The EMBO Journal vol.9 no.7 pp.2079 - 2084, 1990

Identification and characterization of C-terminal fragments of the f-amyloid precursor produced in cell culture

David Wolf', Diana Quon, Yu Wang and Barbara Cordell2 California Biotechnology Inc., 2450 Bayshore Parkway, Mountain View, CA 94043, USA 'Present address: Cor Therapeutics, Inc., 256 E. Grand Avenue, South San Francisco, CA 94080, USA. 2To whom correspondence should be addressed. Communicated by K.Beyreuther

The mechanism of amyloid formation in Alzheimer's disease is unknown but appears to involve proteolytic processing of the amyloidogenic peptide from a larger precursor. When the C-terminus containing the amyloidforming and cytoplasmic domains of the precursor was recombinantly expressed in cultured mammalian cells, a 16 kd protein accumulated which had a tendency to aggregate and form deposits. These deposits had physical characteristics resembling those of preamyloid. Recombinant expression of the full-length precursor was found to produce a similar, cell-associated 16 kd C-terminal fragment as well as a 12 kd fragment, neither of which formed detectable aggregates suggesting efficient catabolism of these fragments. Identification of these two naturally occurring metabolic products of the ,B-amyloid precursor provides a system permitting the characterization of the proteolytic processing events of the amyloid precursor protein.

Introduction Alzheimer's disease (AD) is a progressive dementia involving loss of memory and cognitive functions. Accompanying these symptoms is a distinct pathological lesion, the formation of extracellular amyloid deposits. These amyloid deposits are also seen in older individuals with Down's syndrome (DS) who invariably become afflicted with AD (Katzman, 1986). The 4.2 -4.5 kd amyloid protein subunit associated with AD and DS plaques and meningeal vessels, termed or A4-protein, has been isolated and characterized (Glenner and Wong, 1984; Wong et al., 1985; Masters et al., 1985). Further understanding of the -4 kd fl-protein has come from analyses of its encoding cDNAs. Results from these studies indicate that the fi-amyloid protein is contained within a larger, post-translationally modified precursor which has alternative molecular weight forms of 695, 751, and 770 amino acids (Kang et al., 1987; Ponte et al., 1988; Tanzi et al., 1988; Kitaguchi et al., 1988). The deduced amino acid sequence of the fi-amyloid precursor protein (fl-APP) displays features predictive of a membrane-associated molecule although a soluble form of the precursor has been identified which appears to be derived from the membraneassociated form (Palmert et al., 1989; Schubert et al., 1989; Weidemann et al., 1989). A proteolytic cleavage near the f-

(©) Oxford University Press

C-terminus has been proposed as a mechanism for the release of soluble (3-APP. This postulate is based on several observations. Only membrane-associated fl-APP, but not soluble precursor, is recognized by antibodies directed against the C-terminal domain of the precursor (Weidemann et al., 1989; Selkoe et al., 1988). The molecular weight of the protein component from both native and recombinant derived soluble precursor is 17-18 kd lower than its fulllength theoretical coding value (Weidemann et al., 1989). A C-terminal fragment of - 11 kd has been observed in human brain by Western blot analysis using antibodies raised to this domain (Selkoe et al., 1988). The putatiye cleavage of the precursor liberating a Cterminal fragment is highly relevant since proteolytic cleavage events are required to release the amyloidogenic f-peptide from its precursor. Because the fl-protein is located in close proximity to the membrane surface, cleavage to solubilize the precursor may generate one terminus of the amyloid domain. A second cleavage is then needed to produce the -4 kd amyloid subunit. In vitro transcription-translation of the precursor's C-terminus containing the ,B-peptide and adjacent cytoplasmic domain produces a protein with a high tendency to self-aggregate, an important characteristic of amyloid forming proteins (Dyrks et al., 1988). In an intact mammalian cell, however, the fate of such a C-terminal protein fragment is unknown. We are interested in whether a C-terminal fragment of fAPP containing the ,B-peptide displays characteristics of amyloid formation, i.e., self-polymerization, stable deposition and fl-pleated sheet conformation when expressed in a cellular milieu. Moreover, we sought to identify and characterize the putative C-terminus liberated during the formation of the soluble form of the precursor.

Results Preparation of ,3-APP recombinant vaccinia viruses A recombinant vaccinia virus has several features which make it a suitable expression system, such as, high level expression, a wide host range (permitting rapid investigation of expression in many different host cell types) and the ability to control expression levels simply by varying the infectious dose. A recombinant vaccinia virus carrying the C-terminal 99 amino acids of fl-APP was constructed using a fl-APP75 1 cDNA that we isolated previously (Ponte et al., 1988). A cDNA was assembled which encodes 99 amino acids of the C-terminus of the precursor, the first amino acid of which corresponds to the N-terminal residue of the f-peptide. This cDNA was inserted adjacent to the P11 late protein promoter of the vaccinia virus recombination vector, pUV 1. The resultant construct employs the initiator methionine of the P11 viral protein, plus 2 additional adjacent amino acids, joined in-frame to the 99 residues of the fl-APP C-terminus. This construct was used to generate a recombinant vaccinia virus, referred to as VV:A99 (Figure 1).

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Fig. 1. Schematic representation of f-APP recombinant vaccinia viruses' coding sequences and location of antisera epitopes. The epitopes are marked by short horizontal lines. Other features of f-APP are indicated. A4i refers to the Kunitz proteinase inhibitor domain within ,3-APP751. The open box designates the highly anionic region of the precursor; the stippled box, a cysteine-rich domain; the N-terminal black box, the signal peptide; the C-terminal black box, the amyloid subunit; and the branch-like structures, probable N-linked glycosylation sites.

Recombinant vaccinia viruses were also engineered to express the 695 and 751 amino acid forms of fl-APP, which we refer to as VV:695 and VV:75 1, respectively. Specifically, a cDNA fragment containing the entire coding region, 8 bp of 5'-untranslated region and 290 bp of 3'-untranslated region was inserted into a vaccinia virus recombination vector and used to make VV:751 virus. Preparation of VV:695 was identical to that for VV:751 except that the 3APP751 cDNA was modified to delete the 168 bp Kunitz proteinase inhibitor domain prior to cloning into the pSC 11 vaccinia virus recombination vector. Both full-length f-APP constructs utilize the vaccinia 7.5 kd late protein promoter. Protein expression by /3-APP recombinant vaccinia viruses The fl-APP recombinant vaccinia viruses were each characterized for production of their predicted encoded protein after infection of CV-1 monkey fibroblasts grown in culture. Cells infected with VV:99 or control vaccinia virus (VV:CON) were labeled with [35S]methionine for 3 h after which cell lysates and conditioned media were prepared. The samples were then subjected to immunoprecipitation with polyclonal sera raised against different synthetic peptide epitopes within the C-terminal domain of fl-APP. The location of the synthetic peptide immunogens in fl-APP is shown in Figure 1. One antiserum was directed to an epitope in the ,B-peptide (CORE) and the other two (B3 and C5) to epitopes within the intracellular cytoplasmic domain. From cell lysates infected with VV:99, one protein of 16 kd was immunoprecipitated by both B3 and C5 antisera (Figure 2A). No proteins were immunoprecipitated from the VV:99 -

conditioned medium (Figure 2A). Based on our previous experience with recombinant vaccinia viruses, we anticipated that maximal f-APP protein expression would occur late in the infection cycle (18-24 h). In Figure 2B, continuously labeled [35S]methionine cell lysates from VV:99 and VV:CON infections were harvested early (3 h) and late (18 h). The 16 kd protein was observed to accumulate during the course of VV:99 infection. B3 serum proved to be the most efficient of the three f-APP antibodies for immunoprecipitation of the 16 kd protein; C5 serum was less efficient and CORE serum was the least reactive (Figure 2C). The expression of the 695 and 751 amino acid f-APPs encoded by VV:695 and VV:751, respectively, was 2080

examined in a manner identical to that employed for VV:99. CV- I cell cultures were infected with each virus and labeled with [35S]methionine. Lysates and conditioned media were harvested at 18 h, immunoprecipitated, then analyzed by electrophoresis on polyacrylamide gels. VV:751 lysates contained two proteins of 115 and 140 kd which were specifically precipitated with B3 immune serum but not with control serum (Figure 3A). Analysis of VV:695 lysates gave a similar result except that the expressed proteins were smaller, migrating at 107 and 120 kd (Figure 3B). These fl-APP species were nearly identical in size to those reported by Weidemann et al. (1989) who determined that posttranslational modifications accounted for the molecular weight differences among species of each f-APP. fl-APP protein in conditioned medium from both VV:695 and VV:751 infections could not be immunoprecipitated with any of the three C-terminal antisera. However, soluble forms of fl-APP were found to be abundant in the media. When a polyacrylamide gel of total protein from conditioned media was stained with Coomassie blue, a 120 kd protein from VV:751 infection and a protein of slightly lower molecular weight from VV:695 infection were present at higher levels than in the VV:CON sample (which also contained endogeneous CV-1 fl-APP) (Figure 4). In the case of VV:751, N-terminal sequence analysis of the - 120 kd band verified that it was f-APP. A single homogeneous sequence (LEVPTDGNA) was obtained and was the expected amino terminal sequence for fl-APP lacking its 17 amino acid signal peptide. Because the conditioned media contained fl-APP which did not react with antibodies directed to the fi-peptide or cytoplasmic domain, we analyzed the VV:695 and VV:751 cell lysates for a C-terminal fragment(s). Unlike VV:99 lysates in which the C-terminal protein was readily detected, minor amounts of a 16 kd protein were immunoprecipitated with B3 antiserum from VV:751 and VV:695 lysates (Figure 3C). In addition, these lysates contained an 12 kd protein which was specifically recognized by the f-APP immune serum but not by control sera. CORE antiserum did not immunoprecipitate the 12 or 16 kd proteins. Either this serum is too weakly reactive to allow detection of these rare fragments, or the corresponding epitopes to this portion of the fi-peptide are missing. VV:CON lysates did not contain detectable levels of either the 12 or the 16 kd proteins by this method of analysis. -

(3-Amyloid precursor C-terminal fragments

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Fig. 2. Panel A: 17.5% SDS-PAGE analysis of continuously [35S]methionine labeled VV:99 C-terminal protein in conditioned media and cell lysates prepared 3 h after infection. Lane 1, C5 antiserum reacted with VV:99 conditioned medium; lane 2, B3 antiserum reacted with VV:99 conditioned medium; lane 3, C5 antiserum reacted with VV:99 cell lysate; lane 4, B3 antiserum reacted with VV:99 cell lysate; lane 5, C5 antiserum reacted with VV:CON conditioned medium; lane 6, B3 antiserum reacted with VV:CON conditioned medium; lane 7, C5 antiserum reacted with VV:CON cell I sate; lane 8, B3 antiserum reacted with VV:CON cell lysate. Internal molecular weight standards are indicated. Panel B: 17.5% SDS-PAGE of [5S]rmethionine labeled VV:99 protein produced early and late during infection. Lanes 1 and 2, W:CON 3 h lysates reacted with nonimmune and B3 sera, respectively; lanes 3 and 4, VV:CON 18 h lysates reacted with nonimmune and B3 sera, respectively; lanes 5 and 6, VV:99 3 h lysates reacted with nonimmune and B3 sera, respectively; lanes 7 and 8, VV:99 18 h lysates reacted with nonimmune and B3 sera, respectively. Panel C: 17.5% SDS-PAGE of [35S]methionine labeled VV:99 protein harvested 18 h after infection and reacted with (3-APP antisera. Lane 1, normal rabbit serum; lane 2, C5 antiserum; lane 3, B3 antiserum; lane 4, CORE antiserum. Arrowheads denote the C-terminal 16 kd protein and its aggregated higher molecular weight forms.

Immunofluorescent analysis of viral infected cells Expression of VV:99, VV:695, and VV:751, and of the control virus, VV:CON, was investigated by indirect immunofluorescent staining using B3, C5 and CORE antisera. Major morphological changes in all vaccinia infected cultures were apparent due to the cytopathic effects of this lytic virus. However, at the time selected for harvesting the cultures (18 h), cell lysis was minimal. Cultures of virus-infected CV-1 cells were stained with B3 antiserum then with rhodamine-conjugated anti-rabbit IgG. Faint punctate staining was observed with both the mock and VV:CON cultures (Figure 5A and B). The VV:751infected CV-1 cells displayed intense staining uniformly distributed over the cell (Figure 5C). Comparable staining was found with VV:695 (not shown). In contrast to VV:751 and VV:695 staining, the VV:99-infected cultures revealed highly focalized staining of dense deposit-like intracellular structures (Figure 5D). These deposits were irregular in shape and varied in size from - 2-8 ptm. Deposits of a diffuse, granular quality were also seen. C5 or CORE antisera produced identical staining patterns for all cultures and, like B3, staining of the deposit structures could be eliminated by preincubating each fl-APP antiserum with its cognate synthetic peptide (Figure 6A, B and C). Few, small densely staining foci were noticable in the VV:751 and VV:695-infected cultures which might have derived from the C-terminal fragments described in Figure 3C. The human neuronal cell line, SK-N-MC, was also tested and depositlike structures were produced only from VV:99 infection. Figure 6D illustrates these results. Last, we invesigated whether the deposits generated by VV:99 infection could be stained with the classical amyloid dyes such as Congo red, thioflavine S, and silver salts. We could not detect staining of these structures with any of the dyes used.

Fig.

3.

Panel

A:

7%

SDS-PAGE

VV:695 18 h infected cell

prior

to

harvesting.

lysates

lysate

3-APP

Lanes 1 and 2, VV:751

nonimmune and B3 antisera,

VV:695

of

from

labeled with

VV:751

lysate

respectively. Panel

nonimmune sera,

respectively;

with B3 and nonimmune sera,

lysate

B: Lanes 3 and 4,

respectively.

reacted with B3 and

lanes 7 and 8, VV:695

respectively.

16 kd and 12 kd (3-APP C-terminal

for 4 h

reacted with

reacted with nonimmune and B3 sera,

Panel C: Lanes 5 and 6, VV:751

and

[35S]methionine

lysate reacted identify the

Arrowheads

proteins.

Discussion

knowledge of the amyloid-forming peptide deposits in the brains of AD and DS individuals indicates that proteolytic cleavage events are necessary to release the fi-peptide from its larger membrane-anchored precursor. Several groups have reported the presence of soluble forms of fl-APP which lack C-terminal epitopes and 17 kd. which are reduced in molecular weight by Weidemann et al.(1989) and Palmert et al. (1989) both Our current

which forms

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kD

Mr

2

200 68-UUbt 42

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Fig.

4. 7% SDS- PAGE of soluble

13-APP present in conditioned

media from VV:695 and VV:751 infections harvested at 18 h and

stained with Coomassie blue. Lane 1,

VVCON; lane 2, VV:751,

lane 3, VV:695 media. The arrowhead identifies

13-APP.

11I

I

Fig. 6. Rhodamine-labeled photomicrographs of VV:99 infected cultures stained with ,3-APP antisera. (A) CV-l cells stained with C5 antiserum; (B) CV-1 cells stained with C5 antiserum preincubated with C5 synthetic peptide; (C) CV-1 cells stained with CORE antiserum; (D) SK-N-MC cells stained with CORE antiserum. Inset shows 3.1-3.4 Am diameter beads. All photographs are at 165x magnification.

LL Fig.5. Rhodamine-labeled photomicrographs of CV-1 cultures harvested 18 h post infection stained with B3 serum; (A) mock infected; (B) VV:CON infected; (C) VV:751 infected; (D) VV:99 infected. Magnification is 165 x.

describe such soluble precursor proteins in human cerebrospinal fluid; the latter group also describes these forms in human brain. Schubert et al. (1988), Weidemann et al. (1989) and now we describe the existence of a secreted, 2082

soluble form of f-APP recombinantly produced in cultured cells. In addition, the data from this study indicate that a single primary cleavage occurs within fl-APP near the cell surface to liberate a 16 kd C-terminal fragment. The 16 kd C-terminus might be further processed to a 12 kd fragment since this smaller protein was also produced. The possibility of two-step C-terminal processing has important ramifications with regard to the generation of the 4 kd amyloidogenic potein. Alternatively, there may exist two different proteolytic events leading to soluble fl-APP resulting in either a 16 kd or 12 kd by-product. Irrespective of which scenario is operative, it is conceivable that either site of cleavage may differ in the normal and pathological states creating either a benign or an amyloidogenic product. The amino acid sequence of these two fragments, their possible interrelationship and relationship to the generation of the fpeptide are currently being investigated. It is relevant that Selkoe et al. (1988) have observed an 11 kd fragment from the C-terminal domain of lO-APP in human brain homogenates. It seems unlikely that the 11 kd fragment in -

,B-Amyloid precursor C-terminal fragments

human brain contains the entire (-peptide sequence since the antibodies used for detection were directed to the terminal residues of (-APP and the C-terminus containing the (3peptide has an apparent mol wt. of 16 kd. These data suggest that a C-terminal fragment of ,3-APP is naturally produced which may, under certain conditions, be potentially amyloidogenic. In fact, it has been shown by Dyrks et al. (1988) that when an artifical C-terminal fragment harboring the ,B-peptide domain is synthesized in vitro by a rabbit reticulocyte cell-free system, a protein was produced which self-aggregates; this is one of the hallmarks of amyloid. Whether this C-terminal protein domain is stable and has the capacity for aggregation in vivo, in addition to displaying other features of amyloid (i.e., deposit formation with a 1-pleated sheet configuration capable of staining with amyloid-specific dyes) was investigated in this study. We find that when the C-terminal 99 amino acids of (3-APP are recombinantly expressed, a 16 kd protein is produced which is not readily degraded and which has a tendency to aggregate. The 16 kd recombinant protein has an anomalous behavior on SDS gels, appearing larger than the predicted 11 kd protein encoded by the cDNA fragment. This apparent mol wt. of 16 kd was also observed for the C-terminus produced in cell free systems (Dyrks et al., 1988). These results differ from those of Yanker et al. (1989) who expressed an analogous C-terminal (3-APP region in PC-12 cells and did not see a 16 kd protein. Rather, they report a high molecular weight (> 110 kd) smear of protein. The discrepancy between these data is unclear. We find that the C-terminal 99 amino acid protein of (3APP also accumulates into deposit-like structures. Marotta et al. (1989) have described punctate immunostaining from the recombinant expression of a much larger C-terminal fragment of the (3-APP cDNA but did not characterize the protein produced or the structures stained. In our study, these structures were readily stained with antisera directed against the ,B-peptide and the cytoplasmic domain, indicating that both regions are included in these structures. Other characteristics of the deposit-like structures include irregular shape of either granular or highly compact appearance, random location within the cell, absence of classical amyloid staining properties, and a size of 2-8 Izm. These are all characteristics described for preamyloid deposits found in abundance in the brains of Alzheimer's (Davies et al., 1988; Yamaguchi et al., 1988; Tagliavini et al., 1988; Ikeda et al., 1989a; Ishii et al., 1989) and aged Down's individuals (Ikeda et al., 1989b; Giaccone et al., 1989). It is noteworthy that preamyloid has also been described using an antiserum to an epitope located in the cytoplasmic domain of (-APP (Ishii et al., 1989). One important difference exists between preamyloid and the structures that we have observed. The deposits produced recombinantly are intracellular, whereas preamyloid is extracellular. Attaching either the homologous -

-

-

(-APP or a heterologous (human growth hormone) secretory

signal peptide sequence

to the C-terminal constructs also resulted in intracellular accumulation of the 16 kd protein, albeit at a much reduced level. In contrast to preamyloid-like formation when only the C-terminus of 1-APP is expressed, high level expression of the full-length precursor itself does not produce extensive deposits even though a similar (or perhaps indentical) 16-kd cell-associated C-terminal fragment is generated. Few, very

small, intensely staining foci were noted in the full-length (-APP expressing cells. This raises the possibility that plaque formation might reflect increased levels of (-APP expression and, in turn, unmanageable excesses of a C-terminal, amyloidogenic fragment. It is also possible that catabolism of the C-terminal fragment is aberrant in the pathological condition. While we have not made quantitative measurements of the recombinant proteins, the relative abundance of the precursor-derived fragments compared to the artifically generated C-terminus appears substantially lower. Thus, it is possible that we have not reached a threshold level of the natural precursor fragment necessary to initiate formation of preamyloid-like deposits. The intracellular pathways for maturation of nascent full-length precursor and artificial C-terminal fragment probably differ, leaving each C-tenninal fragment in potentially very different subcellular compartments. The final localization of each fragment within the cell would clearly influence its catabolism. Therefore, it seems probable that the naturally cleaved C-terminus from (3-APP is turned over more rapidly than the artifical C-terminal fragment. Lastly, it is important to consider that the naturally produced C-terminal fragment may lack part (in the case of the 16 kd fragment) or all (in the case of the 12 kd fragment) of the (3-peptide domain and hence such fragments would not be expected to aggregate efficiently and form preamyloid-like structures. Further characterization of these natural C-terminal proteins is critical to our understanding of amyloidogenesis and is in progress.

Materials and methods Preparation of recombinant vaccinia viruses Two vaccinia virus insertion vectors, pSC1 1 (Chakrabarti et al., 1985) and pUVI (Falkner et al., 1987) were used to generate the (3-APP-vaccinia recombinants. VV:99 recombination plasmid was constructed using the ,B-APP cDNA isolated by Ponte et al. (1988). A 590 bp DdeI-PvuJI (Positions 2101-2511) fragment was isolated from the C-terminal 1 kb EcoRI fragment, ligated with a 27 bp EcoRI-DdeI adaptor sequence and cloned into the EcoRI-SmaI digested pUVI. The 27 bp adaptor altered the 3-peptide amyloid domain such that the first amino acid of the (3-APP sequence corresponded to the aspartate at position 653, i.e. the first amino acid of the ,B-peptide. The initiator methionine was supplied by the vaccinia p 1 coding sequence. Two additional vaccinia amino acids, asparagine and serine, follow the initiator methionine prior to the ,B-APP codons. The fidelity of the construction was determined by DNA sequence analysis. VV:751 recombination plasmid was generated using a SmaI -XnnI (positions 133 -2665) fragment of the original (3-APP cDNA (Ponte et al., 1988). This fragment, containing 8 bp of 5'-untranslated region and 290 bp of 3'-untranslated region, was inserted into the pSC 11 vaccinia vector at the SmiaI site using blunt-end ligation. The original ,B-APP751 isolate was used to reconstruct a ,B-APP6595 cDNA by deletion mutagenesis using restriction endonuclease digestion and synthetic oligonucleotides according to standard methodologies. The accuracy of the reconstruction was confirmed by DNA sequence analysis. The SmnaI-XinnI (positions 133-2497) fragment of the (3-APP695 cDNA was cloned into the SmaI site of pSC1 1 creating the VV:695 recombination vector. The three recombination plasmids were used to introduce the recombinant gene into the vaccinia virus genome using methods described by Cochran et al. (1985) and Chakrabarti et al., (1985). VV:99, VV:751, and VV:695 virus isolates were each plaque-purified several times, amplified into stocks, and tested for the presence of inserted (-APP sequences by Southern blot analysis (Southern, 1975). The control virus, VV:CON, is a recombinant virus prepared using a pUVI recombination plasmid lacking foreign sequences.

Preparation of antibodies Peptides were synthesized with an Applied Biosystems model 430A automated peptide solid phase synthesizer. Purification of crude peptides was

accomplished

with

gel

filtration followed

by ion-exchange

and

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D.Wolf et al.

preparative reverse-phase liquid chromatography. Each peptide was fully characterized by amino acid composition and sequence analysis. The CORE peptide corresponds to amino acids 653 -680 (DAEFRHDSGYEVHHQKLVFFAEDVGSNK), the B3 peptide to amino acids 705 -719 (KKKQYTSIHHGVVEV) and C5 to amino acids 729-742 (HLSKMQQNGYENPT) of /3-APP751 (Ponte et al., 1988). New Zealand white rabbits were immunized intradermally with 500 yg of peptide conjugated to keyhole limpet hemocyanin in Freund's complete adjuvant. Booster injections were made with 250 /Ag of conjugated peptide in incomplete Freund's. Antibody titers against the appropriate peptide were determined by ELISA analysis. Titers of 7.4 x I04, 9.1 x 10 , and 2.5 x 105 were obtained for CORE, B3 and C5, respectively.

Immunoprecipitation analysis CV-l cells infected with the recombinant vaccinia viruses were labeled with

250,uCi/ml [35S]methionine (New England Nuclear, Boston, MA)

at

various times after infection. Cell layers were washed with phosphate buffered saline (PBS) and extracted with lysis buffer (150 mM NaCl, 10 mM NaH2PO4, 1 mM EDTA, 1% Triton X-100, 0.5% SDS, 0.5% Na deoxycholate, 2 mM phenylmethylsulfonyl fluoride (PMSF) and 5 Ag/ml leupeptin). Conditioned medium was harvested, treated with 2 mM PMSF, concentrated 10-fold by precipitation with 10% trichloroacetic acid (TCA) and resuspended in lysis buffer. All samples were clarified by centrifugation at 10 000 x g and the supematants were precleared with protein A -Sepharose. Antiserum (2 ul) was added to an aliquot of each cell extract containing 107 cpm or to an aliquot of medium containing 105 cpm followed by incubation of the sample at 4°C for 18 h then for 1 h with protein A-sepharose (50 Mi). Immune complexes were washed several times in 500 mM NacI, 50 mM Tris-HCl pH 7.5, 5 mM EDTA, 0.5% NP-40, then in the same buffer but with NaCl at 150 mM, and once in 10 mM Tris-HCI pH 7.5 before solubilizing in SDS electrophoresis sample buffer

(Laemmli, 1970). Indirect immunofluorescence and amyloid staining CV-1 and SK-N-MC cells were obtained from the American Type Culture Collection (Rockville, MD). Viral infections were carried out in Earles minimal serum-free media with cells plated on microscope slides divided into 4 chambers (Lab-Tek, Miles Scientific, Naperville, IL) with 5 x I04 cells per chamber. At 18 h post infection, medium was removed, monolayers washed with PBS and the chamber housing removed. Slides were fixed with 4% paraformaldehyde (Fluka, Buchs, Switzerland) for 10 min, washed 5 times with PBS, then permeabilized with 0.2% Triton X-100 for 5 min and washed again with PBS. The primary antisera were diluted 1:200 with PBS with 0.2% gelatin (PBS-gelatin) and incubated with the slides for 30 min at 37°C. After washing 5 times with PBS-gelatin, slides were inbubated with a 1:200 dilution of rhodamine-conjugated goat anti-rabbit IgG (Cappel, West Chester, PA) made in PBS-gelatin and incubated at 37°C for 20 min. Slides were washed 5 times with PBS-gelatin before mounting with Cytoseal 60 (Stephens Scientific, Denville, NJ). Peptide blocking experiments were conducted by incubating 50 /Ag of synthetic peptide with the cognate diluted serum sample at 37 °C for 1 h prior to application to the slide. Stained cells were examined and photographed using an Olympus BH-2 microscope equipped for fluorescent microscopy. Fluorescent photomicrographs were shot with Kodak Ectachrome film at an ASA of 1600 and at an equivalent exposure time of 40 s for all samples. Size estimates were made by photographing 3.1 -3.4 Mim diameter polystyrene beads (Pandex, Mundelein, IL) at an identical magnification to that used for cell samples. Slides fixed and permeabilized as described above were stained with Congo red Accustain kit (Sigma Diagnostics, St. Louis, MO) or with 1% thioflavine S in 70% ethanol for 30 min or with silver nitrate salts for 7 min.

Amino-terminal sequence analysis ,3-APP present in conditioned media of VV:751 infected CV-1 cell was concentrated by precipitation with 10% TCA. Protein samples were electrophoresed on a 7% polyacrylamide gel then stained with Coomassie blue. Total protein was transferred by electroblotting onto a polyvinylenedifluoride membrane, the 1-APP band was excised and subjected to Edman degradation using an Applied Biosystems A77A protein sequencer (Foster City, CA) with an online Applied Biosystems model 120A

phenylthiodantoin analyzer.

Acknowledgements We would like to thank Line Hawes for support with tissue culture, Ken Lau for protein sequence analysis, Sharon Kaye Spratt for construction of VV:751, Eric Stoelting for art work, and all the Cal Bio research facility

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groups. This work was supported through a commercial collaboration with Daiichi Pharmaceutical Co., Tokyo, Japan.

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Identification and characterization of C-terminal fragments of the beta-amyloid precursor produced in cell culture.

The mechanism of amyloid formation in Alzheimer's disease is unknown but appears to involve proteolytic processing of the amyloidogenic peptide from a...
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