Proteolytic Processing of Human Amyloid /3 Protein Precursor in ...

Vol. 268, No. 3, Issue of January 25, pp. 2009-201’2,1993 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 8 1993 by The American Society for Biochemistry and Molecular Biology, Inc

Proteolytic Processing of Human Amyloid/3 Protein Precursor in Insect Cells MAJORCARBOXYL-TERMINALFRAGMENT

IS IDENTICALTOITSHUMANCOUNTERPART* (Received for publication, October 6, 1992)

Triprayar V. RamabhadranSS,Samuel E. GandyStl, Jorge Ghisoll, AndrewJ. Czernik$, David Ferris$, Ramaninder Bhasin**, Dmitry Goldgaber**,Blas Frangionell , and Paul Greengard$ From the $Laboratory of Molecular and Cellular Neuroscience, the Rockefeller University and the TDepartment of Neurology and Neuroscience, the New York Hospital-Cornel1 Medical Center, New York, New York 10021, the IIDepartment of Pathology, New York University Medical Center, New York, New York 10016, and the **Departmentof Psychiatry and Behavioral Science, State University of New York, Stony Brook, New York 11794

The predominant component of amyloid plaques of the extracellular and transmembrane domains of APP, with Alzheimer’s disease is the amyloid protein (AB), a 28 amino-terminal residues in the extracellularor intralumi39-42-amino-acid peptide derived by proteolysis of a nal space (8).In the course of standard metabolic processing family of precursors known asamyloid precursor pro- of APP, the extracellular domain is released from cells by a teins (APP).In mammalianbrain and in cultured mam- proteolytic cleavage within the Ap domain (9, 10). malian cells, the release of APP amino-terminal fragDefining the mechanisms of the normal processing and ments into the extracellular medium occurs by a pro- abnormal proteolysis of APP is crucial to understanding the teolyticcleavagewithin the AB domain, thereby pathogenesis of Alzheimer’s disease. APP is expressed and precluding amyloidogenesis. Infection of Sf9 insect released by most mammalian cells, but the steady-statelevels cells with baculovirus vectors containing APP cDNAs of the cell-associated carboxyl-terminal fragments generated results in high levels of APP expression. The concom- in this proteolytic event are comparatively low in several cell itant release of amino-terminal fragments of APP and the productionof carboxyl-terminal, cell-associated types (10-13). Several groups have recently shown that insect cleavage products are observed. Here we demonstrate cells, Spodopteru frugiperda, infected with recombinant bacby direct protein microsequencing that the proteolytic ulovirus vectors engineered to express human APP cDNAs, quantities of APP (14-17). The processing of APP in the Sf9 cells generates a promi- producerelativelylarge nent carboxyl-terminal species that is identical to that amino-terminal portionof APP is released into thecell culture produced in human cells, suggesting that the major medium, and electrophoretically and antigenically heterogeincluding potentially pathway for proteolytic processing of APP is con- neouscarboxyl-terminalfragments, amyloidogenic fragments, are foundassociated with cells (18). served among metazoans. High level production of the APP and its carboxyl-terminal fragments by this system might facilitate the study of APP proteolysis and the identification of the sites of alternative The deposition of amyloid plaques in the brain parenchymacleavages. Therefore, we have undertaken the detailed chemand around cerebralvessels is the histopathological hallmark ical characterization of the cell-associated APP fragments in of Alzheimer’s disease (1).The major component of the plaque order to compare the proteolytic products with those generis the amyloid P protein (AP), a 39-42-amino-acid peptide, ated in mammalian cells (9, 10, 12, 19-21). Here we report which results from the proteolytic degradation of a family of that the amino-terminal sequence of the major 15-kDa carlarger proteinsreferred to generically as theamyloid precursor boxyl-terminal fragment of human APP produced in insect protein(APP).’APPconsists of three major alternately cells is identicalto thatgenerated by the constitutivecleavage spliced isoforms of 695, 751, and 770 amino acids, referred to of APP in mammalian cells (9, 10, 19-21). as APPeg6,APP751, and APPT70,respectively (for review, see Refs. 2, 3). MATERIALSANDMETHODS APP is a membrane glycoprotein (4)with a single transCell Culture and Virus Infection-Cells of S. frugiperda (Sf9) were membrane domain and isN - and 0-glycosylated (4, 5), tyro- maintained asdescribed (17). Cells wereinfected with human APP75,sine sulfated (5), and phosphorylated (6, 7). The sequence containing baculovirus in 150-cm2 T flasks at a multiplicity of 1-2 corresponding to theAP domain is located at the junctionof plaque forming units/cell and incubated for 48-50 h in serum-free * This work was supported by United States Public Health Service Grants AG 09464 and AG 10491 (to P. G.), AG 11508 and AG 080581 (Pilot Project) (to S. G.), AG 05891 and AG 08721 (to B. F.), and Alzheimer Association Grant IIRG-90-193 (to D. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” inaccordance with 18U.S.C. Section 1734 solely to indicate this fact. 5 To whom correspondence shouldbe addressed. The abbreviations usedare: APP, amyloid precursorprotein; PAGE, polyacrylamide gel electrophoresis; APPL, amyloid precursor protein-like.

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medium ExCell 400 (JRH Bioscience, Lenexa, KS). Cells were dislodged, pelleted, and washed twice with phosphate-buffered saline containing protease inhibitors, asdescribed (10). Detection of A P P Holoprotein and Carboxyl-terminal FragmentsAntibody Ab369, raised against a peptide corresponding to amino acids 645-694 at the carboxyl terminus of human APP696, was used for detection of carboxyl-terminal fragments (11).Cell lysates and columnfractions were subjected toSDS-PAGE(22) followed by immunoblotting (23). APP-related bands were detected using secondary antibody coupled to horseradish peroxidase followed bycolor or by the enhanced development with 4-chloro-naphthol (Sigma), chemiluminescence method (Amersham Corp.). Purification of A P P Fragments-Initial steps of cell lysis and

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FIG. 1. Fractionation o f r e c o m b i n a n t h u m a n A P P in baculovirus-infected Sf9 cell lysates by DEAE-Sephacel chromatography. Lysate was applied and thecolumn was eluted batch-wise with buffersof increasing NaCl concentration as described in the text. 20pl aliquots of 50-drop fractions (numbersabove the lanes) were separated by 12% SDS-PAGE and analyzed on immunoblotsusing Ab369. T and S represent, respectively, 5-pl aliquots of the total lysate and the100,000 X g supernatant.

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through was circulated for 8-10 h through a 1.5-ml column equilibrated with 10 mM Tris, pH 7.5, 0.15 M NaCI, 5 mM EDTA. The column was washed with 20-30 ml of equilibration buffer containing 97 0.5 M NaCI, followed by washes with 10 ml each of TBST (50 mM Tris, pH 7.5, 0.15 M NaC1, 0.1% Tween-20, 0.2% NaNd, BBST (25 mM sodium borate, pH 8.3, 1 M NaCI, 0.1% Tween-PO), and 20 mM 46 "g 46-a sodium phosphate, pH 7, 0.5 M NaCI. Fragments were eluted from the column using 5-6 ml of 5 M sodium thiocyanate followed by 30 30 - 4 desalting of the eluate through a column of Sephadex G-25 equili21.5 21.5-4 brated with 10 mM Tris, pH 7.5, 0.1% SDS. The fractions containing 14.3 I +15 kD 14.3 - 4 6.5 C the APP carboxyl-terminal peptides were detected by dot immunoas6.5 - 4 say, pooled, dried, and resuspended in1/50 volume of distilled water, 4 5% 2-mercaptoethanol, 0.02% bromphenol blue, 15% glycerol, for SDS-PAGE analysis. FIG. 2. Immunoaffinitypurification of t h e 15-kDa carSemipreparative Immunoprecipitation-APP carboxyl-terminal boxyl-terminal APP fragment. a, immunoblot of samples sepafragments in theformic acidextract preparation were immunopreciprated on a 6-1596 gradient SDS-PAGEgel, using Ab369; b, Coomassie itated using 1 mg of affinity purified antibody Ab369 as described Brilliant Blue-stained gel. Lanes 1-6 in a and b represent the same (11). Immune complexes were collectedusing 0.4 g of protein Asamples with 2.5-10-fold higher amounts used in b to enhance detec- coupled CL-4B beads (Pharmacia, Uppsala, Sweden). tion. 1, 100,000 X g supernatant; 2, DEAE-Sephacel flow-through Protein Sequence Determination-Proteinsseparated on 12% prior to storage; lanes 3-6, Ab369 immunoaffinity column fractions; SDS-PAGE gels were electroblotted onto ProBlott membranes(Ap3 , loading material; 4, flow-through; 5, wash; 6, 5 M NaSCN eluate. plied Biosystems, Foster City, CA) and stained with Coomassie BrilIg denotes the immunoglobulin heavy chain released by degradation liant Blue as recommended by the manufacturer. The desired fragof the column. Numbers on the left in a and b represent molecular ments, located by immunostaining of flanking lanes, were excised and masses in kDa. Lane M W (b) representsmolecular weight markers. sequenced. Automatic Edman degradation analyses were carried out on a 477A protein sequencer (Applied Biosystems), asdescribed (25). fractionation were performed as described (15). Infected cells were resuspended a t a density of lo7cells/ml in buffer A (10 mM Tris, pH RESULTS 7.5, 5 mM EDTA, 1% Nonidet P-40) containing 0.5 M NaCl and the Infection of Sf9 cells with recombinant human APPbacuproteaseinhibitorspepstatin (0.5 pg/ml),leupeptin,chymostatin, antipain (5 pg/ml each), benzamidine (5 mM), phenylmethylsulfonyl lovirus results in high levels of APP production (14-17). As fluoride (1 mM), aprotinin (100 KIU/ml). The cell lysate prepared by observed in mammalian cells (5, 7), the APP ectodomain is Dounce homogenization was centrifuged a t 100,000 X g for 30 min. released into themedium and a 14-15-kDa carboxyl-terminal T h e supernatant was diluted with 10 mM Tris, pH 7.5, 5 mM EDTA fragment (referred to as "15-kDa fragment") remains in the to lower the final NaCl concentration to 0.15 M and applied toa 10cell. We have purified and sequenced the amino terminusof ml column of DEAE-Sephacel (Pharmacia AB, Uppsala, Sweden). the 15-kDafragment fromAPP751-expressingbaculovirusT h e flow-through from this column containing the APP carboxylinfected insect cells to determine thefidelity of APP processterminal fragments was used for further purification. A modification (19) of the formic acid extraction procedure (10) ing. was also used in some experiments. Cell pellet derived from 10' cells Isolation of 15-kDa Fragment-For efficient immunoaffinwas resuspended in 10 volumes of0.2 M NaCI, 10 mM sodium ity purification, the carboxyl-terminal fragmentswere sepaphosphate, pH 7.2, homogenized using a Teflon homogenizer, and rated from the large quantities of APP holoprotein present in centrifuged a t 50,000 X g. The resulting pellet was washed with the same buffer, collected by recentrifugation, and extractedwith 1 ml of cell lysate by DEAE-Sephacel chromatography. The elution 99% formic acid. The formic acid extract was diluted to 15 ml with pattern of the APP holoprotein and carboxyl-terminal fragdistilled water, neutralized using saturated (-5 M) Trizma base so- ments of APP is shown in Fig. 1.The lane S shows the 100,000 lution, and dialyzed against 4 liters (2 x 2 liter) of 10 mM Tris, pH X g supernatant (S-100) applied to thecolumn. This sample 7.5, 0.15 M NaC1, 5 mM EDTA, 0.3% Nonidet P-40. The precipitate contains large quantities of APP holoprotein and the major, resulting from dialysis was collected by centrifugation a t 18,000 X g 15-kDa carboxyl-terminal fragment well as as larger carboxylfor 20 min, and dissolved by sonication and boiling in 2 ml of 10 mM terminalfragments derived by alternate cleavage of APP Tris, pH 8,50mM NaC1, 1% SDS. ImmunoaffinityPurification-Antibody 369 immunoaffinity col- holoprotein (18).Carboxyl-terminal fragmentsof APP eluted umns were prepared by coupling affinity purified antibody Ab369 in theflow-through fractions 6-33, in 0.15 M NaCl, alongwith (24) to Affi-Prep Hydrazide matrix according to the manufacturer's trace amounts of APP holoprotein. The bulk of the APP recommendations(Bio-Rad). The DEAE-Sephacelcolumn flow- holoprotein, elutedin fractions 50-80 at NaCl concentrations a

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TABLEI A m i n o acid seauence of the 15-kDa fragment

gation of the cell lysate resulted in the preferentialrecovery of carboxyl-terminal APP fragments (45 kDa and below) in the pellet fraction. The precipitate resulting upon neutraliIsolated by DEAE-Sephacel Isolated by and Ab369 affinity zation anddialysis contained the carboxyl-terminal fragments immunoprecipitation chromatography which were subsequently immunoprecipitated. PTH Yield Cycle PTH Yield Sequence Determination-A sample containing30 times as Cycle much material as thatused in lane 6 of Fig. 2a was separated pmol pmol by SDS-PAGEandtransferredto a ProBlott membrane. L 86.8 1 L 13.0 1 76.0 V Sequencing was performed asdescribed under “Materials and 2 2V 11.4 F 67.4 3 3F 16.9 Methods.” Alternately, the immunoprecipitate of the formic 62.4 F 4F 12.6 4 acid extracts was also separated, and the 15-kDa band was 64.3 A 5A 16.0 5 sequenced as described above. 26.4 E 6E 6.5 6 Theamino-terminal sequences obtained for the15-kDa 16.8 D 7 7D 4.2 fragment derived from independent experiments using two V 38.6 8 8V 5.7 49.7 9 G isolation methods were identical (Fig. 3). The agreement on 9 X 12.9 3.1 10 S S 10 the identityof the amino-terminalresidue (Leu-) between the 12.4 5.6 11 N 11 N two methods suggests that the amino terminus was not genK 15.9 12 12 X erated as a cleavage artifact during the purification proceG 51.3 10.6 13 G13 dures. Using the APP645-694 peptide as standard, the samples A 31.7 6.2 14 A 14 used for sequencing were estimated to contain 40 (immunoaf26.6 6.5 15 I I 15 31.7 I I 8.3 16 finity column) and 600 pmol (immunoprecipitation) of the 16 7.3 17 X 17 G 15-kDa fragment. Considering the probable recovery losses, 5.8 L 29.2 L18 18 the initial yield of phenylthiohydantoin amino acid deriva13.3 1.2 19 M M 19 tives obtained for each sequencing run (TableI) is consistent 25.5 V 20 V20 2.6 withtheamount of fragment loaded, indicatingthatthe sequences obtained represented themajor peptide in theorigbetween 0.25-0.35 M as described (15).Theflow-through inal sample. Also shown in Fig. 3 is the sequence of the fractionscontainingthecarboxyl-terminal APP fragments carboxyl-terminal fragment generated by processing of human were pooled and incubatedwith 5 ml of DEAE-Sephacel; this APP in transfected human293 cells (10). batch adsorption step removed virtually all remaining APP holoprotein (compare lanes 1 and 2 in Fig. 2, a and b ) . The DISCUSSION material was stored a t -20 “C prior to further purification. In all mammalian species examined to date, secreted APP The 15-kDa fragmentwas further purified by affinity chromatographyon acolumn of immobilized, affinity purified appears to be cleaved at the junction between Lys611 and processing of Ab369 (Fig. 2a). The immunoreactive material shown inlane Led1’ (APP695 numbering sequence). Thus, the 6 corresponds to the amount recovered from 100 times the APP in COS cells of simian origin (19), in PC12 cells of rat hamster (21) volume of the samples analyzed in lanes 1-3. The eluate of origin (20),’ and in ovarycellsfromChinese results in cleavage at the same site as in the human 293 cells. t h e Ab369 immunoaffinity column (lane 6 ) containedan additional prominent immunoreactive band of about 45 kDa The identityof the Sf9-derived sequence withthe mammalian not present in the original lysate. This band represents the sequence indicates that Sf9 cells of insect origin, which do heavy chain of the IgG released by degradation of the Ab369 notexpress endogenous APP,nonetheless process human APP ina manneridenticaltothat used inthe secretory immunoaffinity column matrix. The total protein composition of the fractions in Fig. 2a pathway by mammalian cells. The position of the cleavage observation by was determined by staining with Coomassie Brilliant Blue site identified here is consistentwiththe lane 6 corre- Lowery et al. (16) who determinedthecarboxyl-terminal (Fig. 2b). Total protein in the NaSCN eluate in sponds to the amountrecovered from a volume of 100,000 x residues of the human APP ectodomainreleased into the medium by recombinant baculovirus-infected insect cells. Our g supernatant equivalent to approximately 400 times that shown in lane 1. Together, Fig. 2, a and b, shows that use of results together with those of Lowery et al. (16) suggest that the affinity column results in a high degree of enrichment of the majority of APP released from insect cells occurs through a specific singlecleavage event. Processing of human APP by carboxyl-terminal APP fragments. The 15-kDa fragmentwas also isolated by semipreparative immunoprecipitation from a neutralized formic acidextracted J. Buxbaum, S. Gandy, and P. Greengard, unpublished observasample described under “Materials and Methods.” Centrifu- tions.

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Major Human

APP C-terminal Fragment

in Insect Cells

6. Gandy, S. E., Czernik, A. J., and Greengard, P. (1988) Proc. Natl. Acad. insect cells in a manner identical to that used by mammalian Sci. U. S. A . 85,6218-6221 cells suggests that the processing apparatus for APP may be 7. Oltersdorf, T., Ward, P. J.,Henriksson, T., Beattie, E. C., Neve, R., Lieherburg, I., and Fritz, L. (1990) J. Biol. Chem. 265,4492-4497 widely distributed inmetazoans. Thisis of interestsince 8. Kang, J., Lemaire, H.-G., Unterbeck, A., Salbaum, J. M., Masters, C. L., Grzeschik, K. H., Multhaup, G., Beyreuther, K., and Miiller-Hill, B. insect cells are not known to express proteins withsequence (1987) Nature325,733-736 homology to the APP cleavage site. A protein related to APP, 9. Sisodia, S. S., Koo, E. H., Beyreuther, K., Unterheck, A,, and Price, D. L. (1990) Science 248,492-495 termed APPL (APP-like), has beenidentified in Drosophila. Esch, F. S., Keim, P. S., Beattie, E. C., Blacher, R. W:, Culwell, A. R., 10. APPL contains three regions of significant homology with Ol!e,rsdorf, T., McClure, D., and Ward, P. J. (1990) Sczence 2 4 8 , 1122APP, ranging between 37 and 47% (26). However, no homolI124 J. D., Gandy, S. E., Cicchetti, P., Ehrlich, M., Czernik, A. J., ogy exists between APP and APPL in theregion containing 11. Buxbaum, Fracasso, R. P., Ramabhadran, T. V., Unterbeck, A,, and Greengard, P. the A@ peptide. An amino-terminal fragment of APPL is (1990) Proc. Natl. Acad. Sei. U. S. A. 87,6003-6006 S., Golde, T. E., Kunishita, T., Blades, D., Lowery, D., Eisen, M., released by embryonic, primary, and transfectedcells derived 12. Estus, Usiak, M., Qu, X., Tabira, T., Greenberg, B. D., and Younkin, S. G. (1992) Science 255,726-728 from Drosophila, by cleavage within APPL (27). It is possible G. L., Gandy, S. E., Buxbaum, J. D., Ramabhadran, T. V., and that Sf9 cells, like Drosophila cells, express an APPL homolog 13. Caporaso, Greengard, P. (1992) Proc. Natl. Acad. Sci. U. S. A: 89,3055-3059 which is released from the cell by proteolysis. Then, the same 14. Ramakrishna, N., Saikumar, P., Potempska, A,, Wlsnlewski, H. M., and Miller, D. L. (1991) Biochern. Biophys. Res. Comrnun. 174,983-989 enzyme(s)that process insectAPPL mayalsocleave the 15. Knops, J., Johnson-Wood,K., Schenk, D. B., Sinha, S., Lieberburg, I., and McConlo e, L.(1991) J. Biol. Chem. 266,7285-7290 expressed human APPs. This implies that the APPcleavage 16. Lowery, D. f?, Pasternack, J. M., Gonzales-DeWhitt, P. A,, Zurcher-Neely, enzyme(s) have abroad substratespecificity, as alsosuggested H., Tomich, C-S. C., Altman, R. A., Fairbanks, M. B., Heinrikson, R. L., Younkin, S. G., and Greenberg, B. D. (1991) J.Btol. Chem. 266,19842by experiments using mammalian cells(19, 28). Our data, 19850 together with those of Lowery et al. (16), suggest that pro- 17. Bhasin, R., Van Nostrand, W. E., Saitoh, T., Donets,M. A., Barnes, E. A., Quitschke, W. W., and Goldgaher, D. (1991) Proc. Natl. Acad. Scz. U. S. A. teases with APP secretase-like properties(10) may be widely 88, 10307-10311 distributed inmetazoans. Occurrence of correct intra-amyloid 18. Gandy, S. E., Bhasin, R., Ramabhadran, T. V., Koo, E. H., Price, D. L., Goldgaber, D., and Greengard, P. (1992) J. Neurockrn. 58,383-386 cleavage in insect cells also strengthens the possibility that 19. Maruyama, K., Kametani, F., Usaml, M., Yamao-Harlgaya, W., and Tanthe alternative cleavage of human APP by Sf9 cells (18)may aka, K. (1991) Biochern. Bzophys. Res. Commun. 179,1670-1676 berelevanttothepotentially amyloidogenic pathway for 20. Anderson, J. P., Esch, F. S., Keim, P. S., Sambamurti, K., Leiherburg, I., and Robakis, N. K. (1991) Neurosci. Lett. 12.8, 126-128 metabolism of human APP in humancells (12). 21. Wang, R., Meschia, J. F., Cotter, R. J., and Slsodla, S. S. (1991) J. Biol.

REFERENCES 1. Tomlinson, B. E., and Corsellis, J. A. N. (1984) in Greenfield’sNeuropathology (Adams, H., Corsellis, J. A. N., and Duchen, L. W., eds) pp. 9511021, John Wiley and Sons, New York 2. Selkoe, D. J. (1991) Neuron 6,487-498 3. Hardv. J.. and Allsou. D. (1991) Trends Pharmucol. Sci. 12.383-388 4. Dyrks,’T.; Weideminn, A:, Muithaup, G., Salbaum, J. M., Lemaire, H.-G., Kang, J., Muller-Hill, B., Masters, C.L., andBeyreuther, K. (1988) EMBO J. 7,949-957 5. Weidemann, A,, Konig, G., Bunke, D., Fischer, P., Salbaum, J. M., Masters, C. L., and Beyreuther, K. (1989) Cell 57,115-126

Chem. 266,16960-16964 22. Laemmli, U. (1970) Nature 227,680-685 23. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76,4350-4354 24. Czernik, A. J., Girault, J-A., Nairn, A. C., Chen, J., Snyder, G., Kebabian, J., and Greengard, P. (1991) Methods Enzymol. 201,264-283 25. Ghiso, J., Tagliavini, F., Timmers, W. F., and FranGone,B. (1989)Biochem. Bwphys. Res. Commun. 163,430-437 26. Rosen, D. R., Martin-Morris, L.,Luo, L., and White, K. (1989) Proc. Natl. Acad. Sci. U. S. A . 86,2478-2482 27. Luo, L., Martin-Morris, L. E., and White, K. (1990) J.Neurosci. 10,38493861 28. Sisodia, S. S. (1992) Proc. Natl. Acad. Sei. U. S. A. 8 9 , 6075-6079