Characterization of the Glycosylation Profiles of Alzheimer's beta -Secretase Protein Asp-2 Expressed in a Variety of Cell Lines*

Joanne CharlwoodDagger , Colin Dingwall§, Rosalie Matico, Ishrut Hussain§, Kyung Johanson, Stephen Moore§, David J. Powell||, J. Mark SkehelDagger , Steve Ratcliffe**, Brian Clarke**, John TrillDagger Dagger , Sharon SweitzerDagger Dagger §§, and Patrick CamilleriDagger ¶¶

From the Dagger  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 Dagger Dagger  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


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Amyloid 39-42 beta  -peptides are the main components of amyloid plaques found in the brain of Alzheimer's disease patients. Amyloid 39-42 beta -peptide is formed from amyloid precursor protein by the sequential action of beta - and gamma -secretases. Asp-2 is a transmembrane aspartic protease expressed in the brain, shown to have beta -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -peptides (Abeta )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 beta -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 Abeta via cleavage of APP (5).

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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 alpha -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


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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.

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).


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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).

                              
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Table I
Structure of glycans referred to in the figures and text
The following symbols have been used to identify the various sugar residues: , N-acetylglucosamine; , mannose; , galactose; octagon , fucose; diamond , sialic acid. Linkages between sugar residues have been removed for simplicity.

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.


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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).

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.



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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 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).

<UP><SC>Structure</SC> I</UP>
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.

<UP><SC>Structure</SC> II</UP>
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 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).

                              
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Table II
Activity of Asp-2 mutants
Asp-2Fc mutants which lack specific glycosylation sites were analyzed for proteolytic activity against an APP Swedish variant peptide (ISEVNLDAEFRHDK(dnp)G). Proteolytic activity is the mean of duplicate incubations.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -secretase properties either due to direct interaction with APP or indirectly by ensuring its correct folding in the endoplasmic reticulum.

In conclusion, we have shown that the beta -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.

Glycosylation of Asp-2 seems to be essential in the maintenance of an active conformation of this protein, ensuring optimum interaction and beta -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.

    ACKNOWLEDGEMENTS

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

    FOOTNOTES

* 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.

    ABBREVIATIONS

The abbreviations used are: Abeta , amyloid beta -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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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