Proc. Natl. Acad. Sci. USAVol. 88, pp. 10302-10306, November 1991Biochemistry

The predominant form of the amyloid ,8-protein precursor in humanbrain is protease nexin 2

(protease inhibitor/Alzheimer disease/proteolytic processing)

WILLIAM E. VAN NOSTRAND*t, JEFFREY S. FARROW*¶, STEVEN L. WAGNER*¶, RAMANINDER BHASINt,DMITRY GOLDGABERt, CARL W. COTMAN§, AND DENNIS D. CUNNINGHAM*Departments of *Microbiology and Molecular Genetics and §Psychobiology, University of California, Irvine, CA 92717; and tDepartment of Psychiatry andBehavioral Sciences, State University of New York, Stony Brook, NY 11794

Communicated by D. Carleton Gajdusek, August 6, 1991

ABSTRACT The amyloid f3 protein and the amyloida-protein precursor (APP) are major constituents of senileplaques and cerebrovascular deposits in patients with Alzhei-mer disease and Down syndrome. Most human tissues containmRNA that encodes forms of APP that contain the Kunitzprotease inhibitor (KPI+) domain. A major 120-kDa proteincorresponding to this KPI+ mRNA is also found in thesetissues. This protein is identical to the protease inhibitorprotease nexin 2. Brain contains an additional mRNA speciesthat encodes a form of APP that lacks the KPI domain (KPI-).This latter mRNA has been suggested to encode a 105-kDaKPI- form of APP protein also found in brain. Using proteaseinhibitory functional assays, we show that both the 105-kDaand 120-kDa APP proteins in normal and Alzheimer diseasebrain contain the KPI domain. Moreover, KPI domain-specificprecipitation assays reveal that KPI- forms of APP proteinrepresent <14% of total brain APP. Lastly, an enrichedfraction from total brain homogenate contains proteolyticactivity that can process the purified 120-kDa KPI+ form ofAPP into a 105-kDa form, resulting in a high-molecular-massdoublet identical to that seen in brain. These rmdings indicatethat although KPI- APP mRNA is abundant in brain, littlecorresponding protein is present. Thus, KPI+ APP protein(equivalent to protease nexin 2) is the predominant form ofAPPin human brain.

The amyloid (3 protein is a 4.2-kDa peptide that is depositedin senile plaques and the cerebrovasculature of individualsafflicted with Alzheimer disease (AD) and Down syndrome(1-5). This protein is derived from a larger precursor protein,the amyloid (-protein precursor (APP) (6-9). Previous stud-ies showed that APP can be translated from, at least, threealternatively spliced mRNAs to yield proteins of 695, 751,and 770 amino acids (10-12). The latter two species containan additional insert that encodes a domain homologous toKunitz-type protease inhibitors (KPIs) (10-12). The secretedform ofAPP protein, which contains the KPI domain (KPI+),was shown to be identical to the protease inhibitor proteasenexin 2 (PN-2) (13, 14). PN-2 is a potent inhibitor of certainserine proteases, including factor XIa, trypsin, chymotryp-sin, epidermal growth factor-binding protein (EGF-BP), andthe y subunit of nerve growth factor (13, 15-17). Inhibitionby PN-2 involves the catalytic-site serine of the protease andresults in a stoichiometric protease-PN-2 complex.

Previous studies have shown that KPI+ APP mRNA ispresent, at various levels, in most tissues, whereas KPI- APPmRNA is found primarily in brain (10-12, 18). Two majorAPP proteins with estimated molecular masses of 120 kDaand 105 kDa were found in human brain (19-21). Immunoblot

analysis with a polyclonal antiserum prepared against asynthetic peptide corresponding to a region of the KPIdomain indicated that the 120-kDa form of APP protein isKPI' (19) and is, thus, PN-2. Lack of immunoreactivity withthis same polyclonal antiserum suggested that the 105-kDaform ofAPP protein in brain is devoid ofthe KPI domain (19).These qualitative studies also suggested that the 105-kDaputative KPI- form of APP is the predominant species ofAPP protein in brain, apparently consistent with the presenceof abundant KPI- APP mRNA in this tissue.The proteolytic events leading to the formation of amyloid

(3protein from APP remain unclear. Recent studies showedthat release of the large extracellular domain of APP is theresult of proteolytic cleavage through the amyloid (3-proteindomain (22, 23). This result indicates that altered proteolyticmechanisms occur in AD such that the integrity of theamyloid ,B protein is maintained and it is ultimately liberatedfrom APP. It was suggested that because KPI- forms ofAPPprotein lack the potent protease inhibitory domain, they maybe more susceptible to altered proteolytic mechanisms im-plicated in AD. The present studies were designed to providequalitative and quantitative analyses of the different forms ofAPP protein in human brain.

MATERIALS AND METHODSMaterials. The anti-PN-2 mouse monoclonal antibody

(mAb) P2-1 was prepared as described (13). cDNAs encodingAPP-695 (KPI-) and APP-751 (KPI+) were expressed in thebaculovirus insect system (24, 25). PN-2 was purified fromserum-free culture medium from human foreskin fibroblastsand human platelet lysates as described (15, 26, 27). Humanbrains of control individuals and AD patients were removedat autopsy and bisected midsagitally. One hemisphere wasfixed in 10%o (vol/vol) buffered formalin; the AD diagnosiswas confirmed by using the autopsy criteria of Khachaturian(28). The other hemisphere was dissected, frozen immedi-ately in dry ice, and stored at -70°C. Twenty-five-gramsections of cortical tissue were homogenized at 4°C in 10 volof 20 mM potassium phosphate/0.2 M NaCl, pH 7.4/20%(vol/vol) glycerol/2% Triton X-100/5 mM EDTA/1 mMphenylmethanesulfonyl fluoride/1 ,uM antipain/1 ,uM leu-peptin/10 ,M chymostatin. The homogenates were centri-fuged for 20 min at 15,000 x g. APP was purified from thehomogenates by using anion-exchange chromatography overDEAE-Sepharose and affinity chromatography over dextran

Abbreviations: AD, Alzheimer disease; APP, amyloid ,B-proteinprecursor; PN-2, protease nexin 2; KPI, Kunitz protease inhibitor;mAb, monoclonal antibody; EGF-BP, epidermal growth factor-binding protein.tTo whom reprint requests should be addressed.VPresent address: Salk Institute Biotechnology and Industrial Asso-ciates, 505 Coast Blvd. South, La Jolla, CA 92037.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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sulfate-Sepharose (26). Alternatively, an APP-processingprotease was enriched from brain homogenates by 50-75%ammonium sulfate precipitation, anion-exchange chromatog-raphy over Q-Sepharose, and preparative SDS/PAGE.Mouse EGF-BP was purified as described (29). Goat anti-mouse IgG1 that was adsorbed with human serum wasobtained from Sigma. EGF-BP and goat anti-mouse IgG1were labeled with Na125I by means of the chloroglycourilmethod (30); specific activities were 5.0 and 2.5 x 106cpm/pmol, respectively.Immunoblot Analyses. Samples containing APP proteins

were electrophoresed on SDS/7.5% polyacrylamide gelsaccording to Laemmli (31). Nonreducing conditions wereused for immunoblots with mAb P2-1, and reducing condi-tions were used for immunoblots with the anti-KPI peptidepolyclonal antiserum. The proteins were then electroblottedonto poly(vinylidene difluoride) membranes (Millipore), andthe unoccupied sites were blocked overnight with a solutionof phosphate-buffered saline/0.25% gelatin. The membraneswere incubated with mAb P2-1 monoclonal hybridoma cul-ture supernatant or 1:100 dilution of the anti-KPI peptiderabbit polyclonal antiserum, which was obtained from S.Younkin (Case Western Reserve University). Bound mousemAb or rabbit polyclonal antibody was detected with abiotinylated-sheep anti-mouse IgG (1:200) or a biotinylated-donkey anti-rabbit IgG (1:200), respectively, followed by a1:400 dilution of a streptavidin-peroxidase conjugate (Am-ersham). The immunoblots were developed in a solution of 17mM 4-chloronaphthol dissolved in 16 ml of ice-cold methanoladded to 80 ml of ice-cold Tris-buffered saline/0.024% H202.

Protease-PN-2 Complex Formation Assays. Samples con-taining APP proteins were incubated with 20 ng of 12 I-labeledEGF-BP for 15 min at 22°C; the reactions were terminated bythe addition of an equal volume of nonreducing LaemmliSDS-sample buffer. Without prior boiling, the samples wereelectrophoresed on SDS/7.5% polyacrylamide gels undernonreducing conditions. The completed gels were stained,destained, and dried; autoradiograms were exposed for 1-2 hrat -70°C.

Trypsin-Agarose Precipitation ofAPP Protein. Twenty-fivemicroliter aliquots of packed trypsin-agarose (Sigma) orcontrol agarose (Bio-Rad BioGel A1.5M) were washed with1 ml of 20 mM potassium phosphate/0.5 M NaCl, pH7.4/bovine serum albumin at 200 ,g/ml. The agarose waspelleted by centrifugation in a microcentrifuge, and the washbuffer was removed by aspiration. Purified baculovirus-expressed APPs or brain APP (20-40 nM) in a vol of 25 ,ul ofthe above buffer were incubated with the trypsin-agarose orcontrol agarose for 15 min on ice with intermittent mixing.The agarose was pelleted by centrifugation in a microcentri-fuge, and 12.5-,lI aliquots were subjected to SDS/PAGE andimmunoblotting with mAb P2-1 as described above. Forquantitative immunoblots, bound mouse mAb P2-1 was de-tected with a solution (50 ng/ml) of I251-labeled goat anti-mouse IgG1 (2.5 x 106 cpm/pmol). Autoradiograms wereexposed at -70°C for 1- and 2-hr exposures; each autoradio-gram was quantitated by scanning with an LKB Ultrascanlaser densitometer.

RESULTSSpecificity of Immunochemical and Protease Inhibitor Func-

tional Assays. To validate the specificity ofthe immunochem-ical and functional assays used in these studies, experimentswere conducted with baculovirus expression products en-coded by cDNAs for KPI- (APP-695) and KPI+ (APP-751)forms of APP protein. Although PN-2 is identical to thesecreted form of APP(KPI+), the anti-PN-2 mAb P2-1 rec-ognizes both APP(KPI+) and APP(KPI-) (Fig. 1A). Thisrecognition is due to an amino-terminal epitope common to

A B CKPIT KPit-

KPI- KPI* dPI' KPI+' r-- PI_ K4.

200K -116K- - --_97K - _1i66K -

43K -

30K -

t:=2 4 Z r -7'

FIG. 1. Immunoblot and protease inhibitor functional assays withbaculovirus-expressed KPI- and KPI+ APP proteins. (A) Approxi-mately 25 ng of KPI- APP-695 protein (lane 1) or KPI+ APP-751protein (lane 2) was immunoblotted with mAb P2-1. (B) Approxi-mately 50 ng of KPI- APP-695 protein (lane 3) or KPI+ APP-751protein (lane 4) was incubated with 20 ng of 125I-labeled EGF-BP andsubjected to SDS/PAGE with subsequent autoradiography. (C)Approximately 100 ng of KPI- APP-695 protein or KPI+ APP-751protein was subjected to precipitation with control agarose (lanes 5and 7, respectively) or trypsin-agarose (lanes 6 and 8, respectively),and the resulting supernatants were immunoblotted with mAb P2-1.K, kDa.

both forms of APP protein; in fact, this mAb has been usedto immunopurify both forms of baculovirus-expressed APPprotein (W.E.V.N., J.S.F., and R.B. unpublished data). Incontrast, Fig. lB shows that only the KPI+ form of APPprotein forms SDS-stable complexes with the protease EGF-BP. Similarly, the KPI+ form ofAPP protein was precipitatedby trypsin-agarose, whereas the KPI- form was not (Fig.1C). These latter two experiments show that only the KPI+form ofAPP protein (identical to PN-2) was detected in thesefunctional protease inhibitor assays based on presence of theKPI domain.Immunochemical and Functional Analysis of APP Proteins

from Different Sources. The immunoblot and protease com-plex formation assays described above were used to charac-terize the forms of APP protein in human fibroblasts, humanplatelets, and human brain. Immunoblot analysis showed thatonly one major form of APP protein at '='120 kDa wassecreted by cultured human fibroblasts and contained inhuman platelets (Fig. 2A, lanes 1 and 2). In contrast, theimmunoblot analysis showed that human brain contained twomajor forms ofAPP protein at 120 kDa and 105 kDa (Fig. 2A,lane 3), consistent with previous reports (19-21). Most brain

A N~4§\sNt~V ( .r

B s

e< ,

200K-116K ~ _97K66K-

43K-30K-

2 4

FIG. 2. Immunoblot and autoradiogram of 1251-labeled EGFBP-PN-2 complex formation assays with APP proteins purified fromvarious sources. Approximately 75 ng of APP protein purified fromcultured human fibroblasts, human platelets, and control humanbrain was immunoblotted with mAb P2-1 (A) or incubated with 20 ngof 125I-labeled EGF-BP and subjected to SDS/PAGE with subse-quent autoradiography (B). K, kDa.

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APP proteins could be extracted from tissue without deter-gents (W.E.V.N. and J.S.F. unpublished data) and exhibitedsimilar electrophoretic mobilities as APP secreted by fibro-blasts and platelets (Fig. 2A), indicating that these are likelysecreted forms ofAPP. Fig. 2B shows that APP proteins fromfibroblasts (lane 1) and platelets (lane 2) contained the KPIdomain based on the functional assay of its ability to formstable complexes with 125I-labeled EGF-BP, consistent withour previous findings that these cells secrete PN-2 (13, 15, 26,27). Unexpectedly, both forms of APP protein from humanbrain complexed with 1251-labeled EGF-BP (Fig. 2B, lane 6),demonstrating the presence of the KPI domain. These find-ings indicate that the 105-kDa form of APP protein in humanbrain contains KPI domain.

Similar studies were conducted with APP preparationspurified from cortical tissue obtained from two control brainsand two confirmed AD brains. APP purifications from brainhomogenates were conducted in the presence of proteaseinhibitors. It is noteworthy that purifications conducted in theabsence of protease inhibitors increased the 105-kDa band ofAPP (W.E.V.N. and J.S.F. unpublished data). It should beemphasized that immunoblotting of all fractions obtainedduring APP purification from brain-tissue homogenates en-sured that we did not select for only certain forms of theprotein during isolation procedures. In fact, the KPI' andKPI- forms of APP protein expressed in the baculovirussystem showed similar binding properties on DEAE-Sepharose and dextran sulfate-Sepharose columns (25), thesame columns used to purify APP from brain-tissue homoge-nates. Immunoblotting studies show that two prominent APPproteins, with approximate molecular masses of 120 kDa and105 kDa, were purified from the two control brains (Fig. 3A,lanes 1 and 2) and the two AD brains (Fig. 3A, lanes 3 and 4).In the protease inhibitor functional assay, both APP proteinsfrom the two control brains (Fig. 3B, lanes 5 and 6) and thetwo AD brains (Fig. 3B, lanes 7 and 8) formed SDS-stablecomplexes with 125I-labeled EGF-BP. This functional activ-ity, based on the presence of the KPI domain, indicates thatthe 120-kDa and the 105-kDa APP proteins contain the KPIdomain and are, thus, PN-2.To further evaluate properties of the 120-kDa and 105-kDa

APP proteins in control and AD brain we conducted precip-itation studies with trypsin-agarose, an assay that distin-guishes KPI+ from KPI- forms of APP (Fig. 1C). Proteinsamples were precipitated with trypsin-agarose, and the APPproteins remaining in the supernatants were immunoblottedwith mAb P2-1 and an 125I-labeled secondary antibody. Theautoradiogram in Fig. 4A confirmed that the baculovirus-expressed KPI- form of APP was not precipitated (lane 2),whereas the KPI+ form of APP was completely removedfrom the supernatant (lane 4). In addition, this proceduredemonstrated that KPI- forms ofAPP were not degraded bytrypsin-agarose under the conditions used in the precipitation

AkDa Control AD

200 -

116- UI97 -

66 -

43 -

30 -

BControl AD-Fr

.... m_

2 3 4 5 6 7 8

FIG. 3. Immunoblot and autoradiogram of 125I-labeled EGF-BP-PN-2 complex formation assays with APP proteins purified fromcontrol andAD brain. Approximately 100 ng ofAPP proteins purifiedfrom control and AD brains was immunoblotted with mAb P2-1 (A)or incubated with 20 ng of 125I-labeled EGF-BP and subjected toSDS/PAGE with subsequent autoradiography (B).

AI'_I -!

_

I6 <-

B

W

FIG. 4. Autoradiogram of trypsin-agarose precipitations of puri-fied APP proteins from control and AD brains. (A) Approximately100 ng of baculovirus-expressed KPI- APP-695 protein (lanes 1 and2) or KPI+ APP-751 protein (lanes 3 and 4) were precipitated withcontrol agarose (lanes 1 and 3) or trypsin-agarose (lanes 2 and 4), andthe supernatants were immunoblotted with mAb P2-1. (B) Approx-imately 100 ng of APP proteins purified from control brains (lanes5-8) orAD brains (lanes 9-12) were precipitated with control agarose(lanes 5, 7, 9, and 11) or trypsin-agarose (lanes 6, 8, 10, and 12), andthe resulting supernatants were immunoblotted with mAb P2-1. In Aand B, mAb P2-1 was detected with an 1251-labeled goat anti-mouseIgG, and autoradiograms were prepared; the autoradiograms werequantitated with an LKB scanning laser densitometer. K, kDa.

experiments. When this assay was conducted with the APPpurified from control and AD brains, most protein in eachpreparation was precipitated (Fig. 4B, lanes 6, 8, 10, and 12).The amount ofAPP protein in solution after precipitation wasquantitated from the autoradiograms by scanning laser den-sitometry. This analysis revealed that =12% and 14% of thetotal APP protein remained in the supernatants of control andAD brain samples, respectively. These experiments wereconducted with two different concentrations of brain APP,and two different autoradiographic exposures were preparedand quantitated by laser densitometry, yielding nearly iden-tical results. Because this assay specifically precipitatesKPI+ forms of APP protein, these analyses indicate that nomore than 14% of total APP protein in brain is the KPI- form.Immunoblot analyses with mAb P2-1 (13) and a polyclonal

antiserum raised against a synthetic peptide corresponding toa region of the KPI domain (19) were conducted with twopreparations of APP protein purified from different controlbrains by using the procedures described above (Fig. 5). Theimmunoblots in lane 1 of Fig. 5 A and B show that mAb P2-1detects what appears to be abundant 120-kDa and 105-kDaAPP proteins in each of the different preparations. When anequivalent amount of APP was immunoblotted with theanti-KPI domain polyclonal antiserum, the 120-kDa and105-kDa APP proteins were recognized in the particularpreparation presented in lane 2 of Fig. 5A, consistent with ourfindings in the functional assays described above. However,in the other APP preparation shown in lane 2 of Fig. SB onlythe 120-kDa APP protein was recognized with this anti-KPIantiserum, consistent with described results (19) that sug-gested the 105-kDa form of the protein is KPI-. To furtherunderstand these differences, each preparation of purifiedbrain APP was serially diluted and then immunoblotted withmAb P2-1. The immunoblot in lanes 3-7 of Fig. 5A demon-strates a relatively even distribution of the 120-kDa and105-kDa forms of APP protein in this particular preparation.In contrast, the immunoblot shown in lanes 3-7 of Fig. 5Breveals that this preparation contains predominantly the120-kDa APP protein. The findings presented in Fig. 5B mayexplain the lack of anti-KPI immunoreactivity with the 105-kDa form of APP protein in some preparations of brain APP(19). The dilution and immunoblotting experiment showedthat this preparation actually contains little 105-kDa APP.Thus, the small amount of this form is insufficient to be

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A kDa

200116 - _97

66

43 -

30 --

1 2 3 4 5 6 7

B kDa

200 --116-_97 --

66 -

43 -

30.... ...

1 2 3 4 5 6 7

FIG. 5. Immunoblots of purified APP proteins from controlbrains. Approximately 100 ng of APP proteins purified from twocontrol brains (designated A and B) was immunoblotted with mAbP2-1 (lanes 1) or rabbit anti-KPI antiserum (lanes 2). In addition, thepurified APP proteins were serially diluted and immunoblotted withmAb P2-1. Lanes: 3, 100 ng; 4, 50 ng; 5, 25 ng; 6, 12.5 ng; and 7, 6.25ng.

detected with the anti-KPI antiserum but is readily detectedwith a more sensitive antibody, such as mAb P2-1. Thesefindings indicate that the observed differences in the relativeamounts of the 120-kDa and 105-kDa APP proteins reflectvariability of these two species in different brain prepara-tions.

Biochemical Basis for the 105-kDa Form of APP Protein inBrain. Our findings in the functional assays indicated thatmuch of the 105-kDa form ofAPP was KPI+, suggesting thatit may result from proteolysis of the 120-kDa form of theprotein. To test this hypothesis, total brain homogenate wasfractionated and screened for proteolytic activity by usingpurified PN-2 from human fibroblasts as the substrate. APN-2-processing activity was prepared from total brain ho-mogenate using ammonium sulfate precipitation, anion-exchange chromatography on Q-Sepharose, and preparativeSDS/PAGE. Immunoblot analysis of this enriched proteasefraction with mAb P2-1 confirmed that no endogenous brainAPP protein was present in this fraction (W.E.V.N. andJ.S.F. unpublished data). The immunoblot in Fig. 6 showsthat incubation of the 120-kDa KPI+ form of APP fromhuman fibroblasts (lane 1) with an aliquot of this enrichedbrain protease fraction resulted in formation of a doublet thatcontains a KPI+ APP protein with an apparent molecularmass of 105 kDa (lane 2). Moreover, this protease-generateddoublet is very similar to the high-molecular-mass PN-2doublet found in human brain (lane 3). These findings indicatethat human brain contains protease activity that can generatea 105-kDa form of PN-2 from the 120-kDa PN-2.

DISCUSSIONThe present studies show that the predominant form of APPprotein in control and AD brain contains the KPI domain andis, thus, PN-2. This fact was demonstrated by two proteaseinhibitor functional assays based on the presence of the KPI

2. -10OK ---

6K -37 K --

P.,'.a.u

32 K

FIG. 6. Immunoblot of PN-2 processed by enriched brain prote-ase fraction. Purified PN-2 was incubated with an aliquot of anenriched protease fraction from brain and analyzed by immunoblot-ting with mAb P2-1. Lanes: 1, 50 ng of purified PN-2; 2, 50 ng ofpurified PN-2 incubated with the enriched brain protease fraction(-10 ng of protein) for 10 min at 370C; 3, 50 ng of purified brain APP.K, kDa.

domain. The first assay was SDS/PAGE-stable complexformation with 125I-labeled EGF-BP (15, 26, 27). The secondassay involved specific precipitation of KPI' forms of APPprotein with trypsin-agarose. Quantitation with this latterassay revealed that no more than 14% ofthe total APP proteinin brain is KPI-.

Previous studies (10-12, 18) demonstrated that brain con-tains abundant levels ofmRNA that encode KPI' and KPI-forms of APP protein. The presence of abundant KPI' andKPI- APPmRNA in brain would predict that abundant levelsof each form of protein exist in brain. Seemingly consistentwith this expectation, brain-tissue homogenates were shownto contain two major soluble APP proteins with estimatedmolecular masses of 120 kDa and 105 kDa (19-21). Immuno-blot analysis with a polyclonal antiserum prepared against asynthetic peptide corresponding to a sequence of the KPIdomain indicated that the 120-kDa form ofAPP is KPI+ (19);lack ofimmunoreactivity with this same polyclonal antiserumsuggested that the 105-kDa form ofAPP in brain is KPI- (19).Together, these findings have suggested that the predominantform ofAPP protein in brain lacks the KPI domain. However,these conclusions were based on lack of immunoreactivitywhen using an antipeptide antibody. The present studiesshown in Fig. 5 suggest that these previous qualitativefindings may have resulted from variability in the relativeamounts of the 120-kDa and 105-kDa forms ofAPP protein inconjunction with immunoblots that used antibodies of differ-ent levels of sensitivity.The present findings, on the other hand, were based on

positive results with functional assays that demonstrated theKPI domain in both 105-kDa and 120-kDa forms of APPprotein. Control experiments unequivocally showed thatKPI- forms ofAPP protein did not complex with 125I-labeledEGF-BP and were not precipitated by trypsin-agarose (Fig.1). Therefore, analyses of the APP preparations from thecontrol and AD brains convincingly show that the 105-kDaform of APP protein contains the KPI domain (Fig. 3). Itshould be emphasized that immunoblotting analysis of eachstep in the purification of APP protein from brain-tissuehomogenates ensured that KPI- forms ofAPP were not beingsegregated during isolation procedures. Furthermore, whenthe Triton X-100-insoluble material, resulting from the brain-homogenate preparations, was further solubilized with 2%SDS and analyzed by immunoblotting, no detectable APPprotein was found. Quantitation of the purified brain APPprotein remaining in solution after trypsin-agarose precipita-tion indicated that no more than 14% of the total APP wasKPI-. It is noteworthy that some of the purified brain APPprotein not precipitated by trypsin-agarose could represent

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additional KPI' APP that was inactive or in complex withendogenous brain proteases. The finding that an enrichedprotease activity from brain homogenates can process thepurified 120-kDa KPI+ form ofAPP to a 105-kDa KPI' formof the protein offers a likely explanation for the KPI+ APPdoublet in brain (Fig. 6). Together, these analyses indicatethat most APP protein in brain is KPI', or PN-2.Our results indicate that APP protein levels do not corre-

late with APP mRNA levels in human brain. Several expla-nations exist for these disparate findings. In certain tissuesthere may be additional regulation at the translational level ofthe specific types of APP mRNA, resulting in abundantmRNA levels and little corresponding protein. This type ofregulation has been shown with ferritin mRNA (32, 33).Alternatively, KPI- APP protein may be turned over muchmore rapidly in brain than forms of the protein that containthe KPI domain. Different rates ofturnover have been shownfor different forms ofthe protooncogene product p53 (34, 35).It is noteworthy that forms ofAPP protein that lack the KPIdomain may be more susceptible to proteolysis, leading to amore rapid turnover. In AD, altered proteolysis ofboth KPI-and KPI' APP proteins could contribute to the formation anddeposition of the amyloid a-protein. Therefore, changes inthese APP protein levels in the microenvironment of certainregions in AD brain may be involved in the pathology of thatdisease.

Note Added in Proof. We have extended our functional analyses toAPP proteins that were purified from normal and Alzheimer cere-brospinal fluid and found that the predominant form of APP incerebrospinal fluid is KPI-, contrary to what is observed in braintissue.

We thank Dr. Steve Younkin for the anti-KPI domain antiserumand Raymond Chung for technical assistance. This work was sup-ported by National Institutes of Health Grants to W.E.V.N.(AG00538), D.D.C. (AG00538 and GM31609), and C.W.C.(AG00538), and an Alzheimer's Association grant to D.G. (IIRG-90-193). S.L.W. was supported by a postdoctoral fellowship from theGeorge E. Hewitt Foundation for Medical Research. This work wasalso supported by a Pew Charitable Trust Grant.

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