type full length Asp-2 expressed in
baculovirus-infected SF9 cells; Asp-(1-460Fc), Asp-2Fc expressed in
COS and CHOE1a cells; Asp-(1-460), Asp-2Fc, expressed in CHO E1a
cells, purified and proteolytically processed to remove the Fc.
Expression and Purification of Asp-2 in Mammalian Cells--
The
various Asp2 Fc expression constructs were transiently transfected into
COS-1 cells using LipofectAMINE Plus (Life Technologies, Inc.)
according to the manufacturer's instructions with minor modifications.
For stable cell line selection, the plasmids were linearized by
digestion with NotI (15 µg of DNA, 37 °C, overnight), precipitated and resuspended into 50 µl of 1× TE buffer (10 mM Tris, 1 mM EDTA, pH 7.5). The DNA was
electroporated, using a Bio-Rad Gene Pulser into a CHO E1A cell line
(derived from DG-44 (15) adapted for growth in suspension in
maintenance medium) using the technique of Hensley et al.
(16). The cells were plated into 96-well culture plates at 5 × 105 cells/plate in maintenance medium for 24 h prior
to selection. Cells were selected in maintenance medium without
nucleosides (selection medium). Conditioned medium from individual
colonies was assayed using an electrochemiluminescence detection method on an Origen analyzer (IGEN) the technology reviewed by Yang et al. (17). A colony expressing high levels of Asp-2 was used to
inoculate 100 ml of selection medium to generate conditioned medium
from which Asp-2 Fc was purified by affinity chromatography on
immobilized Protein A.
Expression and Purification of Asp-2 in Baculovirus-infected SF9
Cells--
The cDNA encoding Asp2 (amino acids 1-501) with a
COOH-terminal six-histidine tag was cloned into pFastbac (Life
Technologies, Inc.). Recombinant virus was generated according to
manufacturer's protocols. Protein was purified from SF9 cells (Life
Technologies, Inc.), which were infected with virus as described, and
Asp2 protein was purified by metal chelating and lentil lectin
chromatography.2
Generation of Expression Constructs and Site-directed
Mutagenesis--
The cDNA encoding amino acids 1-460 of Asp-2
(GenBankTM accession no. AF204943) was subcloned upstream of the
cDNA encoding amino acids 99-330 of human IgG1, and this construct
was subcloned into the mammalian expression vector pCDN (18). To
generate the appropriate glycosylation mutants (see Table I for
mutations), the Quikchange site-directed mutagenesis kit (Stratagene,
La Jolla, CA) was used according to manufacturer's instructions. All
mutations were confirmed by sequence analysis, and the mutated cDNA
was recloned into the parental vector.
Activity Analysis--
Peptide (Swedish variant
ISEVNLDAEFRHDK(dnp)G) (50 µM) was incubated with Asp2-Fc
and Asp2-Fc mutants (50 nM) in buffer containing 50 mM sodium acetate, 20 mM NaCl, pH 4.5, for
30 min in a final assay volume of 100 µl. The reaction was stopped by
addition of four volumes of 5% trifluoroacetic acid. The assay
components were loaded onto a POROS R1 column (Applied Biosystems) in
0.08% trifluoroacetic acid and eluted with a linear gradient of 0.08% trifluoroacetic acid in acetonitrile (linear gradient from 5% to 50%
acetonitrile over 15 min). The chromogenic dnp group on the peptide and
product was followed by monitoring at 360 nm. Enzyme concentrations
were determined by absorbance at 280 nm in each case.
Sodium Dodecyl Sulfate (SDS)-Polyacrylamide Gel
Electrophoresis--
Proteins (10-20 µg) were separated on
one-dimensional SDS-polyacrylamide gels using 4-20% gradient
Tris-glycine polyacrylamide slab gels (19) (10 cm × 8 cm × 1.0 mm; Novex, San Diego, CA). Proteins in the gels were stained with
Coomassie Brilliant Blue R250 dye.
Protein Reduction and Alkylation--
The Coomassie-stained
bands corresponding to the proteins of interest were excised from the
gel and washed for 30 min in distilled water. The gel slice was
dehydrated in acetonitrile for 10 min. The acetonitrile was removed and
the gel slice dried for 10 min in a vacuum centrifuge. The dried gel
slice was incubated first in 20 mM Tris-HCl, pH 8.0, containing 10 mM dithiothreitol for 1 h at 55 °C,
and second with 20 mM Tris-HCl, pH 8.0, containing 55 mM iodoacetamide for 45 min in the dark at room
temperature. The supernatant was removed and the gel slice washed in 20 mM Tris-HCl, pH 8.0, for 10 min. After removal of the
solution, the gel was dehydrated in acetonitrile and dried as before.
Enzymatic Release of Glycans--
The reduced and alkylated gel
slice was then re-hydrated for 30 min in 50 mM sodium
phosphate buffer, pH 7.2, and dehydrated again with acetonitrile. The
gel piece was then incubated with 40 µl of 50 mM sodium
phosphate buffer, pH 7.2, containing 1 unit of PNGase F (Roche
Molecular Biochemicals) overnight at 37 °C.
Extraction of Glycans after in Situ Digestion--
Glycans were
extracted from gel slices in 5% v/v formic acid for 1 h, followed
by three extractions with 60% v/v acetonitrile in 5% v/v formic acid
for 1 h (with intermittent sonication). The combined extracts were
dried in a vacuum centrifuge.
Derivatization of Glycans with 3-(Acetylamino)-6-aminoacridine
(AA-Ac)--
Extracted glycans were labeled with AA-Ac by incubating
them with 10 µl of an AA-Ac solution (2.5 mg in 200 µl of dimethyl sulfoxide/acetic acid (17:3)) and 10 µl of sodium cyanoborohydride (12.5 mg in 200 µl of dimethyl sulfoxide/acetic acid (17:3)) for 30 min at 80 °C. The reaction was stopped by drying in a vacuum centrifuge. Excess reagent was removed on an OASIS cartridge (Waters). The column was primed with 2 ml of acetonitrile followed by 2 ml of
water. The sample was loaded in 200 µl of water and washed with 1 ml
of water. The derivatized glycans were eluted with 2× 600 µl of 20%
v/v acetonitrile/water and dried in a vacuum centrifuge.
Digestion of Glycan Pools with Sialidase--
AA-Ac-labeled
oligosaccharides were digested with sialidase by incubating them with
20 µl of Arthrobacter ureafaciens sialidase (0.2 units in
100 µl of 100 mM sodium acetate, pH 5) for 18 h at
37 °C. The resulting derivatives were freeze-dried and desalted using an OASIS cartridge.
Hydrophilic Interaction Liquid Chromatography (HILC)--
HILC
was carried out on a Waters Alliance 2690 Separations module using a
GlycoSep N column (Oxford GlycoSciences, 25 cm × 3.9 mm, inner
diameter). The mobile phases were acetonitrile (solvent A) and 250 mM ammonium formate, pH 4.4 (solvent B).
The following gradient elution conditions were used: step 1, 65% A,
35% B (equilibration), 0.4 ml/min; step 2, 35-39% B for 50 min,
linear gradient; step 3, 39-58% B for 30 min, linear gradient; step
4, 58% B to 100% B for 3 min; step 5, 100% B for 10 min at 1 ml/min;
step 6, re-equilibration of column at 65% A, 35% B at 0.4 ml/min for
15 min. AA-Ac-derivatized oligosaccharides were detected on a Waters
474 fluorescence detector at an excitation wavelength of 442 nm and an
emission wavelength of 525 nm.
Mass Spectrometry--
MALDI-TOF-MS of the derivatized glycans
was carried out using a TofSpec 2E mass spectrometer (Micromass,
Manchester, UK) operated in the reflectron mode. Photon
irradiation from a 337-nm pulsed nitrogen laser and 20-kV accelerating
voltage was used. The instrument was externally calibrated using the
[M + H]+ ion peaks of the peptides substance P
(Mr 1347) and adrenocorticotropic hormone
fragment 18-39 (Mr 2465) using an
Polyacrylamide Gel Electrophoresis and In-gel Enzymatic Digestion
of Asp-2--
The recombinant proteins expressed in
baculovirus-infected SF9 cells, COS, and CHO E1a cells are shown in
Fig. 1A. The
baculovirus-expressed material has a COOH-terminal six-histidine tag
and an apparent molecular mass of 65 kDa, whereas the mammalian
expressed material has a COOH-terminal Fc tag and an apparent
molecular mass of 94 kDa. The recombinant Asp2-Fc is found as an
Asp2-Fc/Fc heterodimer as described (8). Under the reducing conditions
employed for electrophoresis, the disulfide bond of the Fc moiety is
reduced, leading to two major protein bands being visible on the gel.
The 30 kDa represents the heavy chain of the Fc domain, and the band at
~94 kDa represents the recombinant Asp-2Fc protein.
The purified recombinant protein from CHO cells after removal of the Fc
moiety has an apparent molecular mass of 65 kDa and is shown in Fig.
1B. Analysis of the carbohydrate from this material allowed
for direct comparison to the baculovirus-infected material, which
contained no Fc tag. The major band at about 65 kDa is a mixture of
both the mature protein and mature protein in which the pro-domain has
been removed. The minor protein bands migrating at ~38 and 40 kDa are
recombinant protein degraded at the carboxyl terminus (present at
<10%, as estimated from staining intensities). This was confirmed by
MALDI-TOF-MS peptide mapping (data not shown). All recombinant Asp-2 is
present as a mixture of the pro-form (amino terminus is
Thr22) and the mature form of the protein (amino terminus
is Glu46) as determined by amino-terminal sequence analysis.
After separation by electrophoresis, protein bands
corresponding to recombinant Asp-2 were excised directly from the
gel and oligosaccharides were released by incubating with PNGase
F.3 Following in-gel
enzymatic digestion, the released glycans were extracted, dried down,
and labeled with AA-Ac. This probe is highly fluorescent and is
suitable for the analysis of reducing carbohydrates by normal- and
reverse-phase high performance liquid chromatography techniques and by
mass spectrometric methods (21). The labeled oligosaccharides were
divided into three portions, two of which were digested with sialidase
overnight for comparison with the original (nondesialylated) mixture.
HILC Analysis of the AA-Ac-derivatized Glycans--
The mixture of
AA-Ac labeled glycans released from Asp-2 and the corresponding Fc
fusion protein were resolved by HILC as shown in Fig.
2. In this chromatographic method, the
order of elution is related approximately to the size of the
derivatized oligosaccharides; thus, smaller glycans have a shorter
retention time, and others containing sialic acid residues elute later. The AA-Ac glycan profile obtained from expression of Asp-2 in the
baculovirus-infected insect cells (Fig. 2A) differs from
that of the glycoprotein expressed in CHO E1a cells (Fig.
2D). A comparison of the retention time of the derivatized
oligosaccharides from the Asp-2 expressed in the baculovirus-infected
cells with those from standard ribonuclease B glycan pool revealed that
the components with an elution time of 50 min and later were the
neutral high mannose structures Man 5 to Man 9 (Table
I). This was confirmed by MALDI-TOF-MS
(described below). The major peak at ~35 min was due to the presence
of the glycan core, Man3GlcNAc2, containing a
fucose attached to the N-acetylglucosamine residue on the
reducing end of the sugar. Other lower intensity signals seen at
retention times less than 45 min are due to the addition of fucose
and/or N-acetylglucosamine to the "core" structure (see
Table I).
We established that the highest proportion of glycans released from
either COS or CHO E1a cells were acidic, containing one or more sialic
acid residues (Fig. 2, B-D). These glycan structures were
identified as A1F and A2F. A noticeable difference between the glycan
profiles from Asp-2 and the Fc fusion protein, both expressed in CHO
E1a (Fig. 2, C and D), is the appearance of the neutral glycans G1F and G0F, containing one or no galactose residues at
the nonreducing end. Our previous analysis of glycans released from a
number of therapeutic antibodies and other fusion proteins expressed in
this cell line indicates these two biantennary oligosaccharides are
attached to an asparagine residue on the Fc
moiety.3
On digestion with sialidase, the glycan mixture released from Asp-2
expressed in baculovirus-infected insect cells was identical to that
before treatment (compare Figs. 2A and 3A). This
confirmed that only neutral glycans are produced in this expression
system. In contrast, the chromatographic profile of the derivatized
glycans from Asp-2 (or the Fc fusion protein) synthesized in the CHO
E1a and COS cell lines was considerably simplified after sialidase treatment (compare chromatograms in Figs. 2 and
3). Based upon the retention times of the
peaks compared with those of AA-Ac-derivatized standards, these glycans
were provisionally identified as NA2F, NA3F, and NA4F. The schematic
structures of these oligosaccharides are shown in Table I.
MALDI-TOF Mass Spectrometric Analysis of AA-Ac-derivatized
Glycans--
Molecular weight analysis of the individual components in
the mixtures of the AA-Ac-derivatized neutral glycans by MALDI-TOF mass
spectrometry provided detailed structural information on the
corresponding heterogeneity and the identity of the individual glycans.
The mass spectrometric analysis of the neutral glycan mixtures released
from Asp-2 and its Fc fusion protein is shown in Fig. 4. The majority of AA-Ac glycans were
observed as the proton adducts, [M + H]+, although
in the case of glycans from the COS cell expression a sodium adduct was
also identified (Fig. 4C). The profile obtained for the
oligosaccharide mixture from the protein expression in baculovirus-infected insect cells confirmed the presence of most of the
glycans identified by their chromatographic behavior. Thus, the major
glycans from this cell line were confirmed as oligomannose-type oligosaccharides.
The profiles of the desialylated glycan mixtures released from
the Fc fusion protein synthesized in COS and CHO E1a, are shown in Fig.
4 (B and C, respectively). From the
chromatographic and MALDI data, it is clear that the distribution of
components in the two mixtures are very similar and contain bi-, tri-,
and tetra-antennary complex-type glycans, which can be sialylated at
the galactose residues on the nonreducing ends of these molecules. The
heterogeneity of the major glycans released from Asp-2 expressed in CHO
E1a is also similar to that of the Fc fusion protein from the same cell
line (Fig. 4D). The MALDI-TOF-MS data are summarized in
Table I.
Three minor signals with m/z values of 1657.7, 2226.8, and
2591.7 were observed in the MALDI-TOF-MS of the glycan profile released
from Asp-2 (Fig. 3D). These signals were largely absent in
the Fc fusion protein. The low level increase in heterogeneity is most
likely due to small differences in the fermentation conditions used to
prepare a larger batch of Asp-2 Fc before proteolytic cleavage of the
Fc moiety.
Loss of an N-acetylglucosamine residue (203 mass units) from
G1F (m/z 1860.7) gives m/z 1657.7. This probably
has the structure shown below (Structure I), an uncommon glycan that
has also recently been identified as one of the glycan components
released from gelatinase B (22).
Activity Analysis of Glycosylated and Unglycosylated
Asp-2--
Site-directed mutagenesis was used to alter four asparagine
residues (amino acids 153, 172, 223, and 354) to glutamines, within the
consensus glycosylation sites of Asp-2Fc. The mutants were expressed
and purified from COS cells, and the activity was compared with the
wild-type Asp-2Fc (Table II). Three
double mutants, which eliminated two of the four glycosylation sites,
showed reduced proteolytic activity (between 30% and 40% of
wild-type).
The glycosylation state of Asp-2 plays a critical role in
maintaining its proteolytic activity. In our initial experiments, tunicamycin treatment of a stable CHO cell line expressing Asp-2Fc was
used to obtain unglycosylated Asp-2Fc. This protein exhibited ~40%
of the activity of the Asp-2Fc isolated from untreated cells (data not
shown). This protein is unaltered in its amino acid sequence but has
greatly reduced carbohydrate content (data not shown). These results
suggest that glycosylation may play an important role in the
proteolytic activity of Asp-2. Although glycosylation could not be
detected from the tunicamycin-treated Asp-2Fc, there was a possibility
that this sample was not homogeneous in its glycosylation state.
Therefore, to address this question more rigorously, we undertook to
mutate the glycosylation sites on Asp-2Fc.
Mutating two of the four consensus glycosylation sites of Asp-2 had a
significant effect on proteolytic activity. The effect of glycosylation
on enzyme activity could be indirect, perhaps by increasing the
solubility of the Asp-2 protein. However, it is also possible that the
glycosylation could have a direct effect on substrate binding.
According to the crystal structure of Asp-2, asparagine 172 is within
10 Å of the unprime side of the binding pocket and asparagine 223 is
within 13 Å (23). A complex carbohydrate, such as A2F, has dimensions
in excess of 30 Å, allowing for the possibility of a direct
interaction with peptide substrates and a likely interaction with the
larger surface of the natural substrate, APP.
A number of other observations support the effect of glycosylation on
activity. The baculovirus-expressed Asp2, which has high mannose
glycosylation, showed ~50% of the activity of the mammalian
expressed material, which contains complex carbohydrates (data not
shown). This may suggest that it is not only important to have
carbohydrate at these sites, but that specific sugar moieties may be
important for a direct interaction with substrate. In addition, the
refolded E. coli expressed Asp-2, which is not glycosylated (24), was assayed at a 10-fold higher concentration than our experiments, suggesting that its activity is significantly lower than
the mammalian expressed protein. The E. coli expressed
material contains asparagine residues at all four consensus
glycosylation sites, again supporting the hypothesis that the lack of
glycosylation and not the alteration of the asparagine residues is
leading to the decrease in activity of Asp-2.
Each of the three double mutants analyzed shows a significantly reduced
activity, suggesting that multiple glycosylation sites play a role in
maintaining optimal activity of Asp-2. Although this analysis does not
address the individual contributions of each of the glycosylation
sites, it does show that more than one carbohydrate effects activity
and implies that some of the glycosylation effect is indirect. Initial
attempts at mutating all four of the consensus glycosylation sites in
Asp-2 were problematic due to low levels of protein expression. This
could be due to a retardation in the rate of secretion of this protein,
or an increase in the lability of the protein once it is secreted.
The chromatographic behavior of the intact and desialylated mixture of
glycans released from Asp-2 expressed in mammalian cell reveals that
the majority of these glycans are of the complex type, containing one
or more sialic acid residues. Retention time measurements of the
neutral glycans obtained after desialylation gave a preliminary
indication of the identity of these oligosaccharides as bi-, tri-, and
tetra-antennary parents. Molecular weight measurements by MALDI-TOF
mass spectrometry determined the molecular weight of the components in
these mixtures, providing further evidence for their identity.
The use of AA-Ac in preference to other fluorescent labels, such as
2-aminoacridone, has provided an increase of approximately an order of
magnitude in sensitivity to MALDI-TOF-MS measurements (21). Due to this
higher sensitivity, it has been possible to reveal the presence of
other lower level carbohydrates attached to the
N-glycosylation sites of Asp-2.
In contrast to the expression of Asp-2 in mammalian cell lines,
synthesis in baculovirus-infected insect cells produced only neutral
oligosaccharides containing three or more mannose residues. This result
is in agreement with the preponderance of evidence in the literature
that N-glycosylation in insect cells is limited to almost
exclusively oligomannose-type glycans
(Man3-9GlcNAc2) (25).
Limited information on the glycosylation of Asp-2 has been published to
date. One study reported that enzymatic digestion of this glycoprotein
with endoglycosidase H did not lead to deglycosylation (12). This is
not surprising as endoglycosidase H will only hydrolyze high mannose
carbohydrates, whereas the glycans identified in this study are of the
complex type when expression of the protein is carried out in a
mammalian cell line. In another study it was shown that all the four
N-glycosylation sites on Asp-2 are occupied by a
heterogeneous mixture of carbohydrate (8). These authors found that
four oligosaccharides were linked to Asn172. The molecular
weights of these glycans differed from one another by 162 and 366 mass
units, which indicated the loss of a hexose and of a disaccharide made
up of hexose and N-acetylhexosamine, respectively. These
differences are in agreement with the type of bi-, tri-, and
tetra-antennary complex structures we identified in our investigation,
as shown in Table I. For example, the difference in molecular weight
between a bi- and a tri-antennary glycan is 366, as is the case for
tri- and tetra-antennary glycans. The presence and extent of
sialylation of these complex-type glycans could not be estimated from
this recent study (8).
Knowledge of the type of carbohydrate covalently attached to asparagine
residues in Asp-2 has also been useful in the design of a purification
protocol. It has allowed a rational choice of the correct lectin column
for the purification of recombinant Asp-2, and has helped to establish
a suitable protocol for the crystallization of this protease.
It is now widely accepted that the biological functions of carbohydrate
covalently attached to proteins are known to be diverse (9). They can
ensure the correct folding of a protein by stabilizing folded domains,
they can enhance the solubility of a protein by providing polar groups
on its surface, they can prevent or diminish aggregation of the
glycoprotein, they can protect the protein from protease degradation,
and can modulate intracellular routing and intercellular recognition. A
key question is whether the large size of the glycan moieties in Asp-2
compared with the protein domain has any influence on its In conclusion, we have shown that the Glycosylation of Asp-2 seems to be essential in the maintenance of an
active conformation of this protein, ensuring optimum interaction and
-peptides are the main
components of amyloid plaques found in the brain of Alzheimer's
disease patients. Amyloid 39-42
-peptide is formed from amyloid
precursor protein by the sequential action of
- and
-secretases.
Asp-2 is a transmembrane aspartic protease expressed in the brain,
shown to have
-secretase activity. Mature Asp-2 has four
N-glycosylation sites. In this report we have characterized
the carbohydrate structures in this glycoprotein expressed in three
different cell lines, namely Chinese hamster ovary, CV-1 origin of
SV40, and baculovirus-infected SF9 cells. Biantennary and triantennary
oligosaccharides of the "complex" type were released from
glycoprotein expressed in the mammalian cells, whereas mannose-rich
glycans were identified from glycoprotein synthesized in the
baculovirus-infected cells. Site-directed mutagenesis of the asparagine
residues at amino acid positions 153, 172, 223, and 354 demonstrate
that the protease activity of Asp-2 is dependent on its glycosylation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-peptides
(A
)1 (1, 2). The formation
of these peptides by the action of proteases on amyloid precursor
protein (APP) has been the subject of several studies (3, 4), as the
identification of these proteases could lead to the discovery of
inhibitors useful in the treatment of Alzheimer's disease. Over the
last year there have been a number of reports detailing the discovery,
purification, and characterization of a transmembrane aspartic
proteinase (Asp-2). This glycoprotein has been shown to cleave the APP
at the
-secretase site (5-9). High levels of this proteinase were
identified in the human brain, and the enzyme activity was found in
cells associated with the central nervous system (7) and in cell lines
known to produce A
via cleavage of APP (5).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cyano-4-hydroxycinnamic acid matrix (10 mg/ml in acetonitrile:water
(1:1)). The AA-Ac-derivatized glycans were dissolved in 10 µl of
water and purified on a C18 ZipTip (Millipore Corp., Bedford, MA) for
MALDI-TOF-MS analysis. Following conditioning of the ZipTip and binding
of the derivatized glycans to the support according to the
manufacturer's protocol, the bound glycans were washed once with 10 µl of 0.1% v/v trifluoroacetic acid and then eluted in 3 µl of
20% v/v acetonitrile containing the matrix 2,5-dihydroxybenzoic acid
at a concentration of 10 mg/ml, directly onto the MALDI target.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Recombinant Asp-2 produced in different
expression systems. The positions of the marker proteins and their
estimated molecular masses are indicated on the left.
A, lane 1, wild-type
Asp-2-His6 expressed in baculovirus-infected SF9 cells;
lane 2, Asp-2Fc expressed in COS cells;
lanes 3 and 4, Asp2Fc expressed in CHO
E1a cells. B, lane 1, purified Asp-2Fc
in CHO E1a cells and proteolyzed to remove the Fc. The protein bands at
~38 and 40 kDa have been generated by minor COOH-terminal degradation
of the recombinant protein in both mature and proprotein forms.
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Fig. 2.
Hydrophilic interaction liquid chromatography
with fluorescence detection of glycan pools released from Asp
samples. The glycans used for HILC analysis were released from
wild-type Asp-2-His6 expressed in baculovirus-infected SF9
cells (A), Asp-2Fc expressed in COS cells (B),
Asp-2Fc expressed in CHO E1a cells (C), and purified
wild-type Asp-2 expressed as an Fc fusion in CHO E1a cells with
subsequent removal of the Fc moiety (D).
Structure of glycans referred to in the figures and text
, N-acetylglucosamine;
, mannose;
,
galactose;
, fucose;
, sialic acid. Linkages between sugar
residues have been removed for simplicity.
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Fig. 3.
Hydrophilic interaction liquid chromatography
with fluorescence detection of desialylated glycan pools released from
Asp samples. The glycans used for HILC analysis after
desialylation were released from wild-type Asp-2-His6
expressed in baculovirus-infected SF9 cells (A), Asp-2Fc
expressed in COS cells (B), Asp-2Fc expressed in CHO E1a
cells (C), and purified Asp-2 expressed as an Fc fusion in
CHO E1a cells with subsequent removal of the Fc moiety
(D).
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Fig. 4.
MALDI-TOF spectra of desialylated glycan
pools released from Asp samples. The glycans used for MALDI-TOF-MS
analysis were released from wild-type Asp-2-His6 expressed
in baculovirus-infected SF9 cells (A), Asp-2Fc expressed in
COS cells (B), Asp-2Fc expressed in CHO E1a cells
(C), and purified wild-type Asp-2 expressed as an Fc fusion
in CHO E1a cells with subsequent removal of the Fc moiety
(D).
The component with an [M + H]+ value of 2225.8 is
203 mass units higher than that of the neutral biantennary glycan NA2F. This is most probably related to the presence of low levels of a
bisected glycan (Structure II), although the exact position of the
N-acetylglucosamine position cannot be determined from these
measurements.
The very low level glycan component at m/z 2591.7 is
about 162 mass units (galactose residue) lower in molecular weight than NA4F. The presence of this oligosaccharide may be due either to the
incomplete processing of the tetra-antennary complex-type glycan or to
the formation of triantennary complex-type glycan with an
N-acetylglucosamine residue attached to the central mannose residue in the mannose core.
Activity of Asp-2 mutants
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-secretase
properties either due to direct interaction with APP or indirectly by
ensuring its correct folding in the endoplasmic reticulum.
-secretase activity of Asp-2
is dependent on the extent of N-glycosylation. This result was derived by expressing Asp-2 in the presence of tunicamycin and was
further confirmed when the protease activity of mutants were assessed
using a peptide substrate. It is well known that a number of
glycoproteins need one or more of their N-linked
oligosaccharides during folding (12). The fact that protease
activity was affected by the occupancy of glycosylation sites, close
and further away from the site of action, appears to indicate that all
four glycosylation sites in Asp-2 must be occupied by oligosaccharide
and act in a cooperative manner to make sure that this glycoprotein has
the correct folded conformation (12, 20). We will be carrying out
future studies to find out whether one or more of the glycosylation sites plays a crucial role in the correct folding of Asp-2. As two of
the N-glycosylation sites, namely Asn223 and
Asn354, are spatially close to the disulfide bridges
Cys216-Cys420 and
Cys278-Cys443, respectively, the presence of
the correct type of oligosaccharide may also play an active role in the
formation of disulfide bridges and hence can have an effect on the
tertiary structure of the glycoprotein. To fully appreciate the impact
of glycosylation on protease activity, it will also be important to
ascertain the glycosylation states of the mammalian-expressed
full-length Asp-2, as well as native Asp-2. The technology presented
here makes analysis of the native protein possible because small
amounts of protein can be easily analyzed.
-secretase reactivity with APP. Clearly establishing the precise
role of glycosylation in the protease activity of Asp-2 will be
important in the design of specific inhibitors that may more
effectively interfere with the function of Asp-2. This knowledge will
be useful in structure-activity relationships, which will be important
in optimizing the therapeutic value of drug treatments for Alzheimer's disease.
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ACKNOWLEDGEMENTS |
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We thank the Department of Trade and Industry (UK) for a grant to develop novel methodology for the analysis of oligosaccharides. We thank our colleagues at SmithKline Beecham for helpful discussions
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FOOTNOTES |
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* 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 may be addressed: Dept. of Gene Expression Sciences, SmithKline Beecham Pharmaceuticals, 709 Swedeland Rd., King of Prussia, PA 19406. Tel.: 610-270-7208; Fax: 610-270-4091; E-mail: sharon_m_sweitzer@sbphrd.com.
¶¶ To whom correspondence may be addressed: Dept. of Analytical Sciences, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, United Kingdom. Tel.: 44-1279-622026; Fax: 44-1279-627427; E-mail: patrick_camilleri@sbphrd.com.
Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M009361200
2 R. Matico, S. Sweitzer, D. J. Powell, and K. Johanson, manuscript in preparation.
3 J. Charlwood, M. Skehel, and P. Camilleri, submitted for publication.
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ABBREVIATIONS |
---|
The abbreviations used are:
A, amyloid
-peptide(s);
AA-Ac, 3-(acetylamino)-6-aminoacridine;
APP, amyloid
precursor protein;
Asp-2, transmembrane aspartic protease;
CHO, Chinese
hamster ovary;
COS, CV-1 origin of SV40;
HILC, hydrophilic interaction
liquid chromatography;
MALDI-TOF, matrix-assisted laser
desorption/ionization-time of flight;
Man3-9GlcNAc2, neutral glycans containing 3-9
mannose and 2 N-acetylglucosamine residues;
PNGase F, peptide N-glycanase;
MS, mass spectrometry.
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