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

Glycosylphosphatidylinositol-anchored Proteins Play an Important Role in the Biogenesis of the... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 38, Issue of September 17, pp. 26810 –26814, 1999 © 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Glycosylphosphatidylinositol-anchored Proteins Play an Important b-Protein* Role in the Biogenesis of the Alzheimer’s Amyloid (Received for publication, January 28, 1999, and in revised form, July 13, 1999) Kumar Sambamurti‡, 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 The Alzheimer’s amyloid protein (Ab) is released from (amyloid b-peptide, Ab) that is derived from a larger protein b-protein precursor (APP) by uniden- referred to as the amyloid b-protein precursor (APP) (1, 2). APP the larger amyloid b- and g-secretase. b-Secre- tified enzymes referred to as is a type I integral membrane glycoprotein with a large N- b producing a tase cleaves APP on the amino side of A terminal extracellular domain, a single transmembrane do- b) and an Ab-bearing large secreted derivative (sAPP main, and a short cytoplasmic tail. The Ab peptide begins 99 C-terminal derivative that is subsequently cleaved by amino acids from the C terminus of APP, and it extends from g-secretase to release Ab. Alternative cleavage of the the extracellular region to a point half-way through the APP a-secretase at Ab16/17 releases the secreted de- APP by membrane-spanning domain (1). Ab is released from APP by a. In yeast, a-secretase activity has been rivative sAPP cleavage on its N- and C-terminal ends by b- and g-secretase, attributed to glycosylphosphatidylinositol (GPI)-an- respectively. b-Secretase cleavage before residue 1 of Ab (672 of chored aspartyl proteases. To examine the role of GPI- APP770) also releases the secreted derivative sAPPb, whereas anchored proteins, we specifically removed these pro- an alternative cleavage before residue 17 by a-secretase re- teins from the surface of mammalian cells using leases sAPPa. In most culture systems tested, the predominant phosphatidylinositol-specific phospholipase C (PI-PLC). cleavage product is sAPPa, and this may serve to prevent the PI-PLC treatment of fetal guinea pig brain cultures sub- production of Ab (1). The proteolytic processing of APP to sAPP b40 and Ab42 in the stantially reduced the amount of A and Ab is regulated by protein kinase C (3), protein tyrosine a. A mutant CHO cell medium but had no effect on sAPP kinase (4), muscarinic receptors (5), and estrogens (6). The line (gpi85), which lacks GPI-anchored proteins, se- regulatory pathways involved are cell type-dependent, have b40, Ab42, and sAPPb than its creted lower levels of A little or no effect in some cell types, and normally stimulate the parental line (GPI1). When this parental line was secretion of sAPPa while simultaneously reducing the secre- b40, Ab42, and sAPPb decreased treated with PI-PLC, A tion of Ab (2). A metalloproteinase related to the tumor necro- to levels similar to those observed in the mutant line, sis factor-a converting enzyme (7, 8) can cleave APP to sAPPa and the mutant line was resistant to these effects of PI-PLC. These findings provide strong evidence that one upon activation of PKC by phorbol esters (9, 10). or more GPI-anchored proteins play an important role Strong evidence that Ab plays an important role in AD b-secretase activity and Ab secretion in mammalian in pathogenesis has come from the study of mutations in the APP cells. The cell-surface GPI-anchored protein(s) involved (11–14), presenilin 1 (15), and presenilin 2 (16) genes that are b biogenesis may be excellent therapeutic target(s) in A known to cause early onset familial AD (FAD) (17). A funda- in Alzheimer’s disease. mental effect of all the FAD-linked mutations is to increase the concentration of Ab42 or of both Ab40 and Ab42 (18 –22). Ab42 is deposited early and selectively in the senile plaques that are The amyloid that is invariably deposited in Alzheimer’s dis- observed in all forms of AD, so these findings provide strong ease (AD) is composed of an approximately 4-kDa peptide evidence that the FAD-linked mutations all act to cause AD by increasing the extracellular concentration of Ab42. The evidence implicating Ab in AD pathogenesis has made * This work was supported by National Institutes of Health Grant both b- and g-secretase important therapeutic targets, but 1RO3AG14883 (to K. S.). The costs of publication of this article were neither has yet been identified. It was recently demonstrated, defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 however, that APP is proteolytically processed by an a-secre- U.S.C. Section 1734 solely to indicate this fact. tase-like pathway in the yeast Saccharomyces cerevisiae (23, ‡ To whom correspondence should be addressed: Mayo Clinic, 4500 24). This processing pathway was shown to involve the glyco- San Pablo Rd., Jacksonville, FL 32224. Tel.: 904-953-7383; Fax: 904- sylphosphatidylinositol (GPI)-anchored aspartyl proteases 953-7380; E-mail: [email protected]. YAP3 and MKC7 (25, 26). In mammals, GPI-anchored proteins § Present address: Nathan Kline Institute, 140 Old Orangeburg, Orangeburg, NY 10962. are a relatively small class of membrane proteins that are The abbreviations used are: AD, Alzheimer’s disease; FAD, familial anchored to the outer leaflet of the cell membrane by a glyco- AD; Ab, Alzheimer’s amyloid b-protein; Ab40, Ab ending at residue 40; lipid anchor consisting of ethanolamine, mannose, glucosa- Ab42, Ab ending at residue 42; sAPP, secreted APP derived from APP mine, and phosphatidylinositol (27). These proteins can be by proteolytic cleavage; sAPPa, sAPP product of a-secretase cleavage; sAPPb, sAPP product of b-secretase cleavage; APP, Ab-protein precur- removed from the cell surface by treatment with phosphatidyl- sor; PI, phosphatidylinositol; PI-PLC, phosphatidylinositol-specific inositol-specific phospholipase C (PI-PLC), which cleaves the phospholipase C; PLAP, human placental alkaline phosphatase; GPI1, Chinese hamster ovary cell line transfected with PLAP gpi85 derivative of GPI1 with mutation in N-acetylglucosaminylphosphatidylinositol acid; ELISA, enzyme-linked immunosorbent assay; CHO, Chinese ham- deacetylase responsible for the synthesis of the GPI anchor; PDBu, ster ovary; PBS, phosphate-buffered saline; PKC, protein kinase C; phorbol dibutyrate; BIS, bisindolylmaleimide; DAG, diacylglycerol; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic L. M. Refolo and K. Sambamurti, manuscript in preparation. 26810 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Role of GPI-anchored Proteins in Ab Biogenesis 26811 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 Ab biogenesis. Our data indicate that at least one form of b-secretase or one of its essential, constitutively active regula- tors is GPI-anchored. It appears, however, that a-secretase is not a GPI-anchored protease in mammalian cells. EXPERIMENTAL PROCEDURES Purification of PI-PLC—PI-PLC was expressed in Bacillus subtilis cultures transformed with a plasmid containing the Bacillus thuring- FIG.1. PI-PLC has no effect on mammalian sAPPa. Media from iensis gene and was purified from 8 liters of the culture media (28). triplicate guinea pig brain cultures that were untreated (lanes 1–3)or Briefly, the secreted PI-PLC was concentrated by filtration through a treated with PI-PLC for 3 days (lanes 4 – 6) were probed with the BAN50 Millipore pelican filter, adjusted to 0.2 M NaCl, and loaded onto a 50-ml antibody to residues 1–16 of Ab. Densitometric analysis of the blot octyl-Sepharose column. The column was washed with 100 ml of 20 mM showed that the levels of sAPPa were similar in treated and untreated Na PO , 0.2 M NaCl, pH 7, and eluted with 20 mM Na PO ,pH7. 3 4 3 4 cultures, whereas cultures treated with PI-PLC showed a large decline Fractions containing active PI-PLC were adjusted to pH 8.5 and 0.055% in the amounts of secreted Ab40 and Ab42 (Table I). 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 Ab ELISA—Ab was measured using an established, sensitive, and eluted with 20 mM Tris acetate, 0.55% Triton X-100, pH 5.0. The specific sandwich ELISA (20). Briefly, Nunc immunomax dishes were activity of the fractions was assayed by the measurement of its capacity coated with capture antibodies BAN50 (guinea pig) or BNT77 (CHO) in to hydrolyze H-labeled phosphatidylinositol (PI) in 50 mM Tris acetate, 0.1 M carbonate buffer, pH 9.6, and blocked with 0.5% block ace (Wako) pH 5.5, containing 2 mM 1,2-diheptanoyl-sn-glycero-3-phosphatidylcho- in PBS, pH 7.4. The media samples (100 ml) were incubated overnight line (Avanti Polar Lipids, Inc.). The free inositol released by PI-PLC was in 50 ml of ELISA buffer I (0.02 M sodium phosphate, 2 mM EDTA, 0.4 measured in the aqueous fraction after stopping the reaction in 10:5:0.1 M NaCl, 0.2% bovine serum albumin, 0.05% CHAPS, 0.4% block ace, chloroform:methanol:HCl. The protein concentration was determined 0.05% NaN , pH 7.0), washed three times with PBS, and treated over- by a BCA assay (Pierce). At this stage the PI-PLC is very pure as judged night with the horseradish peroxidase-labeled detection antibodies by the detection of a single band on a Coomassie Blue-stained SDS- BA27 and BC05 diluted in ELISA buffer II (0.02 M sodium phosphate, polyacrylamide gel that was subsequently stained with silver. For most 2mM EDTA, 0.4 M NaCl, 1.0% bovine serum albumin, 0.002% thimer- of our treatments, we used 2 mg/ml of this purified PI-PLC. osal, pH 7.0). After washing twice with PBS and once with PBS 1 0.05% Mixed Brain Cultures—Mixed brain cultures from guinea pig fetuses Tween 20 horseradish peroxidase activity was detected using a Pierce were prepared from 30-day embryos using a method developed by Dr. ELISA kit as described by the manufacturer. Addition of PI-PLC- Yasumasa Ohyagi (see Ref. 29). Briefly, a pregnant guinea pig was containing CCM5 medium did not influence the measurement of sacrificed with carbon dioxide, and six embryos were recovered from the standard Ab. uterus. The brains were dissected and minced into fine pieces. These Western Blots—The human/guinea pig-specific BAN50 antibody and pieces were treated with 10 volumes of 0.03% trypsin and 0.02% EDTA the sAPPb-specific 192 antibody were used to determine the levels of in HEPES-buffered saline, pH 6.4, for 3 min, and the reaction was sAPPa and sAPPb, respectively, by Western blotting using methods stopped by the addition of an equal volume of 5% calf serum in Opti- described earlier (20, 34, 35). We also verified the specificity of the 192 MEM (Life Technologies, Inc.). The cells were collected by centrifuga- antibody by probing peptide spots CSEVKM, CSEVK, and CSEVKMD. tion, and the pellet was resuspended in 20 ml of Opti-MEM containing As expected, strong signals were only obtained for the CSEVKM pep- 5% calf serum and filtered through a 67-mm polyester mesh (Spectrum). tide, which corresponds to sAPPb cleavage, and the other two-peptide The resulting cells were plated at a high density (10 cells per dish) to spots were negative, demonstrating the end specificity of the 192 anti- prevent astrocytic overgrowth. These cultures can be maintained in the body (data not shown). Total sAPP in the medium was detected using same medium for over a month without loss of viability. In addition to the 22C11 antibody (Roche Molecular Biochemicals). 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 Ab40 RESULTS and Ab42 peptides. Furthermore, unlike rodents, the guinea pig Ab PI-PLC Treatment Reduces the Secretion of Ab but Not sequence is identical to that in man, thereby allowing the use of the human-specific antibody against Ab1–16 (BAN50) for the analysis of sAPPa in Guinea Pig Mixed Brain Cultures—Guinea pig mixed sAPPa (30). brain cultures were treated with PI-PLC for 1 and 3 days in Cell Lines and Media—A wild type CHO-K1 cell line (GPI1) express- triplicate. Over 3 days of PI-PLC treatment, sAPPa was not ing GPI-anchored human placental alkaline phosphatase (PLAP) and a reduced in amount (Fig. 1), but both Ab40 and Ab42 were derivative line with a mutation in the GPI biosynthesis pathway (gpi85) substantially reduced (Table I). This large reduction was were kind gifts from Dr. Victoria Stevens (31). The gpi85 mutant is readily observed after 1 day of treatment and was sustained for deficient in N-acetylglucosaminylphosphatidylinositol deacetylase which is responsible for the second step in the biosynthesis of the GPI at least 3 days. Since PI-PLC treatment is known to release anchor (32). These cultures were maintained in Ham’s F-12 media GPI-anchored proteins from the cell surface, these data suggest supplemented with 10% fetal bovine serum and 100 mg/ml G418. that one or more GPI-anchored proteins are involved in Ab Enzyme Assays—Alkaline phosphatase activity was assayed using 24 biogenesis. The lack of an effect on sAPPa indicates that cleav- mM p-nitrophenyl phosphate as a substrate in 1 M diethanolamine age by a-secretase is not affected by the removal of cell-surface buffer, pH 9.6, containing 20 mM homoarginine. The samples were first GPI-anchored proteins and that PI-PLC treatment has no ap- heated to 55 °C for 10 min to increase the specificity for placental alkaline phosphatase activity (33). preciable effect on APP synthesis. Drug and PI-PLC Treatments—All treatments were carried out in Effects of PI-PLC Treatment on Ab Secretion from CHO Cul- the serum-free CCM5 medium (HyClone) after adapting the cultures for tures—To investigate further the role of GPI-anchored proteins 24 h in the same medium. Phorbol dibutyrate (PDBu 1 mM; Sigma) was in Ab biogenesis, we investigated the mutant CHO cell line, dissolved in Me SO, which was also added to controls and other treat- gpi85. This line was derived (31) from a recombinant CHO cell ments. Me SO concentrations were maintained at 0.2% for PDBu treat- line (GPI1), which expresses human PLAP as a GPI-anchored ments. Bisindolylmaleimide (BIS) stocks were prepared in distilled water and directly diluted to 2.5 mM into the culture medium (4). reporter protein. The gpi85 line is deficient in N-acetylglu- Antibodies—Monoclonal antibodies BAN50 (Ab1–16), BNT77 (Ab11– cosaminylphosphatidylinositol deacetylase (4), which is re- 24), BC05(Ab42), and BA27 (Ab40) have been previously described sponsible for the second step in the biosynthesis of the GPI (20) and were gifts from Dr. Nobu Suzuki (Takeda Industries). Ivan anchor (32). The loss of GPI anchor synthesis is neither lethal Lieberburg and John Anderson (Elan Corp.) kindly provided the 192 nor detrimental to the growth of mammalian cells. However, in antibody, specific for sAPPb (34). When appropriate, selected anti- cells (e.g. gpi85) that do not synthesize the GPI anchor, pro- bodies were tagged with horseradish peroxidase, using a kit from Pierce as described by the manufacturer. teins that are normally GPI-anchored are degraded in the 26812 Role of GPI-anchored Proteins in Ab Biogenesis TABLE I PI-PLC reduces Ab40 and Ab42 secretion in guinea pig mixed brain cultures Guinea pig brain cultures prepared as described under “Experimen- tal Procedures” were treated for 1 or 3 days with the indicated dose of PI-PLC. Media were collected and analyzed for Ab40 and Ab42. PI-PLC Days Ab40 Ab42 mg/ml pM 0 1 118 6 13 18 6 2 a a 20 1 16 6 2 3 6 0 0 3 465 6 14 46 6 3 a a 2 3 105 6 27 13 6 2 a a 20 3 36 6 2 5 6 0 p , 0.01. endoplasmic reticulum (36, 37) and are not, therefore, found at the cell surface. FIG.2. Release of human PLAP from GPI1 CHO cells by PI- To determine the concentration of PI-PLC needed to remove PLC. The PLAP release into the medium of untreated GPI1 cultures was 100-fold lower than with PI-PLC treatment. The mutant gpi85 GPI-anchored proteins from the cell surface, we treated GPI1 cultures did not release any PLAP, as reported. By3hof treatment, 2 cultures with two doses of PI-PLC and measured the release of mg/ml PI-PLC released as much PLAP as 20 mg/ml, indicating that all PLAP (Fig. 2). More than 80% of total cellular PLAP was the accessible PLAP was cleaved by the lower dose. released in1hby treatment with 20 mg/ml PI-PLC. The small amount of PLAP remaining after this treatment appears to be TABLE II PI-PLC reduces Ab40 and Ab42 secretion in GPI1 CHO cells but not inaccessible to PI-PLC and is presumably mostly intracellular in the GPI-anchor-deficient mutant, gpi85 because no additional PLAP was released by treating cells for GPI1 and gpi85 CHO cultures were treated for 3 h with PI-PLC. 3 h with 20 mg/ml PI-PLC (Fig. 2). The amount of PLAP re- Media were collected and analyzed for Ab40 and Ab42. leased by treatment with 2 mg/ml PI-PLC for 1 h was 75% of Cell line PI-PLC Hours Ab40 Ab42 that released by 20 mg/ml, but essentially the same amount was released when treatment with 2 mg/ml PI-PLC was extended to mg/ml pM 3 h (Fig. 2). Thus treatment with 2 mg/ml PI-PLC for 3 h GPI1 0 3 193 6 48 8 6 2 a b GPI1 2 3 124 6 31 5 6 2 appears to be adequate to remove virtually all of the GPI- a b gpi85 0 3 113 6 20 5 6 1 anchored protein at the cell surface, and subsequent experi- gpi85 2 3 110 6 25 5 6 1 ments were conducted using 2 mg/ml PI-PLC. GPI1 0 24 550 6 46 21 6 2 a a In 10 independent experiments, secreted Ab was compared GPI1 2 24 251 6 52 8 6 2 b b gpi85 0 24 447 6 13 15 6 1 in gpi85 and GPI1 cells cultured in parallel. In 9 of these gpi85 2 24 391 6 24 14 6 1 experiments, the effect of PI-PLC treatment (2 mg/ml for 3 h) was analyzed in both cell types. The results of this study, which p , 0.01. p , 0.03. 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 signifi- did not increase sAPPa in this preparation (Fig. 1). That Ab cant reduction in Ab40 (p , 0.0008) and Ab42 (p , 0.03) was reduced similarly in PI-PLC-treated GPI1 cells (where relative to untreated GPI1 cells. In gpi85 cells, PI-PLC had no DAG may be released) and in untreated gpi85 cells (where no significant effect on Ab40 or Ab42, as expected. In GPI1 cells, DAG is released) also makes it highly unlikely that DAG re- as in fetal guinea pig brain cultures, removal of cell-surface lease contributes importantly to the Ab reduction observed in GPI-anchored proteins with PI-PLC caused a consistent, highly PI-PLC-treated CHO cells. significant reduction in Ab40 (p , 0.008) and Ab42 (p , 0.03). To address further the question of DAG-induced PKC acti- As shown in Table II, GPI1 cells treated with PI-PLC and vation, the PKC inhibitor BIS (G109203X) was added to GPI1 mutant gpi85 cells produced remarkably similar amounts of cultures prior to treatment with PI-PLC. This compound has Ab40 and Ab42 that were consistently ;40% less than the previously been shown to block the changes in APP metabolism amount produced by normal GPI1 cells. Thus, the removal of (4) that occur when PDBu is used to activate PKC (4). Our GPI-anchored proteins by PI-PLC and by mutation causes a control experiments showed that PDBu reduces Ab secretion in similar, highly significant reduction in Ab40 and Ab42. Con- GPI1 cells, and this reduction was completely blocked by 2.5 firming these findings, we have also observed that total se- mM BIS. In addition, as previously reported (4), PDBu treat- creted Ab (4-kDa band), immunoprecipitated from [ ment elevated total sAPP secretion by about 15-fold, and this S]methi- onine-labeled CHO cells expressing high levels of human was also blocked by the BIS treatment (data not shown). Treat- APP695, is reduced upon PI-PLC treatment (p , 0.05) to ap- ment of GPI1 cultures with BIS did not, however, block the proximately the same extent as shown in Table II without reduction in Ab40 and Ab42 caused by PI-PLC (Table III). significantly changing sAPPa (data not shown). Thus, our results clearly show that the effect of PI-PLC on Ab Activation of PKC by PI-PLC-mediated Hydrolysis of PI Does biogenesis is not due to DAG-induced PKC activation. Not Account for the Reduction in Secreted Ab Levels in GPI1 b-Secretase Processing of APP Is Reduced in PI-PLC-treated Cells—In many cell types, PKC activation results in a substan- CHO Cultures—The PI-PLC-induced reduction in Ab could be tial stimulation of sAPPa release coupled with a reduction in caused by a reduction in either b-or g-secretase activity. To Ab secretion (3). By hydrolyzing PI in membranes, PI-PLC evaluate whether b-secretase activity is reduced when GPI- treatment could, in principle, activate PKC by releasing dia- anchored proteins are removed, several experiments were per- cylglycerol (DAG). It is, however, highly unlikely that PKC formed in which we treated GPI1 and gpi85 cultures in trip- activation contributes significantly to the effect of PI-PLC on licate with PI-PLC for 24 h and then evaluated sAPPb and total guinea pig brain cultures, because PI-PLC decreased Ab but sAPP (sAPPa 1 sAPPb) by immunoblotting (Fig. 3). For spe- Role of GPI-anchored Proteins in Ab Biogenesis 26813 TABLE III Addition of the PKC inhibitor BIS blocks PDBu-mediated but not PI-PLC-mediated Ab reduction in GPI1 cultures The GPI1 and gpi85 cultures were treated for 3 h with PDBu, PI-PLC, and/or BIS. Media were collected and analyzed for Ab40 and Ab42. Ab40 Ab42 Control 147 6 29 12 6 5 BIS 136 6 17 11 6 4 PI-PLC 97 6 22 8 6 4 PI-PLC BIS 86 6 19 7 6 4 PDBu 125 6 18 NT PDBu BIS 168 6 42 NT NT, not tested. cific detection of sAPPb, we used antibody 192, which effi- ciently binds APP cleaved between residues 21 and 11ofthe Ab sequence but shows a low affinity for full-length APP, sAPPa, and other APP fragments (34). We could not specifically analyze the sAPPa secreted from the GPI1 and gpi85 cell lines, because antibodies to human Ab1–16 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 (sAPPa 1 sAPPb) by immunoblotting with monoclonal antibody 22C11, which recognizes both hu- man and rodent sAPP. Densitometric analysis showed that sAPPb was reduced by approximately 90% upon PI-PLC treatment of GPI1 cultures FIG.3. PI-PLC substantially reduces mammalian sAPPb. The (Fig. 3). In contrast, total sAPP (sAPPb 1 sAPPa) was only proteins in media from gpi85 cells (1 untreated; 2– 4 PI-PLC-treated) slightly reduced (;16%). Since sAPPa is the major secreted and GPI1 cells (5–7 untreated; 8 –10 PI-PLC-treated) were separated species in GPI1 cells, the small reduction in total sAPP indi- on 10 –20% Tris-Tricine gels. A, immunolabeled with antibody 192, cates that PI-PLC has little effect on sAPPa. Levels of sAPPb which specifically detects sAPPb (solid arrow). B, immunolabeled with were also highly (71%) reduced in untreated gpi85 cells as antibody 22C11, which detects total sAPP (sAPPa 1 sAPPb, open arrow). Densitometric analysis of lanes from two independent blots compared with GPI1 cells, but again total sAPP was only showed that sAPPb was reduced by 90% when GPI1 cultures were slightly affected. As expected, PI-PLC treatment did not con- treated with PI-PLC. sAPPb was 71% lower in gpi85 than in GPI1 sistently affect sAPPb or the total sAPP secreted by gpi85 cells. cultures, and PI-PLC treatment had no consistent effect on the amount These results provide strong evidence that GPI-anchored cell- of sAPPb secreted by gpi85 cells. Total sAPP was slightly reduced (;16%) when GPI1 cultures were treated with PI-PLC. Total sAPP was surface proteins play an important role in b-secretase activity. also slightly lower in gpi85 than in GPI1 cultures. PI-PLC treatment DISCUSSION 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 The proteins involved in Ab biogenesis are important thera- differences in total sAPP were small, we repeated this study, and the peutic targets in Alzheimer’s disease. To date, none of these data in C are summarized from the two experiments. proteins has been unequivocally identified. In this study, we investigated the role of GPI-anchored cell- surface proteins in surface clearly implicate one or more cell-surface GPI-anchored Ab biogenesis. Our results provide strong evidence that, in proteins in b-secretase activity and Ab biogenesis. This finding mammalian cells, one or more cell-surface GPI-anchored pro- fits well with recent reports that APP and Ab are found in teins play an important role in b-secretase activity and Ab detergent-insoluble membrane domains enriched in GPI-an- secretion. In addition, they indicate that cell-surface GPI-an- chored proteins (38, 39). These domains are rich in cholesterol, chored proteins do not play a major role in a-secretase activity. which has been reported to be important for Ab production These conclusions are based on the following observations. (i) (40, 41). PI-PLC, which removes GPI-anchored proteins from the cell Although reduced, secretion of sAPPb,Ab40, and Ab42 con- surface, substantially reduces the Ab40 and Ab42 secreted tinues both in PI-PLC-treated cells and in mutant gpi85 cells from fetal guinea pig brain cultures and the wild type GPI1 deficient in GPI-anchored proteins. This suggests that normal CHO cell cultures. (ii) The mutant gpi85 line, which lacks b-secretase activity may involve both GPI-dependent and -in- cell-surface GPI-anchored proteins, shows reductions in se- dependent proteins. One should, however, be cautious in draw- creted Ab40 and Ab42 similar to those observed when the ing this conclusion. A small percentage of the GPI-anchored parental GPI1 line is treated with PI-PLC. (iii) The PI-PLC proteins may be resistant to PI-PLC because of fatty acylation treatment that lowers the Ab40 and Ab42 secreted from GPI1 of the GPI anchor (28). PI-PLC treatment removes cell-surface cells has no detectable effect on the residual Ab secretion that GPI-anchored proteins but may not remove intracellular GPI- occurs in gpi85 cells. (iv) sAPPb is substantially reduced in anchored proteins that could continue to produce Ab and gpi85 cells and in PI-PLC-treated GPI1 cells but shows no sAPPb.In gpi85 cells, GPI-anchored proteins continue to be additional reduction in PI-PLC-treated gpi85 cells. (v) PI-PLC synthesized. These proteins are rapidly degraded when they treatment does not affect the levels of sAPPa in fetal guinea pig fail to be GPI-anchored, and they do not accumulate at the cell brain cultures and only slightly reduces total sAPP (sAPPa 1 surface, but some residual protein precursor capable of proc- sAPPb)inGPI1 and gpi85 cultures. essing intracellular APP to sAPPb and Ab may remain in the The coordinate reduction of Ab40 and Ab42, the profound secretory pathway. Thus our findings are consistent with sep- reduction of sAPPb, and the lack of change in sAPPa that arate GPI-anchored protein-dependent and -independent occurs when GPI-anchored proteins are removed from the cell b-secretase pathways, but they do not eliminate the possibility 26814 Role of GPI-anchored Proteins in Ab Biogenesis R. S., Paxton, R. J., March, C. J., and Cerretti, D. P. (1997) Nature 385, that normal b-secretase cleavage is carried out entirely by one 729 –733 or several GPI-anchored proteins. 8. Moss, M. L., Jin, S. L., Milla, M. E., Bickett, D. M., Burkhart, W., Carter, H. L., Given the complexity of the cell surface and intracellular Chen, W. J., Clay, W. C., Didsbury, J. R., Hassler, D., Hoffman, C. R., Kost, T. A., Lambert, M. H., Leesnitzer, M. A., McCauley, P., McGeehan, G., events that are involved in Ab biogenesis, it is likely that some Mitchell, J., Moyer, M., Pahel, G., Rocque, W., Overton, L. K., Schoenen, F., secreted Ab is produced through alternative proteolytic path- Seaton, T., Su, J. L., and Becherer, J. D. (1997) Nature 385, 733–736 ways that do not involve cell-surface GPI-anchored proteins. 9. Merlos-Suarez, A., Fernandez-Larrea, J., Reddy, P., Baselga, J., and Arribas, J. (1998) J. Biol. Chem. 273, 24955–24962 Thus there is likely to be an upper limit to the reduction in Ab 10. Buxbaum, J. D., Liu, K. N., Luo, Y., Slack, J. L., Stocking, K. L., Peschon, J. J., secretion that can be achieved by removing GPI-anchored pro- Johnson, R. S., Castner, B. J., Cerretti, D. P., and Black, R. A. (1998) J. Biol. teins. In the CHO cell lines that we examined, removal of Chem. 273, 27765–27767 11. Goate, A., Chartier-Harlin, M. C., Mullan, M., Brown, J., Crawford, F., Fidani, cell-surface GPI-anchored proteins caused a coordinate ;50% L., Giuffra, L., Haynes, A., Irving, N., James, L., Mani, R., Newton, P., reduction of Ab40 and Ab42 at 24 h that was smaller than the Rooke, K., Roques, P., Talbot, C., Pericak-Vance, M., Roses, A., Williamson, 80 –90% reduction in sAPPb. This sizable difference suggests R., Rossen, M., Owen, M., and Hardy, J. (1991) Nature 349, 704 –706 12. Mullan, M., Crawford, F., Axelman, K., Houlden, H., Lilius, L., Winblad, B., that, in CHO cells, most sAPPb is produced by a GPI-anchored and Lannfelt, L. (1992) Nat. Genet. 1, 345–347 protein-dependent pathway, whereas a substantial percentage 13. Murrell, J., Farlow, M., Ghetti, B., and Benson, M. (1991) Science 254, 97–99 of Ab is produced by alternative pathways that do not result in 14. Kennedy, A., Newman, S., McCaddon, A., Ball, J., Roques, P., Mullan, M., Hardy, J., Chartier-Harlin, M. C., Frackowiak, R. S., and Warrington, E. K. the secretion of sAPPb. This finding agrees well with the pub- (1993) Brain 116, 309 –324 lished evidence for the presence of secondary b-secretase activ- 15. Sherrington, R., Rogaev, E. I., Liang, Y., Rogaeva, E. A., Levesque, G., Ikeda, ities, which cleave APP at alternative locations close to the M., Chi, H., Lin, C., Li, G., Holman, K., Tsuda, T., Mar, L., Foncin, J. F., Bruni, A. C., Montesi, M. P., Sorbi, S., Rainero, I., Pinessi, L., Nee, L., N-terminal end of the Ab sequence. The sequence of the se- Chumakov, I., Pollen, D., Brookes, A., Sanseau, P., Polinsky, R. J., Wasco, creted Ab begins at all known residues ranging from 1 to 11 W., Da Silva, H. A. R., Haines, J. L., Pericak-Vance, M. A., Tanzi, R. E., indicating that the cleavage can occur at any one of these Roses, A. D., Fraser, P. E., Rommens, J. M., and St. George Hyslop, P. H. (1995) Nature 375, 754 –760 residues (42, 43). The sAPPb-specific antibody, 192, will not 16. Levy-Lahad, E., Wasco, W., Poorkaj, P., Romano, D. M., Oshima, J., Pettingell, detect sAPP cleaved at these secondary sites, but the sandwich W. H., Yu, C. E., Jondro, P. D., Schmidt, S. D., Wang, K., Crowley, A. C., Fu, ELISA will detect the Ab generated by these cleavages which Y. H., Guenette, S. Y., Galas, D., Nemens, E., Wijsman, E. M., Bird, T. D., Schellenberg, G. D., and Tanzi, R. E. (1995) Science 269, 973–977 may be GPI anchor-independent. 17. Hardy, J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 2095–2097 The number of cell-surface GPI-anchored proteins that are 18. Citron, M., Oltersdorf, T., Haass, C., McConlogue, L., Hung, A. Y., Seubert, P., involved in b-secretase activity and the precise nature of these Vigo-Pelfrey, C., Lieberburg, I., and Selkoe, D. J. (1992) Nature 360, 672– 674 proteins remain to be determined. It is entirely possible that 19. Cai, X. D., Golde, T. E., and Younkin, S. G. (1993) Science 259, 514 –516 the effects we have observed are all due to the removal of a 20. Suzuki, N., Cheung, T. T., Cai, X. D., Odaka, A., Otvos, L., Jr., Eckman, C., single catalytically active b-secretase that is GPI-anchored to Golde, T. E., and Younkin, S. G. (1994) Science 264, 1336 –1340 21. Borchelt, D. R., Thinakaran, G., Eckman, C. B., Lee, M. K., Davenport, F., the cell surface. Alternatively, a GPI-anchored protein could (i) Ratovitsky, T., Prada, C. M., Kim, G., Seekins, S., Yager, D., Slunt, H. H., be an essential non-catalytic subunit in a multi-subunit Wang, R., Seeger, M., Levey, A. I., Gandy, S. E., Copeland, N. G., Jenkins, b-secretase; (ii) perform a post-translational modification re- N. A., Price, D. L., Younkin, S. G., and Sisodia, S. S. (1996) Neuron 17, 1005–1013 quired to sustain b-secretase activity; (iii) chaperone to bring 22. Scheuner, D., Eckman, C., Jensen, M., Song, X., Citron, M., Suzuki, N., Bird, together APP and b-secretase into one compartment; or (iv) T. D., Hardy, J., Hutton, M., Kukull, W., Larson, E., Levy-Lahad, E., activate another factor that performs one of the functions listed Viitanen, M., Peskind, E., Poorkaj, P., Schellenberg, G., Tanzi, R., Wasco, W., Lannfelt, L., Selkoe, D., and Younkin, S. (1996) Nat. Med. 2, 864 – 870 above. Whatever its precise nature, any cell-surface GPI-an- 23. Hines, V., Zhang, W., Ramakrishna, N., Styles, J., Mehta, P., Kim, K. S., Innis, chored protein involved in b-secretase activity is of great inter- M., and Miller, D. L. (1994) Cell. & Mol. Biol. Res. 40, 273–284 est as a therapeutic target in AD. GPI-anchored proteins can be 24. Zhang, H., Komano, H., Fuller, R. S., Gandy, S. E., and Frail, D. E. (1994) J. Biol. Chem. 269, 27799 –27802 substantially enriched in cell lysates on the basis of their de- 25. Zhang, W., Espinoza, D., Hines, V., Innis, M., Mehta, P., and Miller, D. L. tergent insolubility, and they can be released specifically from (1997) Biochim. Biophys. Acta 1359, 110 –122 intact cells or cell lysates by PI-PLC. In addition, these proteins 26. Komano, H., Seeger, M., Gandy, S., Wang, G. T., Krafft, G. A., and Fuller, R. S. (1998) J. Biol. Chem. 273, 31648 –31651 can be incorporated from the medium into live cultured cells in 27. McConville, M. J., and Ferguson, M. A. (1993) Biochem. J. 294, 305–324 a functionally active form. These unique properties and the 28. Deeg, M. A., Humphrey, D. R., Yang, S. H., Ferguson, T. R., Reinhold, V. N., relatively small number of mammalian GPI-anchored proteins and Rosenberry, T. L. (1992) J. Biol. Chem. 267, 18573–18580 29. Clarke, N. J., Tomlinson, A. J., Ohyagi, Y., Younkin, S., and Naylor, S. (1998) should be highly advantageous in the effort to isolate and FEBS Lett. 430, 419 – 423 characterize the specific GPI-anchored protein(s) that are in- 30. Johnstone, E. M., Chaney, M. O., Norris, F. H., Pascual, R., and Little, S. P. volved in b-secretase activity. (1991). Mol. Brain Res. 10, 299 –305 31. Stevens, V. L., Zhang, H., and Harreman, M. (1996) Biochem. J. 313, 253–258 Acknowledgments—We thank Dr. Victoria Stevens for providing us 32. Nakamura, N., Inoue, N., Watanabe, R., Takahashi, M., Takeda, J., Stevens, V. L., and Kinoshita, T. (1997) J. Biol. Chem. 272, 15834 –15840 with the GPI1 and gpi85 strains, Dr. Ivan Lieberburg and Dr. John 33. Berger, J., Hauber, J., Hauber, R., Geiger, R., and Cullen, B. R. (1988) Gene Anderson of Elan Corp. for the 192 antibody, and Dr. Nobu Suzuki of (Amst.) 66, 1–10 Takeda Industries for antibodies BAN50, BNT77, BA27, and BC05. We 34. Seubert, P., Oltersdorf, T., Lee, M. G., Barbour, R., Blomquist, C., Davis, D. 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Glycosylphosphatidylinositol-anchored Proteins Play an Important Role in the Biogenesis of the Alzheimer's Amyloid β-Protein

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 38, Issue of September 17, pp. 26810 –26814, 1999 © 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Glycosylphosphatidylinositol-anchored Proteins Play an Important b-Protein* Role in the Biogenesis of the Alzheimer’s Amyloid (Received for publication, January 28, 1999, and in revised form, July 13, 1999) Kumar Sambamurti‡, 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 The Alzheimer’s amyloid protein (Ab) is released from (amyloid b-peptide, Ab) that is derived from a larger protein b-protein precursor (APP) by uniden- referred to as the amyloid b-protein precursor (APP) (1, 2). APP the larger amyloid b- and g-secretase. b-Secre- tified enzymes referred to as is a type I integral membrane glycoprotein with a large N- b producing a tase cleaves APP on the amino side of A terminal extracellular domain, a single transmembrane do- b) and an Ab-bearing large secreted derivative (sAPP main, and a short cytoplasmic tail. The Ab peptide begins 99 C-terminal derivative that is subsequently cleaved by amino acids from the C terminus of APP, and it extends from g-secretase to release Ab. Alternative cleavage of the the extracellular region to a point half-way through the APP a-secretase at Ab16/17 releases the secreted de- APP by membrane-spanning domain (1). Ab is released from APP by a. In yeast, a-secretase activity has been rivative sAPP cleavage on its N- and C-terminal ends by b- and g-secretase, attributed to glycosylphosphatidylinositol (GPI)-an- respectively. b-Secretase cleavage before residue 1 of Ab (672 of chored aspartyl proteases. To examine the role of GPI- APP770) also releases the secreted derivative sAPPb, whereas anchored proteins, we specifically removed these pro- an alternative cleavage before residue 17 by a-secretase re- teins from the surface of mammalian cells using leases sAPPa. In most culture systems tested, the predominant phosphatidylinositol-specific phospholipase C (PI-PLC). cleavage product is sAPPa, and this may serve to prevent the PI-PLC treatment of fetal guinea pig brain cultures sub- production of Ab (1). The proteolytic processing of APP to sAPP b40 and Ab42 in the stantially reduced the amount of A and Ab is regulated by protein kinase C (3), protein tyrosine a. A mutant CHO cell medium but had no effect on sAPP kinase (4), muscarinic receptors (5), and estrogens (6). The line (gpi85), which lacks GPI-anchored proteins, se- regulatory pathways involved are cell type-dependent, have b40, Ab42, and sAPPb than its creted lower levels of A little or no effect in some cell types, and normally stimulate the parental line (GPI1). When this parental line was secretion of sAPPa while simultaneously reducing the secre- b40, Ab42, and sAPPb decreased treated with PI-PLC, A tion of Ab (2). A metalloproteinase related to the tumor necro- to levels similar to those observed in the mutant line, sis factor-a converting enzyme (7, 8) can cleave APP to sAPPa and the mutant line was resistant to these effects of PI-PLC. These findings provide strong evidence that one upon activation of PKC by phorbol esters (9, 10). or more GPI-anchored proteins play an important role Strong evidence that Ab plays an important role in AD b-secretase activity and Ab secretion in mammalian in pathogenesis has come from the study of mutations in the APP cells. The cell-surface GPI-anchored protein(s) involved (11–14), presenilin 1 (15), and presenilin 2 (16) genes that are b biogenesis may be excellent therapeutic target(s) in A known to cause early onset familial AD (FAD) (17). A funda- in Alzheimer’s disease. mental effect of all the FAD-linked mutations is to increase the concentration of Ab42 or of both Ab40 and Ab42 (18 –22). Ab42 is deposited early and selectively in the senile plaques that are The amyloid that is invariably deposited in Alzheimer’s dis- observed in all forms of AD, so these findings provide strong ease (AD) is composed of an approximately 4-kDa peptide evidence that the FAD-linked mutations all act to cause AD by increasing the extracellular concentration of Ab42. The evidence implicating Ab in AD pathogenesis has made * This work was supported by National Institutes of Health Grant both b- and g-secretase important therapeutic targets, but 1RO3AG14883 (to K. S.). The costs of publication of this article were neither has yet been identified. It was recently demonstrated, defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 however, that APP is proteolytically processed by an a-secre- U.S.C. Section 1734 solely to indicate this fact. tase-like pathway in the yeast Saccharomyces cerevisiae (23, ‡ To whom correspondence should be addressed: Mayo Clinic, 4500 24). This processing pathway was shown to involve the glyco- San Pablo Rd., Jacksonville, FL 32224. Tel.: 904-953-7383; Fax: 904- sylphosphatidylinositol (GPI)-anchored aspartyl proteases 953-7380; E-mail: [email protected]. YAP3 and MKC7 (25, 26). In mammals, GPI-anchored proteins § Present address: Nathan Kline Institute, 140 Old Orangeburg, Orangeburg, NY 10962. are a relatively small class of membrane proteins that are The abbreviations used are: AD, Alzheimer’s disease; FAD, familial anchored to the outer leaflet of the cell membrane by a glyco- AD; Ab, Alzheimer’s amyloid b-protein; Ab40, Ab ending at residue 40; lipid anchor consisting of ethanolamine, mannose, glucosa- Ab42, Ab ending at residue 42; sAPP, secreted APP derived from APP mine, and phosphatidylinositol (27). These proteins can be by proteolytic cleavage; sAPPa, sAPP product of a-secretase cleavage; sAPPb, sAPP product of b-secretase cleavage; APP, Ab-protein precur- removed from the cell surface by treatment with phosphatidyl- sor; PI, phosphatidylinositol; PI-PLC, phosphatidylinositol-specific inositol-specific phospholipase C (PI-PLC), which cleaves the phospholipase C; PLAP, human placental alkaline phosphatase; GPI1, Chinese hamster ovary cell line transfected with PLAP gpi85 derivative of GPI1 with mutation in N-acetylglucosaminylphosphatidylinositol acid; ELISA, enzyme-linked immunosorbent assay; CHO, Chinese ham- deacetylase responsible for the synthesis of the GPI anchor; PDBu, ster ovary; PBS, phosphate-buffered saline; PKC, protein kinase C; phorbol dibutyrate; BIS, bisindolylmaleimide; DAG, diacylglycerol; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic L. M. Refolo and K. Sambamurti, manuscript in preparation. 26810 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Role of GPI-anchored Proteins in Ab Biogenesis 26811 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 Ab biogenesis. Our data indicate that at least one form of b-secretase or one of its essential, constitutively active regula- tors is GPI-anchored. It appears, however, that a-secretase is not a GPI-anchored protease in mammalian cells. EXPERIMENTAL PROCEDURES Purification of PI-PLC—PI-PLC was expressed in Bacillus subtilis cultures transformed with a plasmid containing the Bacillus thuring- FIG.1. PI-PLC has no effect on mammalian sAPPa. Media from iensis gene and was purified from 8 liters of the culture media (28). triplicate guinea pig brain cultures that were untreated (lanes 1–3)or Briefly, the secreted PI-PLC was concentrated by filtration through a treated with PI-PLC for 3 days (lanes 4 – 6) were probed with the BAN50 Millipore pelican filter, adjusted to 0.2 M NaCl, and loaded onto a 50-ml antibody to residues 1–16 of Ab. Densitometric analysis of the blot octyl-Sepharose column. The column was washed with 100 ml of 20 mM showed that the levels of sAPPa were similar in treated and untreated Na PO , 0.2 M NaCl, pH 7, and eluted with 20 mM Na PO ,pH7. 3 4 3 4 cultures, whereas cultures treated with PI-PLC showed a large decline Fractions containing active PI-PLC were adjusted to pH 8.5 and 0.055% in the amounts of secreted Ab40 and Ab42 (Table I). 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 Ab ELISA—Ab was measured using an established, sensitive, and eluted with 20 mM Tris acetate, 0.55% Triton X-100, pH 5.0. The specific sandwich ELISA (20). Briefly, Nunc immunomax dishes were activity of the fractions was assayed by the measurement of its capacity coated with capture antibodies BAN50 (guinea pig) or BNT77 (CHO) in to hydrolyze H-labeled phosphatidylinositol (PI) in 50 mM Tris acetate, 0.1 M carbonate buffer, pH 9.6, and blocked with 0.5% block ace (Wako) pH 5.5, containing 2 mM 1,2-diheptanoyl-sn-glycero-3-phosphatidylcho- in PBS, pH 7.4. The media samples (100 ml) were incubated overnight line (Avanti Polar Lipids, Inc.). The free inositol released by PI-PLC was in 50 ml of ELISA buffer I (0.02 M sodium phosphate, 2 mM EDTA, 0.4 measured in the aqueous fraction after stopping the reaction in 10:5:0.1 M NaCl, 0.2% bovine serum albumin, 0.05% CHAPS, 0.4% block ace, chloroform:methanol:HCl. The protein concentration was determined 0.05% NaN , pH 7.0), washed three times with PBS, and treated over- by a BCA assay (Pierce). At this stage the PI-PLC is very pure as judged night with the horseradish peroxidase-labeled detection antibodies by the detection of a single band on a Coomassie Blue-stained SDS- BA27 and BC05 diluted in ELISA buffer II (0.02 M sodium phosphate, polyacrylamide gel that was subsequently stained with silver. For most 2mM EDTA, 0.4 M NaCl, 1.0% bovine serum albumin, 0.002% thimer- of our treatments, we used 2 mg/ml of this purified PI-PLC. osal, pH 7.0). After washing twice with PBS and once with PBS 1 0.05% Mixed Brain Cultures—Mixed brain cultures from guinea pig fetuses Tween 20 horseradish peroxidase activity was detected using a Pierce were prepared from 30-day embryos using a method developed by Dr. ELISA kit as described by the manufacturer. Addition of PI-PLC- Yasumasa Ohyagi (see Ref. 29). Briefly, a pregnant guinea pig was containing CCM5 medium did not influence the measurement of sacrificed with carbon dioxide, and six embryos were recovered from the standard Ab. uterus. The brains were dissected and minced into fine pieces. These Western Blots—The human/guinea pig-specific BAN50 antibody and pieces were treated with 10 volumes of 0.03% trypsin and 0.02% EDTA the sAPPb-specific 192 antibody were used to determine the levels of in HEPES-buffered saline, pH 6.4, for 3 min, and the reaction was sAPPa and sAPPb, respectively, by Western blotting using methods stopped by the addition of an equal volume of 5% calf serum in Opti- described earlier (20, 34, 35). We also verified the specificity of the 192 MEM (Life Technologies, Inc.). The cells were collected by centrifuga- antibody by probing peptide spots CSEVKM, CSEVK, and CSEVKMD. tion, and the pellet was resuspended in 20 ml of Opti-MEM containing As expected, strong signals were only obtained for the CSEVKM pep- 5% calf serum and filtered through a 67-mm polyester mesh (Spectrum). tide, which corresponds to sAPPb cleavage, and the other two-peptide The resulting cells were plated at a high density (10 cells per dish) to spots were negative, demonstrating the end specificity of the 192 anti- prevent astrocytic overgrowth. These cultures can be maintained in the body (data not shown). Total sAPP in the medium was detected using same medium for over a month without loss of viability. In addition to the 22C11 antibody (Roche Molecular Biochemicals). 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 Ab40 RESULTS and Ab42 peptides. Furthermore, unlike rodents, the guinea pig Ab PI-PLC Treatment Reduces the Secretion of Ab but Not sequence is identical to that in man, thereby allowing the use of the human-specific antibody against Ab1–16 (BAN50) for the analysis of sAPPa in Guinea Pig Mixed Brain Cultures—Guinea pig mixed sAPPa (30). brain cultures were treated with PI-PLC for 1 and 3 days in Cell Lines and Media—A wild type CHO-K1 cell line (GPI1) express- triplicate. Over 3 days of PI-PLC treatment, sAPPa was not ing GPI-anchored human placental alkaline phosphatase (PLAP) and a reduced in amount (Fig. 1), but both Ab40 and Ab42 were derivative line with a mutation in the GPI biosynthesis pathway (gpi85) substantially reduced (Table I). This large reduction was were kind gifts from Dr. Victoria Stevens (31). The gpi85 mutant is readily observed after 1 day of treatment and was sustained for deficient in N-acetylglucosaminylphosphatidylinositol deacetylase which is responsible for the second step in the biosynthesis of the GPI at least 3 days. Since PI-PLC treatment is known to release anchor (32). These cultures were maintained in Ham’s F-12 media GPI-anchored proteins from the cell surface, these data suggest supplemented with 10% fetal bovine serum and 100 mg/ml G418. that one or more GPI-anchored proteins are involved in Ab Enzyme Assays—Alkaline phosphatase activity was assayed using 24 biogenesis. The lack of an effect on sAPPa indicates that cleav- mM p-nitrophenyl phosphate as a substrate in 1 M diethanolamine age by a-secretase is not affected by the removal of cell-surface buffer, pH 9.6, containing 20 mM homoarginine. The samples were first GPI-anchored proteins and that PI-PLC treatment has no ap- heated to 55 °C for 10 min to increase the specificity for placental alkaline phosphatase activity (33). preciable effect on APP synthesis. Drug and PI-PLC Treatments—All treatments were carried out in Effects of PI-PLC Treatment on Ab Secretion from CHO Cul- the serum-free CCM5 medium (HyClone) after adapting the cultures for tures—To investigate further the role of GPI-anchored proteins 24 h in the same medium. Phorbol dibutyrate (PDBu 1 mM; Sigma) was in Ab biogenesis, we investigated the mutant CHO cell line, dissolved in Me SO, which was also added to controls and other treat- gpi85. This line was derived (31) from a recombinant CHO cell ments. Me SO concentrations were maintained at 0.2% for PDBu treat- line (GPI1), which expresses human PLAP as a GPI-anchored ments. Bisindolylmaleimide (BIS) stocks were prepared in distilled water and directly diluted to 2.5 mM into the culture medium (4). reporter protein. The gpi85 line is deficient in N-acetylglu- Antibodies—Monoclonal antibodies BAN50 (Ab1–16), BNT77 (Ab11– cosaminylphosphatidylinositol deacetylase (4), which is re- 24), BC05(Ab42), and BA27 (Ab40) have been previously described sponsible for the second step in the biosynthesis of the GPI (20) and were gifts from Dr. Nobu Suzuki (Takeda Industries). Ivan anchor (32). The loss of GPI anchor synthesis is neither lethal Lieberburg and John Anderson (Elan Corp.) kindly provided the 192 nor detrimental to the growth of mammalian cells. However, in antibody, specific for sAPPb (34). When appropriate, selected anti- cells (e.g. gpi85) that do not synthesize the GPI anchor, pro- bodies were tagged with horseradish peroxidase, using a kit from Pierce as described by the manufacturer. teins that are normally GPI-anchored are degraded in the 26812 Role of GPI-anchored Proteins in Ab Biogenesis TABLE I PI-PLC reduces Ab40 and Ab42 secretion in guinea pig mixed brain cultures Guinea pig brain cultures prepared as described under “Experimen- tal Procedures” were treated for 1 or 3 days with the indicated dose of PI-PLC. Media were collected and analyzed for Ab40 and Ab42. PI-PLC Days Ab40 Ab42 mg/ml pM 0 1 118 6 13 18 6 2 a a 20 1 16 6 2 3 6 0 0 3 465 6 14 46 6 3 a a 2 3 105 6 27 13 6 2 a a 20 3 36 6 2 5 6 0 p , 0.01. endoplasmic reticulum (36, 37) and are not, therefore, found at the cell surface. FIG.2. Release of human PLAP from GPI1 CHO cells by PI- To determine the concentration of PI-PLC needed to remove PLC. The PLAP release into the medium of untreated GPI1 cultures was 100-fold lower than with PI-PLC treatment. The mutant gpi85 GPI-anchored proteins from the cell surface, we treated GPI1 cultures did not release any PLAP, as reported. By3hof treatment, 2 cultures with two doses of PI-PLC and measured the release of mg/ml PI-PLC released as much PLAP as 20 mg/ml, indicating that all PLAP (Fig. 2). More than 80% of total cellular PLAP was the accessible PLAP was cleaved by the lower dose. released in1hby treatment with 20 mg/ml PI-PLC. The small amount of PLAP remaining after this treatment appears to be TABLE II PI-PLC reduces Ab40 and Ab42 secretion in GPI1 CHO cells but not inaccessible to PI-PLC and is presumably mostly intracellular in the GPI-anchor-deficient mutant, gpi85 because no additional PLAP was released by treating cells for GPI1 and gpi85 CHO cultures were treated for 3 h with PI-PLC. 3 h with 20 mg/ml PI-PLC (Fig. 2). The amount of PLAP re- Media were collected and analyzed for Ab40 and Ab42. leased by treatment with 2 mg/ml PI-PLC for 1 h was 75% of Cell line PI-PLC Hours Ab40 Ab42 that released by 20 mg/ml, but essentially the same amount was released when treatment with 2 mg/ml PI-PLC was extended to mg/ml pM 3 h (Fig. 2). Thus treatment with 2 mg/ml PI-PLC for 3 h GPI1 0 3 193 6 48 8 6 2 a b GPI1 2 3 124 6 31 5 6 2 appears to be adequate to remove virtually all of the GPI- a b gpi85 0 3 113 6 20 5 6 1 anchored protein at the cell surface, and subsequent experi- gpi85 2 3 110 6 25 5 6 1 ments were conducted using 2 mg/ml PI-PLC. GPI1 0 24 550 6 46 21 6 2 a a In 10 independent experiments, secreted Ab was compared GPI1 2 24 251 6 52 8 6 2 b b gpi85 0 24 447 6 13 15 6 1 in gpi85 and GPI1 cells cultured in parallel. In 9 of these gpi85 2 24 391 6 24 14 6 1 experiments, the effect of PI-PLC treatment (2 mg/ml for 3 h) was analyzed in both cell types. The results of this study, which p , 0.01. p , 0.03. 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 signifi- did not increase sAPPa in this preparation (Fig. 1). That Ab cant reduction in Ab40 (p , 0.0008) and Ab42 (p , 0.03) was reduced similarly in PI-PLC-treated GPI1 cells (where relative to untreated GPI1 cells. In gpi85 cells, PI-PLC had no DAG may be released) and in untreated gpi85 cells (where no significant effect on Ab40 or Ab42, as expected. In GPI1 cells, DAG is released) also makes it highly unlikely that DAG re- as in fetal guinea pig brain cultures, removal of cell-surface lease contributes importantly to the Ab reduction observed in GPI-anchored proteins with PI-PLC caused a consistent, highly PI-PLC-treated CHO cells. significant reduction in Ab40 (p , 0.008) and Ab42 (p , 0.03). To address further the question of DAG-induced PKC acti- As shown in Table II, GPI1 cells treated with PI-PLC and vation, the PKC inhibitor BIS (G109203X) was added to GPI1 mutant gpi85 cells produced remarkably similar amounts of cultures prior to treatment with PI-PLC. This compound has Ab40 and Ab42 that were consistently ;40% less than the previously been shown to block the changes in APP metabolism amount produced by normal GPI1 cells. Thus, the removal of (4) that occur when PDBu is used to activate PKC (4). Our GPI-anchored proteins by PI-PLC and by mutation causes a control experiments showed that PDBu reduces Ab secretion in similar, highly significant reduction in Ab40 and Ab42. Con- GPI1 cells, and this reduction was completely blocked by 2.5 firming these findings, we have also observed that total se- mM BIS. In addition, as previously reported (4), PDBu treat- creted Ab (4-kDa band), immunoprecipitated from [ ment elevated total sAPP secretion by about 15-fold, and this S]methi- onine-labeled CHO cells expressing high levels of human was also blocked by the BIS treatment (data not shown). Treat- APP695, is reduced upon PI-PLC treatment (p , 0.05) to ap- ment of GPI1 cultures with BIS did not, however, block the proximately the same extent as shown in Table II without reduction in Ab40 and Ab42 caused by PI-PLC (Table III). significantly changing sAPPa (data not shown). Thus, our results clearly show that the effect of PI-PLC on Ab Activation of PKC by PI-PLC-mediated Hydrolysis of PI Does biogenesis is not due to DAG-induced PKC activation. Not Account for the Reduction in Secreted Ab Levels in GPI1 b-Secretase Processing of APP Is Reduced in PI-PLC-treated Cells—In many cell types, PKC activation results in a substan- CHO Cultures—The PI-PLC-induced reduction in Ab could be tial stimulation of sAPPa release coupled with a reduction in caused by a reduction in either b-or g-secretase activity. To Ab secretion (3). By hydrolyzing PI in membranes, PI-PLC evaluate whether b-secretase activity is reduced when GPI- treatment could, in principle, activate PKC by releasing dia- anchored proteins are removed, several experiments were per- cylglycerol (DAG). It is, however, highly unlikely that PKC formed in which we treated GPI1 and gpi85 cultures in trip- activation contributes significantly to the effect of PI-PLC on licate with PI-PLC for 24 h and then evaluated sAPPb and total guinea pig brain cultures, because PI-PLC decreased Ab but sAPP (sAPPa 1 sAPPb) by immunoblotting (Fig. 3). For spe- Role of GPI-anchored Proteins in Ab Biogenesis 26813 TABLE III Addition of the PKC inhibitor BIS blocks PDBu-mediated but not PI-PLC-mediated Ab reduction in GPI1 cultures The GPI1 and gpi85 cultures were treated for 3 h with PDBu, PI-PLC, and/or BIS. Media were collected and analyzed for Ab40 and Ab42. Ab40 Ab42 Control 147 6 29 12 6 5 BIS 136 6 17 11 6 4 PI-PLC 97 6 22 8 6 4 PI-PLC BIS 86 6 19 7 6 4 PDBu 125 6 18 NT PDBu BIS 168 6 42 NT NT, not tested. cific detection of sAPPb, we used antibody 192, which effi- ciently binds APP cleaved between residues 21 and 11ofthe Ab sequence but shows a low affinity for full-length APP, sAPPa, and other APP fragments (34). We could not specifically analyze the sAPPa secreted from the GPI1 and gpi85 cell lines, because antibodies to human Ab1–16 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 (sAPPa 1 sAPPb) by immunoblotting with monoclonal antibody 22C11, which recognizes both hu- man and rodent sAPP. Densitometric analysis showed that sAPPb was reduced by approximately 90% upon PI-PLC treatment of GPI1 cultures FIG.3. PI-PLC substantially reduces mammalian sAPPb. The (Fig. 3). In contrast, total sAPP (sAPPb 1 sAPPa) was only proteins in media from gpi85 cells (1 untreated; 2– 4 PI-PLC-treated) slightly reduced (;16%). Since sAPPa is the major secreted and GPI1 cells (5–7 untreated; 8 –10 PI-PLC-treated) were separated species in GPI1 cells, the small reduction in total sAPP indi- on 10 –20% Tris-Tricine gels. A, immunolabeled with antibody 192, cates that PI-PLC has little effect on sAPPa. Levels of sAPPb which specifically detects sAPPb (solid arrow). B, immunolabeled with were also highly (71%) reduced in untreated gpi85 cells as antibody 22C11, which detects total sAPP (sAPPa 1 sAPPb, open arrow). Densitometric analysis of lanes from two independent blots compared with GPI1 cells, but again total sAPP was only showed that sAPPb was reduced by 90% when GPI1 cultures were slightly affected. As expected, PI-PLC treatment did not con- treated with PI-PLC. sAPPb was 71% lower in gpi85 than in GPI1 sistently affect sAPPb or the total sAPP secreted by gpi85 cells. cultures, and PI-PLC treatment had no consistent effect on the amount These results provide strong evidence that GPI-anchored cell- of sAPPb secreted by gpi85 cells. Total sAPP was slightly reduced (;16%) when GPI1 cultures were treated with PI-PLC. Total sAPP was surface proteins play an important role in b-secretase activity. also slightly lower in gpi85 than in GPI1 cultures. PI-PLC treatment DISCUSSION 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 The proteins involved in Ab biogenesis are important thera- differences in total sAPP were small, we repeated this study, and the peutic targets in Alzheimer’s disease. To date, none of these data in C are summarized from the two experiments. proteins has been unequivocally identified. In this study, we investigated the role of GPI-anchored cell- surface proteins in surface clearly implicate one or more cell-surface GPI-anchored Ab biogenesis. Our results provide strong evidence that, in proteins in b-secretase activity and Ab biogenesis. This finding mammalian cells, one or more cell-surface GPI-anchored pro- fits well with recent reports that APP and Ab are found in teins play an important role in b-secretase activity and Ab detergent-insoluble membrane domains enriched in GPI-an- secretion. In addition, they indicate that cell-surface GPI-an- chored proteins (38, 39). These domains are rich in cholesterol, chored proteins do not play a major role in a-secretase activity. which has been reported to be important for Ab production These conclusions are based on the following observations. (i) (40, 41). PI-PLC, which removes GPI-anchored proteins from the cell Although reduced, secretion of sAPPb,Ab40, and Ab42 con- surface, substantially reduces the Ab40 and Ab42 secreted tinues both in PI-PLC-treated cells and in mutant gpi85 cells from fetal guinea pig brain cultures and the wild type GPI1 deficient in GPI-anchored proteins. This suggests that normal CHO cell cultures. (ii) The mutant gpi85 line, which lacks b-secretase activity may involve both GPI-dependent and -in- cell-surface GPI-anchored proteins, shows reductions in se- dependent proteins. One should, however, be cautious in draw- creted Ab40 and Ab42 similar to those observed when the ing this conclusion. A small percentage of the GPI-anchored parental GPI1 line is treated with PI-PLC. (iii) The PI-PLC proteins may be resistant to PI-PLC because of fatty acylation treatment that lowers the Ab40 and Ab42 secreted from GPI1 of the GPI anchor (28). PI-PLC treatment removes cell-surface cells has no detectable effect on the residual Ab secretion that GPI-anchored proteins but may not remove intracellular GPI- occurs in gpi85 cells. (iv) sAPPb is substantially reduced in anchored proteins that could continue to produce Ab and gpi85 cells and in PI-PLC-treated GPI1 cells but shows no sAPPb.In gpi85 cells, GPI-anchored proteins continue to be additional reduction in PI-PLC-treated gpi85 cells. (v) PI-PLC synthesized. These proteins are rapidly degraded when they treatment does not affect the levels of sAPPa in fetal guinea pig fail to be GPI-anchored, and they do not accumulate at the cell brain cultures and only slightly reduces total sAPP (sAPPa 1 surface, but some residual protein precursor capable of proc- sAPPb)inGPI1 and gpi85 cultures. essing intracellular APP to sAPPb and Ab may remain in the The coordinate reduction of Ab40 and Ab42, the profound secretory pathway. Thus our findings are consistent with sep- reduction of sAPPb, and the lack of change in sAPPa that arate GPI-anchored protein-dependent and -independent occurs when GPI-anchored proteins are removed from the cell b-secretase pathways, but they do not eliminate the possibility 26814 Role of GPI-anchored Proteins in Ab Biogenesis R. S., Paxton, R. J., March, C. J., and Cerretti, D. P. (1997) Nature 385, that normal b-secretase cleavage is carried out entirely by one 729 –733 or several GPI-anchored proteins. 8. Moss, M. 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