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J Biol Chem, Vol. 274, Issue 38, 26810-26814, September 17, 1999


Glycosylphosphatidylinositol-anchored Proteins Play an Important Role in the Biogenesis of the Alzheimer's Amyloid beta -Protein*

Kumar SambamurtiDagger , Daniel Sevlever, Thillai Koothan, Lawrence M. Refolo§, Inga Pinnix, Swetal Gandhi, Luisa Onstead, Linda Younkin, Christian M. Prada, Debra Yager, Yasumasa Ohyagi, Christopher B. Eckman, Terrone L. Rosenberry, and Steven G. Younkin

From the Mayo Clinic, Jacksonville, Florida 32224

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Alzheimer's amyloid protein (Abeta ) is released from the larger amyloid beta -protein precursor (APP) by unidentified enzymes referred to as beta - and gamma -secretase. beta -Secretase cleaves APP on the amino side of Abeta producing a large secreted derivative (sAPPbeta ) and an Abeta -bearing C-terminal derivative that is subsequently cleaved by gamma -secretase to release Abeta . Alternative cleavage of the APP by alpha -secretase at Abeta 16/17 releases the secreted derivative sAPPalpha . In yeast, alpha -secretase activity has been attributed to glycosylphosphatidylinositol (GPI)-anchored aspartyl proteases. To examine the role of GPI-anchored proteins, we specifically removed these proteins from the surface of mammalian cells using phosphatidylinositol-specific phospholipase C (PI-PLC). PI-PLC treatment of fetal guinea pig brain cultures substantially reduced the amount of Abeta 40 and Abeta 42 in the medium but had no effect on sAPPalpha . A mutant CHO cell line (gpi85), which lacks GPI-anchored proteins, secreted lower levels of Abeta 40, Abeta 42, and sAPPbeta than its parental line (GPI+). When this parental line was treated with PI-PLC, Abeta 40, Abeta 42, and sAPPbeta decreased to levels similar to those observed in the mutant line, and the mutant line was resistant to these effects of PI-PLC. These findings provide strong evidence that one or more GPI-anchored proteins play an important role in beta -secretase activity and Abeta secretion in mammalian cells. The cell-surface GPI-anchored protein(s) involved in Abeta biogenesis may be excellent therapeutic target(s) in Alzheimer's disease.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The amyloid that is invariably deposited in Alzheimer's disease (AD)1 is composed of an approximately 4-kDa peptide (amyloid beta -peptide, Abeta ) that is derived from a larger protein referred to as the amyloid beta -protein precursor (APP) (1, 2). APP is a type I integral membrane glycoprotein with a large N-terminal extracellular domain, a single transmembrane domain, and a short cytoplasmic tail. The Abeta peptide begins 99 amino acids from the C terminus of APP, and it extends from the extracellular region to a point half-way through the APP membrane-spanning domain (1). Abeta is released from APP by cleavage on its N- and C-terminal ends by beta - and gamma -secretase, respectively. beta -Secretase cleavage before residue 1 of Abeta (672 of APP770) also releases the secreted derivative sAPPbeta , whereas an alternative cleavage before residue 17 by alpha -secretase releases sAPPalpha . In most culture systems tested, the predominant cleavage product is sAPPalpha , and this may serve to prevent the production of Abeta (1). The proteolytic processing of APP to sAPP and Abeta is regulated by protein kinase C (3), protein tyrosine kinase (4), muscarinic receptors (5), and estrogens (6). The regulatory pathways involved are cell type-dependent, have little or no effect in some cell types, and normally stimulate the secretion of sAPPalpha while simultaneously reducing the secretion of Abeta (2). A metalloproteinase related to the tumor necrosis factor-alpha converting enzyme (7, 8) can cleave APP to sAPPalpha upon activation of PKC by phorbol esters2 (9, 10).

Strong evidence that Abeta plays an important role in AD pathogenesis has come from the study of mutations in the APP (11-14), presenilin 1 (15), and presenilin 2 (16) genes that are known to cause early onset familial AD (FAD) (17). A fundamental effect of all the FAD-linked mutations is to increase the concentration of Abeta 42 or of both Abeta 40 and Abeta 42 (18-22). Abeta 42 is deposited early and selectively in the senile plaques that are observed in all forms of AD, so these findings provide strong evidence that the FAD-linked mutations all act to cause AD by increasing the extracellular concentration of Abeta 42.

The evidence implicating Abeta in AD pathogenesis has made both beta - and gamma -secretase important therapeutic targets, but neither has yet been identified. It was recently demonstrated, however, that APP is proteolytically processed by an alpha -secretase-like pathway in the yeast Saccharomyces cerevisiae (23, 24). This processing pathway was shown to involve the glycosylphosphatidylinositol (GPI)-anchored aspartyl proteases YAP3 and MKC7 (25, 26). In mammals, GPI-anchored proteins are a relatively small class of membrane proteins that are anchored to the outer leaflet of the cell membrane by a glycolipid anchor consisting of ethanolamine, mannose, glucosamine, and phosphatidylinositol (27). These proteins can be removed from the cell surface by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC), which cleaves the phosphatidylinositol in GPI-anchored proteins to release the protein. In this study we used PI-PLC to evaluate the role of GPI-anchored proteins in mammalian secretase activity and Abeta biogenesis. Our data indicate that at least one form of beta -secretase or one of its essential, constitutively active regulators is GPI-anchored. It appears, however, that alpha -secretase is not a GPI-anchored protease in mammalian cells.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Purification of PI-PLC-- PI-PLC was expressed in Bacillus subtilis cultures transformed with a plasmid containing the Bacillus thuringiensis gene and was purified from 8 liters of the culture media (28). Briefly, the secreted PI-PLC was concentrated by filtration through a Millipore pelican filter, adjusted to 0.2 M NaCl, and loaded onto a 50-ml octyl-Sepharose column. The column was washed with 100 ml of 20 mM Na3PO4, 0.2 M NaCl, pH 7, and eluted with 20 mM Na3PO4, pH 7. Fractions containing active PI-PLC were adjusted to pH 8.5 and 0.055% Triton X-100 and loaded onto an 80-ml DEAE column. The column was washed with 70 ml of Tris acetate, pH 8.5, 0.55% Triton X-100 and eluted with 20 mM Tris acetate, 0.55% Triton X-100, pH 5.0. The activity of the fractions was assayed by the measurement of its capacity to hydrolyze 3H-labeled phosphatidylinositol (PI) in 50 mM Tris acetate, pH 5.5, containing 2 mM 1,2-diheptanoyl-sn-glycero-3-phosphatidylcholine (Avanti Polar Lipids, Inc.). The free inositol released by PI-PLC was measured in the aqueous fraction after stopping the reaction in 10:5:0.1 chloroform:methanol:HCl. The protein concentration was determined by a BCA assay (Pierce). At this stage the PI-PLC is very pure as judged by the detection of a single band on a Coomassie Blue-stained SDS-polyacrylamide gel that was subsequently stained with silver. For most of our treatments, we used 2 µg/ml of this purified PI-PLC.

Mixed Brain Cultures-- Mixed brain cultures from guinea pig fetuses were prepared from 30-day embryos using a method developed by Dr. Yasumasa Ohyagi (see Ref. 29). Briefly, a pregnant guinea pig was sacrificed with carbon dioxide, and six embryos were recovered from the uterus. The brains were dissected and minced into fine pieces. These pieces were treated with 10 volumes of 0.03% trypsin and 0.02% EDTA in HEPES-buffered saline, pH 6.4, for 3 min, and the reaction was stopped by the addition of an equal volume of 5% calf serum in Opti-MEM (Life Technologies, Inc.). The cells were collected by centrifugation, and the pellet was resuspended in 20 ml of Opti-MEM containing 5% calf serum and filtered through a 67-µm polyester mesh (Spectrum). The resulting cells were plated at a high density (107 cells per dish) to prevent astrocytic overgrowth. These cultures can be maintained in the same medium for over a month without loss of viability. In addition to being a good cell culture model for events that occur in the brain, this system benefits from the secretion of easily detectable levels of the Abeta 40 and Abeta 42 peptides. Furthermore, unlike rodents, the guinea pig Abeta sequence is identical to that in man, thereby allowing the use of the human-specific antibody against Abeta 1-16 (BAN50) for the analysis of sAPPalpha (30).

Cell Lines and Media-- A wild type CHO-K1 cell line (GPI+) expressing GPI-anchored human placental alkaline phosphatase (PLAP) and a derivative line with a mutation in the GPI biosynthesis pathway (gpi85) were kind gifts from Dr. Victoria Stevens (31). The gpi85 mutant is deficient in N-acetylglucosaminylphosphatidylinositol deacetylase which is responsible for the second step in the biosynthesis of the GPI anchor (32). These cultures were maintained in Ham's F-12 media supplemented with 10% fetal bovine serum and 100 µg/ml G418.

Enzyme Assays-- Alkaline phosphatase activity was assayed using 24 mM p-nitrophenyl phosphate as a substrate in 1 M diethanolamine buffer, pH 9.6, containing 20 mM homoarginine. The samples were first heated to 55 °C for 10 min to increase the specificity for placental alkaline phosphatase activity (33).

Drug and PI-PLC Treatments-- All treatments were carried out in the serum-free CCM5 medium (HyClone) after adapting the cultures for 24 h in the same medium. Phorbol dibutyrate (PDBu 1 mM; Sigma) was dissolved in Me2SO, which was also added to controls and other treatments. Me2SO concentrations were maintained at 0.2% for PDBu treatments. Bisindolylmaleimide (BIS) stocks were prepared in distilled water and directly diluted to 2.5 µM into the culture medium (4).

Antibodies-- Monoclonal antibodies BAN50 (Abeta 1-16), BNT77 (Abeta 11-24), BC05(Abeta 42), and BA27 (Abeta 40) have been previously described (20) and were gifts from Dr. Nobu Suzuki (Takeda Industries). Ivan Lieberburg and John Anderson (Elan Corp.) kindly provided the 192 antibody, specific for sAPPbeta (34). When appropriate, selected antibodies were tagged with horseradish peroxidase, using a kit from Pierce as described by the manufacturer.

Abeta ELISA-- Abeta was measured using an established, sensitive, and specific sandwich ELISA (20). Briefly, Nunc immunomax dishes were coated with capture antibodies BAN50 (guinea pig) or BNT77 (CHO) in 0.1 M carbonate buffer, pH 9.6, and blocked with 0.5% block ace (Wako) in PBS, pH 7.4. The media samples (100 µl) were incubated overnight in 50 µl of ELISA buffer I (0.02 M sodium phosphate, 2 mM EDTA, 0.4 M NaCl, 0.2% bovine serum albumin, 0.05% CHAPS, 0.4% block ace, 0.05% NaN3, pH 7.0), washed three times with PBS, and treated overnight with the horseradish peroxidase-labeled detection antibodies BA27 and BC05 diluted in ELISA buffer II (0.02 M sodium phosphate, 2 mM EDTA, 0.4 M NaCl, 1.0% bovine serum albumin, 0.002% thimerosal, pH 7.0). After washing twice with PBS and once with PBS + 0.05% Tween 20 horseradish peroxidase activity was detected using a Pierce ELISA kit as described by the manufacturer. Addition of PI-PLC-containing CCM5 medium did not influence the measurement of standard Abeta .

Western Blots-- The human/guinea pig-specific BAN50 antibody and the sAPPbeta -specific 192 antibody were used to determine the levels of sAPPalpha and sAPPbeta , respectively, by Western blotting using methods described earlier (20, 34, 35). We also verified the specificity of the 192 antibody by probing peptide spots CSEVKM, CSEVK, and CSEVKMD. As expected, strong signals were only obtained for the CSEVKM peptide, which corresponds to sAPPbeta cleavage, and the other two-peptide spots were negative, demonstrating the end specificity of the 192 antibody (data not shown). Total sAPP in the medium was detected using the 22C11 antibody (Roche Molecular Biochemicals).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PI-PLC Treatment Reduces the Secretion of Abeta but Not sAPPalpha in Guinea Pig Mixed Brain Cultures-- Guinea pig mixed brain cultures were treated with PI-PLC for 1 and 3 days in triplicate. Over 3 days of PI-PLC treatment, sAPPalpha was not reduced in amount (Fig. 1), but both Abeta 40 and Abeta 42 were substantially reduced (Table I). This large reduction was readily observed after 1 day of treatment and was sustained for at least 3 days. Since PI-PLC treatment is known to release GPI-anchored proteins from the cell surface, these data suggest that one or more GPI-anchored proteins are involved in Abeta biogenesis. The lack of an effect on sAPPalpha indicates that cleavage by alpha -secretase is not affected by the removal of cell-surface GPI-anchored proteins and that PI-PLC treatment has no appreciable effect on APP synthesis.


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  		Fig. 1.   PI-PLC has no effect on mammalian sAPPalpha . Media from triplicate guinea pig brain cultures that were untreated (lanes 1-3) or treated with PI-PLC for 3 days (lanes 4-6) were probed with the BAN50 antibody to residues 1-16 of Abeta . Densitometric analysis of the blot showed that the levels of sAPPalpha were similar in treated and untreated cultures, whereas cultures treated with PI-PLC showed a large decline in the amounts of secreted Abeta 40 and Abeta 42 (Table I).

                              
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Table I
PI-PLC reduces Abeta 40 and Abeta 42 secretion in guinea pig mixed brain cultures
Guinea pig brain cultures prepared as described under "Experimental Procedures" were treated for 1 or 3 days with the indicated dose of PI-PLC. Media were collected and analyzed for Abeta 40 and Abeta 42.

Effects of PI-PLC Treatment on Abeta Secretion from CHO Cultures-- To investigate further the role of GPI-anchored proteins in Abeta biogenesis, we investigated the mutant CHO cell line, gpi85. This line was derived (31) from a recombinant CHO cell line (GPI+), which expresses human PLAP as a GPI-anchored reporter protein. The gpi85 line is deficient in N-acetylglucosaminylphosphatidylinositol deacetylase (4), which is responsible for the second step in the biosynthesis of the GPI anchor (32). The loss of GPI anchor synthesis is neither lethal nor detrimental to the growth of mammalian cells. However, in cells (e.g. gpi85) that do not synthesize the GPI anchor, proteins that are normally GPI-anchored are degraded in the endoplasmic reticulum (36, 37) and are not, therefore, found at the cell surface.

To determine the concentration of PI-PLC needed to remove GPI-anchored proteins from the cell surface, we treated GPI+ cultures with two doses of PI-PLC and measured the release of PLAP (Fig. 2). More than 80% of total cellular PLAP was released in 1 h by treatment with 20 µg/ml PI-PLC. The small amount of PLAP remaining after this treatment appears to be inaccessible to PI-PLC and is presumably mostly intracellular because no additional PLAP was released by treating cells for 3 h with 20 µg/ml PI-PLC (Fig. 2). The amount of PLAP released by treatment with 2 µg/ml PI-PLC for 1 h was 75% of that released by 20 µg/ml, but essentially the same amount was released when treatment with 2 µg/ml PI-PLC was extended to 3 h (Fig. 2). Thus treatment with 2 µg/ml PI-PLC for 3 h appears to be adequate to remove virtually all of the GPI-anchored protein at the cell surface, and subsequent experiments were conducted using 2 µg/ml PI-PLC.


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  		Fig. 2.   Release of human PLAP from GPI+ CHO cells by PI-PLC. The PLAP release into the medium of untreated GPI+ cultures was 100-fold lower than with PI-PLC treatment. The mutant gpi85 cultures did not release any PLAP, as reported. By 3 h of treatment, 2 µg/ml PI-PLC released as much PLAP as 20 µg/ml, indicating that all the accessible PLAP was cleaved by the lower dose.

In 10 independent experiments, secreted Abeta was compared in gpi85 and GPI+ cells cultured in parallel. In 9 of these experiments, the effect of PI-PLC treatment (2 µg/ml for 3 h) was analyzed in both cell types. The results of this study, which were analyzed statistically using the Wilcoxon signed rank test, are shown in Table II. In untreated gpi85 cells, which lack GPI-anchored proteins, there was a consistent, highly significant reduction in Abeta 40 (p < 0.0008) and Abeta 42 (p < 0.03) relative to untreated GPI+ cells. In gpi85 cells, PI-PLC had no significant effect on Abeta 40 or Abeta 42, as expected. In GPI+ cells, as in fetal guinea pig brain cultures, removal of cell-surface GPI-anchored proteins with PI-PLC caused a consistent, highly significant reduction in Abeta 40 (p < 0.008) and Abeta 42 (p < 0.03). As shown in Table II, GPI+ cells treated with PI-PLC and mutant gpi85 cells produced remarkably similar amounts of Abeta 40 and Abeta 42 that were consistently ~40% less than the amount produced by normal GPI+ cells. Thus, the removal of GPI-anchored proteins by PI-PLC and by mutation causes a similar, highly significant reduction in Abeta 40 and Abeta 42. Confirming these findings, we have also observed that total secreted Abeta (4-kDa band), immunoprecipitated from [35S]methionine-labeled CHO cells expressing high levels of human APP695, is reduced upon PI-PLC treatment (p < 0.05) to approximately the same extent as shown in Table II without significantly changing sAPPalpha (data not shown).

                              
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Table II
PI-PLC reduces Abeta 40 and Abeta 42 secretion in GPI+ CHO cells but not in the GPI-anchor-deficient mutant, gpi85
GPI+ and gpi85 CHO cultures were treated for 3 h with PI-PLC. Media were collected and analyzed for Abeta 40 and Abeta 42.

Activation of PKC by PI-PLC-mediated Hydrolysis of PI Does Not Account for the Reduction in Secreted Abeta Levels in GPI+ Cells-- In many cell types, PKC activation results in a substantial stimulation of sAPPalpha release coupled with a reduction in Abeta secretion (3). By hydrolyzing PI in membranes, PI-PLC treatment could, in principle, activate PKC by releasing diacylglycerol (DAG). It is, however, highly unlikely that PKC activation contributes significantly to the effect of PI-PLC on guinea pig brain cultures, because PI-PLC decreased Abeta but did not increase sAPPalpha in this preparation (Fig. 1). That Abeta was reduced similarly in PI-PLC-treated GPI+ cells (where DAG may be released) and in untreated gpi85 cells (where no DAG is released) also makes it highly unlikely that DAG release contributes importantly to the Abeta reduction observed in PI-PLC-treated CHO cells.

To address further the question of DAG-induced PKC activation, the PKC inhibitor BIS (G109203X) was added to GPI+ cultures prior to treatment with PI-PLC. This compound has previously been shown to block the changes in APP metabolism (4) that occur when PDBu is used to activate PKC (4). Our control experiments showed that PDBu reduces Abeta secretion in GPI+ cells, and this reduction was completely blocked by 2.5 µM BIS. In addition, as previously reported (4), PDBu treatment elevated total sAPP secretion by about 15-fold, and this was also blocked by the BIS treatment (data not shown). Treatment of GPI+ cultures with BIS did not, however, block the reduction in Abeta 40 and Abeta 42 caused by PI-PLC (Table III). Thus, our results clearly show that the effect of PI-PLC on Abeta biogenesis is not due to DAG-induced PKC activation.

                              
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Table III
Addition of the PKC inhibitor BIS blocks PDBu-mediated but not PI-PLC-mediated Abeta reduction in GPI+ cultures
The GPI+ and gpi85 cultures were treated for 3 h with PDBu, PI-PLC, and/or BIS. Media were collected and analyzed for Abeta 40 and Abeta 42.

beta -Secretase Processing of APP Is Reduced in PI-PLC-treated CHO Cultures-- The PI-PLC-induced reduction in Abeta could be caused by a reduction in either beta - or gamma -secretase activity. To evaluate whether beta -secretase activity is reduced when GPI-anchored proteins are removed, several experiments were performed in which we treated GPI+ and gpi85 cultures in triplicate with PI-PLC for 24 h and then evaluated sAPPbeta and total sAPP (sAPPalpha  + sAPPbeta ) by immunoblotting (Fig. 3). For specific detection of sAPPbeta , we used antibody 192, which efficiently binds APP cleaved between residues -1 and +1 of the Abeta sequence but shows a low affinity for full-length APP, sAPPalpha , and other APP fragments (34). We could not specifically analyze the sAPPalpha secreted from the GPI+ and gpi85 cell lines, because antibodies to human Abeta 1-16 do not detect the hamster APP sequence (GenBank accession number AF030413), which is the same as the sequence in other rodents. For this reason, we analyzed total sAPP (sAPPalpha  + sAPPbeta ) by immunoblotting with monoclonal antibody 22C11, which recognizes both human and rodent sAPP.


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  		Fig. 3.   PI-PLC substantially reduces mammalian sAPPbeta . The proteins in media from gpi85 cells (1 untreated; 2-4 PI-PLC-treated) and GPI+ cells (5-7 untreated; 8-10 PI-PLC-treated) were separated on 10-20% Tris-Tricine gels. A, immunolabeled with antibody 192, which specifically detects sAPPbeta (solid arrow). B, immunolabeled with antibody 22C11, which detects total sAPP (sAPPalpha  + sAPPbeta , open arrow). Densitometric analysis of lanes from two independent blots showed that sAPPbeta was reduced by 90% when GPI+ cultures were treated with PI-PLC. sAPPbeta was 71% lower in gpi85 than in GPI+ cultures, and PI-PLC treatment had no consistent effect on the amount of sAPPbeta secreted by gpi85 cells. Total sAPP was slightly reduced (~16%) when GPI+ cultures were treated with PI-PLC. Total sAPP was also slightly lower in gpi85 than in GPI+ cultures. PI-PLC treatment had no consistent effect on the amount of total sAPP secreted by gpi85 cells. The relative levels of sAPP are shown in the graphs in C. Since the differences in total sAPP were small, we repeated this study, and the data in C are summarized from the two experiments.

Densitometric analysis showed that sAPPbeta was reduced by approximately 90% upon PI-PLC treatment of GPI+ cultures (Fig. 3). In contrast, total sAPP (sAPPbeta  + sAPPalpha ) was only slightly reduced (~16%). Since sAPPalpha is the major secreted species in GPI+ cells, the small reduction in total sAPP indicates that PI-PLC has little effect on sAPPalpha . Levels of sAPPbeta were also highly (71%) reduced in untreated gpi85 cells as compared with GPI+ cells, but again total sAPP was only slightly affected. As expected, PI-PLC treatment did not consistently affect sAPPbeta or the total sAPP secreted by gpi85 cells. These results provide strong evidence that GPI-anchored cell-surface proteins play an important role in beta -secretase activity.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The proteins involved in Abeta biogenesis are important therapeutic targets in Alzheimer's disease. To date, none of these proteins has been unequivocally identified. In this study, we investigated the role of GPI-anchored cell- surface proteins in Abeta biogenesis. Our results provide strong evidence that, in mammalian cells, one or more cell-surface GPI-anchored proteins play an important role in beta -secretase activity and Abeta secretion. In addition, they indicate that cell-surface GPI-anchored proteins do not play a major role in alpha -secretase activity. These conclusions are based on the following observations. (i) PI-PLC, which removes GPI-anchored proteins from the cell surface, substantially reduces the Abeta 40 and Abeta 42 secreted from fetal guinea pig brain cultures and the wild type GPI+ CHO cell cultures. (ii) The mutant gpi85 line, which lacks cell-surface GPI-anchored proteins, shows reductions in secreted Abeta 40 and Abeta 42 similar to those observed when the parental GPI+ line is treated with PI-PLC. (iii) The PI-PLC treatment that lowers the Abeta 40 and Abeta 42 secreted from GPI+ cells has no detectable effect on the residual Abeta secretion that occurs in gpi85 cells. (iv) sAPPbeta is substantially reduced in gpi85 cells and in PI-PLC-treated GPI+ cells but shows no additional reduction in PI-PLC-treated gpi85 cells. (v) PI-PLC treatment does not affect the levels of sAPPalpha in fetal guinea pig brain cultures and only slightly reduces total sAPP (sAPPalpha  + sAPPbeta ) in GPI+ and gpi85 cultures.

The coordinate reduction of Abeta 40 and Abeta 42, the profound reduction of sAPPbeta , and the lack of change in sAPPalpha that occurs when GPI-anchored proteins are removed from the cell surface clearly implicate one or more cell-surface GPI-anchored proteins in beta -secretase activity and Abeta biogenesis. This finding fits well with recent reports that APP and Abeta are found in detergent-insoluble membrane domains enriched in GPI-anchored proteins (38, 39). These domains are rich in cholesterol, which has been reported to be important for Abeta production (40, 41).

Although reduced, secretion of sAPPbeta , Abeta 40, and Abeta 42 continues both in PI-PLC-treated cells and in mutant gpi85 cells deficient in GPI-anchored proteins. This suggests that normal beta -secretase activity may involve both GPI-dependent and -independent proteins. One should, however, be cautious in drawing this conclusion. A small percentage of the GPI-anchored proteins may be resistant to PI-PLC because of fatty acylation of the GPI anchor (28). PI-PLC treatment removes cell-surface GPI-anchored proteins but may not remove intracellular GPI-anchored proteins that could continue to produce Abeta and sAPPbeta . In gpi85 cells, GPI-anchored proteins continue to be synthesized. These proteins are rapidly degraded when they fail to be GPI-anchored, and they do not accumulate at the cell surface, but some residual protein precursor capable of processing intracellular APP to sAPPbeta and Abeta may remain in the secretory pathway. Thus our findings are consistent with separate GPI-anchored protein-dependent and -independent beta -secretase pathways, but they do not eliminate the possibility that normal beta -secretase cleavage is carried out entirely by one or several GPI-anchored proteins.

Given the complexity of the cell surface and intracellular events that are involved in Abeta biogenesis, it is likely that some secreted Abeta is produced through alternative proteolytic pathways that do not involve cell-surface GPI-anchored proteins. Thus there is likely to be an upper limit to the reduction in Abeta secretion that can be achieved by removing GPI-anchored proteins. In the CHO cell lines that we examined, removal of cell-surface GPI-anchored proteins caused a coordinate ~50% reduction of Abeta 40 and Abeta 42 at 24 h that was smaller than the 80-90% reduction in sAPPbeta . This sizable difference suggests that, in CHO cells, most sAPPbeta is produced by a GPI-anchored protein-dependent pathway, whereas a substantial percentage of Abeta is produced by alternative pathways that do not result in the secretion of sAPPbeta . This finding agrees well with the published evidence for the presence of secondary beta -secretase activities, which cleave APP at alternative locations close to the N-terminal end of the Abeta sequence. The sequence of the secreted Abeta begins at all known residues ranging from 1 to 11 indicating that the cleavage can occur at any one of these residues (42, 43). The sAPPbeta -specific antibody, 192, will not detect sAPP cleaved at these secondary sites, but the sandwich ELISA will detect the Abeta generated by these cleavages which may be GPI anchor-independent.

The number of cell-surface GPI-anchored proteins that are involved in beta -secretase activity and the precise nature of these proteins remain to be determined. It is entirely possible that the effects we have observed are all due to the removal of a single catalytically active beta -secretase that is GPI-anchored to the cell surface. Alternatively, a GPI-anchored protein could (i) be an essential non-catalytic subunit in a multi-subunit beta -secretase; (ii) perform a post-translational modification required to sustain beta -secretase activity; (iii) chaperone to bring together APP and beta -secretase into one compartment; or (iv) activate another factor that performs one of the functions listed above. Whatever its precise nature, any cell-surface GPI-anchored protein involved in beta -secretase activity is of great interest as a therapeutic target in AD. GPI-anchored proteins can be substantially enriched in cell lysates on the basis of their detergent insolubility, and they can be released specifically from intact cells or cell lysates by PI-PLC. In addition, these proteins can be incorporated from the medium into live cultured cells in a functionally active form. These unique properties and the relatively small number of mammalian GPI-anchored proteins should be highly advantageous in the effort to isolate and characterize the specific GPI-anchored protein(s) that are involved in beta -secretase activity.

    ACKNOWLEDGEMENTS

We thank Dr. Victoria Stevens for providing us with the GPI+ and gpi85 strains, Dr. Ivan Lieberburg and Dr. John Anderson of Elan Corp. for the 192 antibody, and Dr. Nobu Suzuki of Takeda Industries for antibodies BAN50, BNT77, BA27, and BC05. We also thank Meera Parasuraman for reading the manuscript and providing valuable assistance in editing the text.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant 1RO3AG14883 (to K. S.).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.

Dagger To whom correspondence should be addressed: Mayo Clinic, 4500 San Pablo Rd., Jacksonville, FL 32224. Tel.: 904-953-7383; Fax: 904-953-7380; E-mail: samba@mayo.edu.

§ Present address: Nathan Kline Institute, 140 Old Orangeburg, Orangeburg, NY 10962.

2 L. M. Refolo and K. Sambamurti, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: AD, Alzheimer's disease; FAD, familial AD; Abeta , Alzheimer's amyloid beta -protein; Abeta 40, Abeta ending at residue 40; Abeta 42, Abeta ending at residue 42; sAPP, secreted APP derived from APP by proteolytic cleavage; sAPPalpha , sAPP product of alpha -secretase cleavage; sAPPbeta , sAPP product of beta -secretase cleavage; APP, Abeta -protein precursor; PI, phosphatidylinositol; PI-PLC, phosphatidylinositol-specific phospholipase C; PLAP, human placental alkaline phosphatase; GPI+, Chinese hamster ovary cell line transfected with PLAP gpi85 derivative of GPI+ with mutation in N-acetylglucosaminylphosphatidylinositol deacetylase responsible for the synthesis of the GPI anchor; PDBu, phorbol dibutyrate; BIS, bisindolylmaleimide; DAG, diacylglycerol; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; ELISA, enzyme-linked immunosorbent assay; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; PKC, protein kinase C; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.


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