- Email: [email protected]
The β-amyloid precursor protein is not processed by the regulated secretory pathway
Vol. 181, No. 2, 1991 December 16, 1991
BIOCHEMICAL
THE O-AMYLOID
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 513-519
PRECURSOR PROTEIN IS NOT PROCESSED BY THE REGULATED
SECRETORY
PATHWAY
Caroline C. Overly, Lawrence C. Fritz, Ivan Lieberburg,
and Lisa McConlogue+
Athena Neurosciences, Inc., 8OOFGateway Blvd., South San Francisco, CA 94080 Received
October
16,
1991
The i3-amyioid peptide is derived from a larger membrane bound protein and accumulates as amyioid in Aizheimer’s diseased brains. O-amyioid precursor protein (BAPP) proteoiyticaiiy processed during constitutive secretion cannot be a source of deposited amyioid because this processing results in cleavage within the amyioidogenic peptide (1). To see if other secretory pathways could be responsible for generating potentially amyioidogenic molecules we tested the possibility that BAPP is Stable AtTPO ceil lines targeted to the regulated secretory pathway. expressing exogenous human DAPP were genetically engineered. These ceils were labeled with [35S]-methionine, and chased in the presence or absence of secretagogue. The RAPP both inside the ceils and released from the ceils was analyzed by immunoprecipitation and gel analysis. Quantitation of autoradiograms showed that virtually ail of the synthesized i3APP was secreted by the constitutive pathway, and that no detectable (4 0 1991Academic %) OAPP was targeted to the regulated secretory pathway. Press, 1°C. In Alzheimer’s disease, a 39 to 43 amino acid peptide is processed from the kmyloid precursor protein (OAPP) and accumulates in the brain as B-amyloid (BAP), (2-5) . The l3APP is synthesized as a membrane bound protein which is subsequently cleaved, leading to the secretion of an N-terminal fragment (secreted BAPP) (6-l 1). In most cell types so far examined, BAPP secretion has occurred via a “constitutive” secretory pathway; in this pathway proteins are released from cells continuously without storage and in a manner that is independent of stimulator-y secretogogues. It has been shown, however, that constitutive secretion of BAPP precludes the deposition of BAP since + To whom correspondence
should be addressed. . . III thrs IIUUIUSCIY~ are;BAFT (B-EIKQ~o~~~.XIXU protein), BAP (Ramyloid peptide), ACTH (adrenocdcotrophic hornme.), KPI (Ku& pmteaseinhibitor), 6956AFT (695 amino acid form of BAPP). 7516APP (75 1 amino acid form of BAPP), DME (Dulbeccols Modifii Eagle’s medium) ,FCS(fetal calf mm), HEPES (4-(2-HydroxyethyQ-l-piperazineethanesulfonic acid), RSV(Rous sarcoma virus long termhal repeat), SDS (Sodium dodfzyl sulfate). *
0006-291X/91
513
$1.50
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 181, No. 2, 1991
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AND BIOPHYSICAL
RESEARCH COMMUNlCATlONS 4
the full-length BAPP is cleaved within the amyloidogenic peptide to release the secreted fragment in this pathway (1). In addition to constitutive secretion, certain specialized cells such as neurons and endocrine cells, have a regulated secretory pathway. The regulated pathway can process and store various secretory proteins which are then specifically released upon stimulation. Recent evidence suggests that BAPP may be processed not only by the constitutive pathway but also by a regulated pathway in certain cells. For instance it has been shown that BAPP is stored in platelet cc-granules, from which it can be released by thrombii stimulation (10-12). Furthermore, full length BAPP in peripheral neurons has been shown to undergo anterograde fast axonal transport (13). Fast axonal transport is a form of microtubule dependent vesicular movement by which organelles reach nerve terminals; microtubule dependant mechanisms are also known to transport regulated secretory vesicles to process tips of certain cells (38,14-17). Three alternatively spliced forms of BAPP that contain the BAP sequence have been characterized: a 695 amino acid form (695BAPP) (22), and two longer forms of 751 (751BAPP), (9,23) and 770 (24) amino acids. The longer forms both contain an exon encoding a Kunitz type serine protease inhibitor (KPI) domain. Each of the three forms can be processed constitutively yielding a secreted N-terminal fragment and a membrane-bound C-terminal fragment, The secreted fragment of 751BAPP has been shown to be identical to protease nexin II (25-26), and to serve as an inhibitor of coagulation factor XIa (27). Direct sequencing of the cleaved fragments derived from 695BAPP and 751BAPP show that the constitutive cleavage occurs withii the amyloidogenic BAP fragment. Therefore, another pathway of BAPP processing must be responsible for the deposition of BAP in Alzheimers disease. The present experiments address whether the regulated secretory pathway could represent this alternative. The AtT20 pituitary cell line has been extensively characterized with respect to the cell biology of regulated secretion. When expressed in these cells, a wide-variety of regulated secretory proteins including B-endorphin, adrenocorticotrophin hormone (ACI’H), insulin, growth hormone, renin and trypsinogen, can be sorted into the regulated pathway, even though they may not be normally expressed in AU’20 cells (14,19-21). However, proteins that are normally secreted constitutively are sorted exclusively into the constitutive pathway in AtT-20 cells (14,33). We have used the AtT20 cell system to assesswhether BAPP has the necessary biochemical properties to be sorted into the regulated pathway. To this end, we transfected the human 695BAPP and 751BAPP cDNAs into AtT20 cells, and measured the amounts of BAPP processed by each pathway. We found that no detectable BAPP is sorted to the classical regulated secretory pathway, and that essentially all of the material is processed by the constitutive pathway.
Materials
and Methods
The expression plasmid, pR2 was constructed by combining the 3.5 k.b.HindIII to HpaI fragment of pRSVcat (29) with a polylinker containing HindIII, SalI, XbaI, SglII and SmaI oriented so that the HindIII site is closest to the promoter. The NruI to SpeI fragments containing the coding sequences for the 695BAPP (22) and 751BAPP (9,23), filled in at the Nru I site were cloned into the filled in HindIII and sticky XbaI sites of pR2. Clones with the correct orientation were chosen and named pR6Al(695BAPP) and pR7A1(751BAPP). 514
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RESEARCH COMMUNICATIONS
AtTU), mouse pituitary cells, were co-transfected with p&41 or pR7Al and pSV2neo (30) by the method of Moore (19). Transfected colonies were isolated in 250 @ml G418 (Geneticin, Gibco). Clones were selected for expression of human 8APP by western blot analysis of cell lysates and maintained in culture medium containing 125 pg/ml G418. 1.3 x 106 cells were plated on 6-well tissue culture dishes and labelled as described (31) with the following modifications. Cells were methionine starved for 30 minutes, and then labelled with 200 pCiiml[35S]-methionine in one of two ways. Cells were labeled for 1.5 hours in methionine free DME containing 10 % FCS, washed, and chased in complete medium for three 2-hour periods to allow the release of all constitutively secreted material. Alternatively, cells were labelled for 15 hours in DME containing l/5 normal methionine plus 10 % dialyzed FCS, and further labelled for an additional hour in the presence of fresh label. They were then washed and chased for two 3-hour plus one 2-hour chase periods in complete medium to allow the release of all constitutively secreted material. The cells were then chased for an additional two or three hours in the presence or absence of secretagogue, 5 mM 8-Br-cAMP alone or with 21 mM KCl. Cell lysates were prepared by washing attached cells two times with PBS and solubilizing the cells in lysis buffer (10 mM Tris pH7.5, 150 mM NaCl, 5 mM EDTA, 2 p.g/ml leupeptin and 1.0 % NP40). Cell lysates and media were spun at 4’ C at 100,000 x g for 10 minutes. They were then immunoprecipitated with either anti-BXS, a polyclonal antibody raised to gAPP (32), or with antiACIH antibody (33). The samples were preadsorbed with 15 mg protein A sepharose beads (Pharmacia) for several hours at 4OC with constant agitation. Beads were removed by centrifugation and the supematants were reacted with 1 pg of affinity purified anti-BX5 or with 2 pl anti-ACI’H serum at 4’ C overnight. Protein A sepharose beads pm-blocked with unlabelled cell lysates were added, and samples were rocked for 20 minutes at room temperature. The beads were recovered by centrifugation and washed three times in the appropriate buffer: wash buffer 1 (10 mM Tris pH. 7.5, 0.5 M NaCl, 0.1% NP-40,5 mM EDTA, 10 pg/ml leupeptin) for anti-BXS, and wash buffer 2 (150 mM NaCl, 50 mM HEPES, 1% BSA, 5 mM EDTA, 0.5% deoxycholate, 1% NP-40,0.1% SDS, 0.5 M KCl, 10 pg/ml leupeptin) for anti-ACTH. The beads were again recovered and resuspended in 40 p.l of SDS polyacrylamide gel sample buffer followed by electrophoresis on a 5-15% SDS polyacrylamide gradient gel (34). Gels were fixed in 7% acetic acid, treated with Amplify (Amersham) and exposed to film at -700 C for autoradiography. Autoradiograms were scanned on a Shimadzu dual wavelength thin layer chromatoscanner, model CS-930.
Results These experiments were designed to determine if 8APP can be sorted to the regulated secretory pathway in AtT20 cells. We transfected the AtI’20 cells with the two most abundant forms of the OAPP in human brain (35), 6958APP and 751BAPP. Plasmids expressing 6958APP and 7518APP using the Rouse sarcoma virus long terminal repeat (RSV) promoter, were constructed as described in Experimental Procedures and are shown in figure 1. Multiple stable lines of AtT20 cells transfected with either plasmid were selected, and assayed for the expression of the human 8APP by Western analysis with the anti-8APP antibody BX5. This antibody is directed to human l3APP and it specifically detects the exogenous human 8APP in the presence of endogenous rodent 8APP. To measure regulated and constitutive secretion of UPP, transfected cells were metabolically labelled with [35Sl-methionine, chased for several hours to remove all the proteins transported by the constitutive pathway, and then chased further either in the presence or in the absence of a sectetagogue to analyze material released on stimulation. Cell lysates and media samples taken at several time points throughout the experiment were immunoprecipitated with anti-BXS, and the precipitated proteins were electrophoresed and analyzed by autoradiography. The results (Figure 2A) show that most or all of the 8APP is constitutively secreted and that no detectable fraction is secreted in a regulated manner. The vast majority of the BAPP is released constitutively during the first 6 hours of chase, (figure 2A, lanes 3-5), and a minor amount of material is released during the 6 515
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AND BIOPHYSICAL RESEARCH COMMUNICATIONS
+
-
Figure1. Two plasmidsexpressing the6958APP(pR6Al) andthe751DAPP(pR7Al). TheRSV LTR &ray arrow)servesasa promoter,andSV40sequences serveasa poly adenylationsignal (gray box). 6APPsequences areshownasa whitebox. The locationof theinsertedKPI domainin plasmidpR7Al isindicatedonthemap.The blacklinesindicateflankingSW0 sequences and pBR322sequences. Figure2. Secretionof 695R4PPfromtransfected AtT20 mousepituitary cells. (A) Cellswere labe-lled with [35S]-methionine for 1.5hours,chased for three2-hourperiodsandstimulatedwith 8Br-cAMP. Celllysatesandmediawere immunoprecipitated with anti-BXS. Samples wererunon a 5-15% SDSpolyacrylamide gradientgelandvisualizedby autoradiography. Lane1) l/3 of cell lysateat theendof thelabeliingperiod(t=O). Lane2) l/3 of celllysateafterthe9 hourchase.Lanes 3.4 and5) total mediumfromO-2hours,2-4 hours,and4-6 hoursrespectively. Lanes6 and7) total mediumbetween6 and9 hoursof chasein thepresence (lane6) andabsence (lane7) of 8-BrCAMP. (B) Cellswerelabelledfor 16hours,chased for 6 hoursandstimulated.Mediasamples collectedin thepresence (Lane1) andin the absence (Lane2) of 8-Br-cAMPwere immunoprecipitated with anti-ACTH andrunon a 5-1596SDSpolyacrylamidegradientgelfor autoradiography.
to 9 hour chase. The amountsof 5APP secretedduring the 6 to 9 hour chaseis not increasedby secretagogue,demonstratingthat no OAPPis secretedby the regulatedsecretorypathway (figure 2A, lanes6 and 7). Furthermore,one would expect that a protein processedby the regulatedpathway would be storedin densecore vesiclesand accumulateinsidethe cell (19,37). In accord with the absenceof regulatedBAPPrelease,very little materialremainsin the cell after 6 (data not shown)and 9 hoursof chase(figure 2A lane 2). The samebehavior wasobservedwith clonesexpressingeither 695BAPPor 7515APP. and under both of the labelingconditions describedin Experimental Procedures.In contrast,incubationof the cells with a secretagoguedramatically enhancesthe release of ACTH (figure 2B), a peptideknown to be releasedin a regulatedmanner. Cell supematantswere immunoprecipitatedwith anti-ACM antibody, and the 4500 Da and 13,000Da maturefoms of ACTH (33,36) went specifically precipitated. Together, theseobservationsdemonstratethat 5APP doesnot exit the cell through the regulatedsecretorypathway of AU20 cells. The amountof 5APP secretedconstitutively from the cells was quautitated by itntntmoprecipitationand gel electmphoresisof labelledsecretedl3APPfollowed by densitotnettic scanningof resulting autoradiogratns.The amountsof material secretedwere converted to 516
Vol. 181, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
808 g
60-
i
40-
2 20-
I HOURS CnAsE
0 0
4
2
6
CWSETlMEtHOURS)
Figunz3. Kineticsof thesecretionof human695DAPPby AtI20 cells. C!&ularandsecreted proteinwasquantitatedby scanning theautomdiogmms from theexperimentdescribed in figure2A. Thepercentage of BAPPsecreted into themediumwascalculatedby dividingtheamountreleased by theamountinsidethecellsat t4 andisplottedasa functionof thelengthof thechaseperiod. Insert. Thepercentage of 6APPremaininginsidethec&s wascalculatedby subtractingthetotal% released at eachtimepointfrom 100.‘Ihe log of thepercentage of BAPPremainingin thecellsis plottedagainsttimeof chase.
percentages of the total labelled BAPP measured inside the cells at t=O and plotted against time of chase (Figure 3A). 71 % of the material quantitated inside the cell at t=O is secreted in the first 2 hours of chase, 24 % in the next 2 hours, and 5.2 % between 4 and 6 hours. Therefore, essentially all of the labelled BAPP present inside the cell is released within a 6hour chase period in the absence of secmtagogue. To examinethe kinetics of this release,the amountof BAPPremainingin the cell was plotted against length of chase. The resulting graph (insert, figure 3) shows that DAPP displays a rapid (tin= 1.1 hours) exponential release of BAPP from the cells, a pattern characteristic of constitutively secreted proteins (2 1,37). There was a small amount of detectable BAPP that was released during the 6 to 9 hour chase period. This residual COnStiNtiVe secretion represents the background against which we were attempting to detect regulated secretion of BAPP. In order to qua&ate this BAPP relative to the initial amounts of BAPP synthesized, autoradiograms exposed for various times were quantitated, corrected for length of exposure and compared to each other. The percent of initial BAPP released during the 6 to 9 hours chase was calculated as 1.1% for the stimulated conditions and 0.84 % for the unstimulated conditions. Therefore, in this experiment at most 0.26 % of the DAPP appeared to be processed by the regulated pathway. We believe that this amount is within the error of these techniques and is therefore insignificant. With this level of background, we could have detected as little as 1% of the initially synthesized BAPP had it been processed by the regulated secretory pathway.
Discussion Constitutive secretionof&VP
has-beenshownto involve a proteolytic cleavagewithin the B-
amyloid sequence (1). However, in Alzheimer’s diseased brains some of the BAPP must avoid this 517
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BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cleavage and be processed in an alternative manner that leads to SAP formation (1,5). Although constitutive secretion of OAPP has been observed in all cells where it has been examined, OAPP has also been shown to be sorted into the vesicular compartment that undergoes fast axonal transport in peripheral neurons (13), and to be released from storage granules in stimulated platelets (lo- 12). These data suggested that alternative secretion pathways could exist for DAPP and the present experiments were designed to address this in a well characterized cellular system. We tested directly whether the classical regulated secretory pathway characterized in AtT20 cells can process DAPP. The AtT20 mouse pituitary cell line is known to correctly target regulated secretory proteins from diverse tissues into its regulated secretory pathway (19-21). The present experiments demonstrate that none (at most 1%) of the DAPP is secreted in a regulated manner in AtT20 cells. Furthermore, we have found that in AtT20 cells, 100% of the OAPP is secreted by the constitutive pathway, and no detectable material accumulates in these cells. Weidemann and colleagues (6) have shown that in PC12 cells at least 30% of the i3APP synthesized is released via the constitutive pathway and is detectable in the supematant. They were not able to detect the remaining material, however, either inside or outside of the cells, and therefore OAPP is not stored in PC12 cells. Thus, although PC12 cells do possess a regulated secretory pathway (39) it does not appear to process BAPP. Recent experiments suggest that intracellular degradation of SAPP without secretion may occur in astrocytes and microglia (40). BAPP has been shown to undergo fast axonal transport (13), however this does not prove that the transport is via regulated secretory vesicles. It has previously been shown, for example, that constitutively secreted kappa light chain protein is detected in the tips of processes in AtT20 and PC12 cells (17), as is the case for regulated proteins. It is possible, therefore, that axonally transported SAPP is destined strictly for constitutive processing in accord with our observations in AtT20 cells. The storage and stimulated release of BAPP from u-granules (lo-12), however, may reflect a fundamental difference between the regulated secretory pathway in platelets and AtT20 cells. Previous studies have shown that constitutive processing of I3APP cannot give rise to the amyloid deposition observed in Alzheimer’s disease. If l3APP is not processed by the regulated secretory pathway in relevant brain cells, other pathways of BAPP metabolism must exist to explain the Alzheimer’s amyloid pathology.
Acknowledmen&We would like to thank Hsiao-Ping Moore for her expert advice and helpful discussions. We are especially grateful to David Quin, Thane Kreiner and Mark Grimes for sharing their experimental expertise and ideas. Mark Grimes and Regis Kelly generously provided the antiACTH antibody. The anti-BXS antibody was generously provided by Pamela Ward. We also thank Dale Schenk, David Agard, and Mark Grimes for their critical reading of the manuscript. References
1. ;:
Esch, F. McClure, Glenner, Glenner, 1135
S. , Keim, P. S., Beattie, E.C., Blather R. W., Culwell, A. R., Oltersdorf, T., D., and Ward, P. J., (1990) Science 248, 1122-l 124 G.G., and Wong, CW., (1984a) Biochem. Biophys. Res. Commun. 120, 885890 G.G., and Wong, C.W. (1984b) Biochem. Biophys. Res. Commun. 122, 1131518
Vol.
4. 2: 7. 8. 9. 10.
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AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Masters, C.L., Simms, G., Weinman, N.A., Multhaup, G., McDonald, L.A., and Beyreuther, K. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 4245-4249 Selkoe, D.J. (1990) Science, 248,1058- 1060. 1990 Weidemann, A., Konig, G., Bunke, D., Fischer, P., Salbaum, J. M., Masters, C. L., Beyreuther, K. (1989) Cell 57,115-126 Dyrks, T., Weidemann, A., Multhaup, G., Salbaum, J.M., Lemaire, H.-G., Kang, J., Milller-Hill, B., Masters, C.L., Beyreuther, K., (1988) EMBO J. 7, 949-957 Selkoe, D. J., Podlisny, M.B., Joachim, C.L., Vickers, E.A., Lee, G., Fritz, L. C., Oltersdorf, T., (1988) Proc. Natl. Acad. Sci. USA 85, 7341-7345 Tanzi, R.E., McClatchey, A.I., Lamperti, E.D., Villa-Komaroff, L., Gusella, J.F., and Neve. R.L. (1988) Nature 331,528-530 Van Nostrand, W.E., Schmaier, A.H., Farrow, J.S., and Cunningham, D.D., (1990) Science 248,745-748
11. 12. 13. 14. ii:
Bush, A.I., Martins, R.N., Rumble, B., Moir, R., Fuller, S., Milward, E., Currie, J., Ames, D., Weidemann, A., Fischer, P., Multhaup, G., Beyreuther, K., Masters, C.L., (1990) J. Biol. Chem. U.S.A. 265,15977-15983 Schlossmacher, M., Ostaszewski, B., Lieberburg, I., Kosik, K., and Selkoe, D., (1991) Sot. Neurosci. A&r., 17, part 2, Abstract # 517.8 Koo, E.H., Sisodia, S.S., Archer, D.R., Martin, L.J., Weidemann, A., Beyreuther, K., Fischer, P., Masters, C.L. & Price, D.L. (1990) Proc. Narl. Acad. Sci. U.S.A. 87, 15611565 Burgess, T.L., Craik, C.S., Kelly, R.B., (1985) J. Cell Biol. 101, 639-645 Buckley, K., and Kelly, R.B., (1985) J. Cell Biol. 100, 1284-1294 Kelly, R.B., Buckley, K.M., Burgess, T.L., Carlson, S.S., Caroni, P., Hooper, J.E., Katzen, A., Moore, H.-P.H., Pfeffer, S., Schroer, T.A., (1983) Cold Spring Harbor Symp. Quant. Biol. 48, 698-705
17. Matsuuchi, L., Buckley, K.M., Lowe, A.W., Kelly, R.B., (1988) J. Cell Biol. 106, 23918. :“o: 21.
22. 23. 24.
251 Burgess, T.L., and Kelly, R.B., (1987) Annu. Rev. Cell Biol. 3, 243-652 Moore, H.P.H., Walker, M.D., Lee, F., Kelly, R.B., (1983a) CeZZ35, 531-538 Fritz, L.C., Haidar, M.A., Arfsten, A.E., Schilling, J.W., Carilli, C., Shine, J., Baxter, J.D., and Reudelhuber, T. L., (1987) J. Biol. Chem.262, 12409- 12412 Moore, H.P.H., and Kelly, R.B., (1985) J. Cell Biol. 101, 1773-178 Kang, J., Lemaire, H.-G., Unterbeck, A., Salbaum, J.M., Masters, C.L., Grzeschik, K.H., Multhaup, G., Beyreuther, K., and Mtiller-Hill, B.(1987) Nature 325,773-736 Ponte, P., Gonzalez-DeWhitt, P., Schilling, J., Miller, J., Hsu, D., Greenberg, B., Davis. K., Wallace, W., Lieberburg, I., Fuller, F., and Cordell, B. (1988) Nature 331, 525-527 Kitaguchi, N., Takahashi, Y., Tokushima, Y., Shiojiri, S., and Ito, H., (1988) Nature 331,
530-532 25. Oltersdorf, T., Fritz, L.C., Schenk, D.B., Lieberburg, I., Johnson-Wood, K.L., Beattie, E.B., Ward, P.J., Blather, R.W., Dovey, H.F., and Sinha, S. (1989) Nature, 341, 144-147 26. Van Norstrand, W.E., Wagner, S.L., Suzuki, M., Choi, B.H., Farrow, J.S., Geddes, J.W., Cotman, C.W., and Cunningham, D.D., (1989) Nature 341,546-549 Smith, R.P., Higuchi, D.A., Broze, G.J., (1990) Science 248, 1126-1128
2 Defeudis, F.V., (1989) TiPS 10, 479-480. 29: Gorman, CM., Merlino, G.T., Willingham, M.C., Pastan, I., Howard, B.H., (1982) Proc. Natl. Acad. Sci., USA, 79, 6777-6781 Southern, P.J., and Berg, P., (1982) J. Mol. Appl. Genet. 1, 327-341 Moore, H.P.H., and Kelly, R.B., (1986) Nature 321, 443-446
E32: Oltersdorf, T., Ward, P.J., Henriksson,T., Beattie, E.C., Neve, R., Lieberburg, I., and Fritz, L.J., (1990) J. Biol. Chem.265, 4492-4497 Moore, H.P., Gumbiner, B., Kelly, R.B., (1983b) J. CeZZBioZ.97, 810-817 Laemmli, U.K. (1970) Nature 227,680-685 Kitaguchi, N., Tokushima, Y., Gishi, K., Takahashi, Y., Shiojiri,S., Nakamura, S., Tanaka, S., Kodaira, R. and Ito, H. (1990) Biochem. Biophys. Res. Commun. 166, 14531459 Gumbiner, B., and Kelly, R.B, (1981) Proc. Natl. Acad. Sci. USA 78, 318-322 Gumbiner, B., and Kelly, R.B., (1982) Cell 2&51-59 Rivas, R.J., and Moore, H.P.H., (1989) J. CeZZBiol. 109, 51-60 Schweitzer, ES. and Kelly, R-B., (1985) J. Cell Biol. 101, 667-676. Haass, C., Hung, A., Koo, E.H., and Selkoe, D.J., (1991) Sot. o?Neurosci. Abs. 17,
1294.
519
BIOCHEMICAL
THE O-AMYLOID
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 513-519
PRECURSOR PROTEIN IS NOT PROCESSED BY THE REGULATED
SECRETORY
PATHWAY
Caroline C. Overly, Lawrence C. Fritz, Ivan Lieberburg,
and Lisa McConlogue+
Athena Neurosciences, Inc., 8OOFGateway Blvd., South San Francisco, CA 94080 Received
October
16,
1991
The i3-amyioid peptide is derived from a larger membrane bound protein and accumulates as amyioid in Aizheimer’s diseased brains. O-amyioid precursor protein (BAPP) proteoiyticaiiy processed during constitutive secretion cannot be a source of deposited amyioid because this processing results in cleavage within the amyioidogenic peptide (1). To see if other secretory pathways could be responsible for generating potentially amyioidogenic molecules we tested the possibility that BAPP is Stable AtTPO ceil lines targeted to the regulated secretory pathway. expressing exogenous human DAPP were genetically engineered. These ceils were labeled with [35S]-methionine, and chased in the presence or absence of secretagogue. The RAPP both inside the ceils and released from the ceils was analyzed by immunoprecipitation and gel analysis. Quantitation of autoradiograms showed that virtually ail of the synthesized i3APP was secreted by the constitutive pathway, and that no detectable (4 0 1991Academic %) OAPP was targeted to the regulated secretory pathway. Press, 1°C. In Alzheimer’s disease, a 39 to 43 amino acid peptide is processed from the kmyloid precursor protein (OAPP) and accumulates in the brain as B-amyloid (BAP), (2-5) . The l3APP is synthesized as a membrane bound protein which is subsequently cleaved, leading to the secretion of an N-terminal fragment (secreted BAPP) (6-l 1). In most cell types so far examined, BAPP secretion has occurred via a “constitutive” secretory pathway; in this pathway proteins are released from cells continuously without storage and in a manner that is independent of stimulator-y secretogogues. It has been shown, however, that constitutive secretion of BAPP precludes the deposition of BAP since + To whom correspondence
should be addressed. . . III thrs IIUUIUSCIY~ are;BAFT (B-EIKQ~o~~~.XIXU protein), BAP (Ramyloid peptide), ACTH (adrenocdcotrophic hornme.), KPI (Ku& pmteaseinhibitor), 6956AFT (695 amino acid form of BAPP). 7516APP (75 1 amino acid form of BAPP), DME (Dulbeccols Modifii Eagle’s medium) ,FCS(fetal calf mm), HEPES (4-(2-HydroxyethyQ-l-piperazineethanesulfonic acid), RSV(Rous sarcoma virus long termhal repeat), SDS (Sodium dodfzyl sulfate). *
0006-291X/91
513
$1.50
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 181, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNlCATlONS 4
the full-length BAPP is cleaved within the amyloidogenic peptide to release the secreted fragment in this pathway (1). In addition to constitutive secretion, certain specialized cells such as neurons and endocrine cells, have a regulated secretory pathway. The regulated pathway can process and store various secretory proteins which are then specifically released upon stimulation. Recent evidence suggests that BAPP may be processed not only by the constitutive pathway but also by a regulated pathway in certain cells. For instance it has been shown that BAPP is stored in platelet cc-granules, from which it can be released by thrombii stimulation (10-12). Furthermore, full length BAPP in peripheral neurons has been shown to undergo anterograde fast axonal transport (13). Fast axonal transport is a form of microtubule dependent vesicular movement by which organelles reach nerve terminals; microtubule dependant mechanisms are also known to transport regulated secretory vesicles to process tips of certain cells (38,14-17). Three alternatively spliced forms of BAPP that contain the BAP sequence have been characterized: a 695 amino acid form (695BAPP) (22), and two longer forms of 751 (751BAPP), (9,23) and 770 (24) amino acids. The longer forms both contain an exon encoding a Kunitz type serine protease inhibitor (KPI) domain. Each of the three forms can be processed constitutively yielding a secreted N-terminal fragment and a membrane-bound C-terminal fragment, The secreted fragment of 751BAPP has been shown to be identical to protease nexin II (25-26), and to serve as an inhibitor of coagulation factor XIa (27). Direct sequencing of the cleaved fragments derived from 695BAPP and 751BAPP show that the constitutive cleavage occurs withii the amyloidogenic BAP fragment. Therefore, another pathway of BAPP processing must be responsible for the deposition of BAP in Alzheimers disease. The present experiments address whether the regulated secretory pathway could represent this alternative. The AtT20 pituitary cell line has been extensively characterized with respect to the cell biology of regulated secretion. When expressed in these cells, a wide-variety of regulated secretory proteins including B-endorphin, adrenocorticotrophin hormone (ACI’H), insulin, growth hormone, renin and trypsinogen, can be sorted into the regulated pathway, even though they may not be normally expressed in AU’20 cells (14,19-21). However, proteins that are normally secreted constitutively are sorted exclusively into the constitutive pathway in AtT-20 cells (14,33). We have used the AtT20 cell system to assesswhether BAPP has the necessary biochemical properties to be sorted into the regulated pathway. To this end, we transfected the human 695BAPP and 751BAPP cDNAs into AtT20 cells, and measured the amounts of BAPP processed by each pathway. We found that no detectable BAPP is sorted to the classical regulated secretory pathway, and that essentially all of the material is processed by the constitutive pathway.
Materials
and Methods
The expression plasmid, pR2 was constructed by combining the 3.5 k.b.HindIII to HpaI fragment of pRSVcat (29) with a polylinker containing HindIII, SalI, XbaI, SglII and SmaI oriented so that the HindIII site is closest to the promoter. The NruI to SpeI fragments containing the coding sequences for the 695BAPP (22) and 751BAPP (9,23), filled in at the Nru I site were cloned into the filled in HindIII and sticky XbaI sites of pR2. Clones with the correct orientation were chosen and named pR6Al(695BAPP) and pR7A1(751BAPP). 514
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AtTU), mouse pituitary cells, were co-transfected with p&41 or pR7Al and pSV2neo (30) by the method of Moore (19). Transfected colonies were isolated in 250 @ml G418 (Geneticin, Gibco). Clones were selected for expression of human 8APP by western blot analysis of cell lysates and maintained in culture medium containing 125 pg/ml G418. 1.3 x 106 cells were plated on 6-well tissue culture dishes and labelled as described (31) with the following modifications. Cells were methionine starved for 30 minutes, and then labelled with 200 pCiiml[35S]-methionine in one of two ways. Cells were labeled for 1.5 hours in methionine free DME containing 10 % FCS, washed, and chased in complete medium for three 2-hour periods to allow the release of all constitutively secreted material. Alternatively, cells were labelled for 15 hours in DME containing l/5 normal methionine plus 10 % dialyzed FCS, and further labelled for an additional hour in the presence of fresh label. They were then washed and chased for two 3-hour plus one 2-hour chase periods in complete medium to allow the release of all constitutively secreted material. The cells were then chased for an additional two or three hours in the presence or absence of secretagogue, 5 mM 8-Br-cAMP alone or with 21 mM KCl. Cell lysates were prepared by washing attached cells two times with PBS and solubilizing the cells in lysis buffer (10 mM Tris pH7.5, 150 mM NaCl, 5 mM EDTA, 2 p.g/ml leupeptin and 1.0 % NP40). Cell lysates and media were spun at 4’ C at 100,000 x g for 10 minutes. They were then immunoprecipitated with either anti-BXS, a polyclonal antibody raised to gAPP (32), or with antiACIH antibody (33). The samples were preadsorbed with 15 mg protein A sepharose beads (Pharmacia) for several hours at 4OC with constant agitation. Beads were removed by centrifugation and the supematants were reacted with 1 pg of affinity purified anti-BX5 or with 2 pl anti-ACI’H serum at 4’ C overnight. Protein A sepharose beads pm-blocked with unlabelled cell lysates were added, and samples were rocked for 20 minutes at room temperature. The beads were recovered by centrifugation and washed three times in the appropriate buffer: wash buffer 1 (10 mM Tris pH. 7.5, 0.5 M NaCl, 0.1% NP-40,5 mM EDTA, 10 pg/ml leupeptin) for anti-BXS, and wash buffer 2 (150 mM NaCl, 50 mM HEPES, 1% BSA, 5 mM EDTA, 0.5% deoxycholate, 1% NP-40,0.1% SDS, 0.5 M KCl, 10 pg/ml leupeptin) for anti-ACTH. The beads were again recovered and resuspended in 40 p.l of SDS polyacrylamide gel sample buffer followed by electrophoresis on a 5-15% SDS polyacrylamide gradient gel (34). Gels were fixed in 7% acetic acid, treated with Amplify (Amersham) and exposed to film at -700 C for autoradiography. Autoradiograms were scanned on a Shimadzu dual wavelength thin layer chromatoscanner, model CS-930.
Results These experiments were designed to determine if 8APP can be sorted to the regulated secretory pathway in AtT20 cells. We transfected the AtI’20 cells with the two most abundant forms of the OAPP in human brain (35), 6958APP and 751BAPP. Plasmids expressing 6958APP and 7518APP using the Rouse sarcoma virus long terminal repeat (RSV) promoter, were constructed as described in Experimental Procedures and are shown in figure 1. Multiple stable lines of AtT20 cells transfected with either plasmid were selected, and assayed for the expression of the human 8APP by Western analysis with the anti-8APP antibody BX5. This antibody is directed to human l3APP and it specifically detects the exogenous human 8APP in the presence of endogenous rodent 8APP. To measure regulated and constitutive secretion of UPP, transfected cells were metabolically labelled with [35Sl-methionine, chased for several hours to remove all the proteins transported by the constitutive pathway, and then chased further either in the presence or in the absence of a sectetagogue to analyze material released on stimulation. Cell lysates and media samples taken at several time points throughout the experiment were immunoprecipitated with anti-BXS, and the precipitated proteins were electrophoresed and analyzed by autoradiography. The results (Figure 2A) show that most or all of the 8APP is constitutively secreted and that no detectable fraction is secreted in a regulated manner. The vast majority of the BAPP is released constitutively during the first 6 hours of chase, (figure 2A, lanes 3-5), and a minor amount of material is released during the 6 515
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+
-
Figure1. Two plasmidsexpressing the6958APP(pR6Al) andthe751DAPP(pR7Al). TheRSV LTR &ray arrow)servesasa promoter,andSV40sequences serveasa poly adenylationsignal (gray box). 6APPsequences areshownasa whitebox. The locationof theinsertedKPI domainin plasmidpR7Al isindicatedonthemap.The blacklinesindicateflankingSW0 sequences and pBR322sequences. Figure2. Secretionof 695R4PPfromtransfected AtT20 mousepituitary cells. (A) Cellswere labe-lled with [35S]-methionine for 1.5hours,chased for three2-hourperiodsandstimulatedwith 8Br-cAMP. Celllysatesandmediawere immunoprecipitated with anti-BXS. Samples wererunon a 5-15% SDSpolyacrylamide gradientgelandvisualizedby autoradiography. Lane1) l/3 of cell lysateat theendof thelabeliingperiod(t=O). Lane2) l/3 of celllysateafterthe9 hourchase.Lanes 3.4 and5) total mediumfromO-2hours,2-4 hours,and4-6 hoursrespectively. Lanes6 and7) total mediumbetween6 and9 hoursof chasein thepresence (lane6) andabsence (lane7) of 8-BrCAMP. (B) Cellswerelabelledfor 16hours,chased for 6 hoursandstimulated.Mediasamples collectedin thepresence (Lane1) andin the absence (Lane2) of 8-Br-cAMPwere immunoprecipitated with anti-ACTH andrunon a 5-1596SDSpolyacrylamidegradientgelfor autoradiography.
to 9 hour chase. The amountsof 5APP secretedduring the 6 to 9 hour chaseis not increasedby secretagogue,demonstratingthat no OAPPis secretedby the regulatedsecretorypathway (figure 2A, lanes6 and 7). Furthermore,one would expect that a protein processedby the regulatedpathway would be storedin densecore vesiclesand accumulateinsidethe cell (19,37). In accord with the absenceof regulatedBAPPrelease,very little materialremainsin the cell after 6 (data not shown)and 9 hoursof chase(figure 2A lane 2). The samebehavior wasobservedwith clonesexpressingeither 695BAPPor 7515APP. and under both of the labelingconditions describedin Experimental Procedures.In contrast,incubationof the cells with a secretagoguedramatically enhancesthe release of ACTH (figure 2B), a peptideknown to be releasedin a regulatedmanner. Cell supematantswere immunoprecipitatedwith anti-ACM antibody, and the 4500 Da and 13,000Da maturefoms of ACTH (33,36) went specifically precipitated. Together, theseobservationsdemonstratethat 5APP doesnot exit the cell through the regulatedsecretorypathway of AU20 cells. The amountof 5APP secretedconstitutively from the cells was quautitated by itntntmoprecipitationand gel electmphoresisof labelledsecretedl3APPfollowed by densitotnettic scanningof resulting autoradiogratns.The amountsof material secretedwere converted to 516
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808 g
60-
i
40-
2 20-
I HOURS CnAsE
0 0
4
2
6
CWSETlMEtHOURS)
Figunz3. Kineticsof thesecretionof human695DAPPby AtI20 cells. C!&ularandsecreted proteinwasquantitatedby scanning theautomdiogmms from theexperimentdescribed in figure2A. Thepercentage of BAPPsecreted into themediumwascalculatedby dividingtheamountreleased by theamountinsidethecellsat t4 andisplottedasa functionof thelengthof thechaseperiod. Insert. Thepercentage of 6APPremaininginsidethec&s wascalculatedby subtractingthetotal% released at eachtimepointfrom 100.‘Ihe log of thepercentage of BAPPremainingin thecellsis plottedagainsttimeof chase.
percentages of the total labelled BAPP measured inside the cells at t=O and plotted against time of chase (Figure 3A). 71 % of the material quantitated inside the cell at t=O is secreted in the first 2 hours of chase, 24 % in the next 2 hours, and 5.2 % between 4 and 6 hours. Therefore, essentially all of the labelled BAPP present inside the cell is released within a 6hour chase period in the absence of secmtagogue. To examinethe kinetics of this release,the amountof BAPPremainingin the cell was plotted against length of chase. The resulting graph (insert, figure 3) shows that DAPP displays a rapid (tin= 1.1 hours) exponential release of BAPP from the cells, a pattern characteristic of constitutively secreted proteins (2 1,37). There was a small amount of detectable BAPP that was released during the 6 to 9 hour chase period. This residual COnStiNtiVe secretion represents the background against which we were attempting to detect regulated secretion of BAPP. In order to qua&ate this BAPP relative to the initial amounts of BAPP synthesized, autoradiograms exposed for various times were quantitated, corrected for length of exposure and compared to each other. The percent of initial BAPP released during the 6 to 9 hours chase was calculated as 1.1% for the stimulated conditions and 0.84 % for the unstimulated conditions. Therefore, in this experiment at most 0.26 % of the DAPP appeared to be processed by the regulated pathway. We believe that this amount is within the error of these techniques and is therefore insignificant. With this level of background, we could have detected as little as 1% of the initially synthesized BAPP had it been processed by the regulated secretory pathway.
Discussion Constitutive secretionof&VP
has-beenshownto involve a proteolytic cleavagewithin the B-
amyloid sequence (1). However, in Alzheimer’s diseased brains some of the BAPP must avoid this 517
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cleavage and be processed in an alternative manner that leads to SAP formation (1,5). Although constitutive secretion of OAPP has been observed in all cells where it has been examined, OAPP has also been shown to be sorted into the vesicular compartment that undergoes fast axonal transport in peripheral neurons (13), and to be released from storage granules in stimulated platelets (lo- 12). These data suggested that alternative secretion pathways could exist for DAPP and the present experiments were designed to address this in a well characterized cellular system. We tested directly whether the classical regulated secretory pathway characterized in AtT20 cells can process DAPP. The AtT20 mouse pituitary cell line is known to correctly target regulated secretory proteins from diverse tissues into its regulated secretory pathway (19-21). The present experiments demonstrate that none (at most 1%) of the DAPP is secreted in a regulated manner in AtT20 cells. Furthermore, we have found that in AtT20 cells, 100% of the OAPP is secreted by the constitutive pathway, and no detectable material accumulates in these cells. Weidemann and colleagues (6) have shown that in PC12 cells at least 30% of the i3APP synthesized is released via the constitutive pathway and is detectable in the supematant. They were not able to detect the remaining material, however, either inside or outside of the cells, and therefore OAPP is not stored in PC12 cells. Thus, although PC12 cells do possess a regulated secretory pathway (39) it does not appear to process BAPP. Recent experiments suggest that intracellular degradation of SAPP without secretion may occur in astrocytes and microglia (40). BAPP has been shown to undergo fast axonal transport (13), however this does not prove that the transport is via regulated secretory vesicles. It has previously been shown, for example, that constitutively secreted kappa light chain protein is detected in the tips of processes in AtT20 and PC12 cells (17), as is the case for regulated proteins. It is possible, therefore, that axonally transported SAPP is destined strictly for constitutive processing in accord with our observations in AtT20 cells. The storage and stimulated release of BAPP from u-granules (lo-12), however, may reflect a fundamental difference between the regulated secretory pathway in platelets and AtT20 cells. Previous studies have shown that constitutive processing of I3APP cannot give rise to the amyloid deposition observed in Alzheimer’s disease. If l3APP is not processed by the regulated secretory pathway in relevant brain cells, other pathways of BAPP metabolism must exist to explain the Alzheimer’s amyloid pathology.
Acknowledmen&We would like to thank Hsiao-Ping Moore for her expert advice and helpful discussions. We are especially grateful to David Quin, Thane Kreiner and Mark Grimes for sharing their experimental expertise and ideas. Mark Grimes and Regis Kelly generously provided the antiACTH antibody. The anti-BXS antibody was generously provided by Pamela Ward. We also thank Dale Schenk, David Agard, and Mark Grimes for their critical reading of the manuscript. References
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