secretion of &amyloid precursor protein involves multiple cleavage

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 1, Issue of January 7, pp. 627-632, 1994 Printed in U.S.A.

Secretion of &Amyloid Precursor Protein Involves Multiple Cleavage Sites*

(Received for publication, July 7, 1993, and in revised form, August 9, 1993)

Ziyang Zhong, Jeffrey Higaki, Kenji MurakamiS, Yu Wang, Rosanne Catalano, Diana Quon, and Barbara Cordells From Scios Nova Inc., Mountain View, California 94043

A major histopathological feature of Alzheimer's disease is deposits of a -4-kDa &amyloid peptide derived by proteolytic processing from a precursor, the B-amyloid precursor protein (B-APP). Proteolytic cleavage of &APP within the -4-kDa &amyloid domain permits the secretion of the amino-terminal portion of @-APP while concomitantly producing a membrane bound -9- kDa carboxyl-terminal fragment. We have characterized the proteolytic cleavage site for D-APP secretion by amino acid sequence analysis of the -9-kDa B-APP carboxyl-terminal cleavage product produced by recombinant and native expression systems. Recombi- nant 8-APP was generated by a vaccinia virus expression system in CV-1 monkey fibroblasts; endogenous &APP was obtained using a fibroblast line derived from an individual with Down's syndrome. The sequences of both unlabeled and metabolically radiolabeled -9-kDa fragment from CV-1 cells reveal that the major (60%) secretory cleavage site is after Lys" of the @-amyloid domain as reported previously; however, an additional cleavage site is seen after Phe" (40%). Radiosequence analysis of the carboxyl-terminal fragment purified from Down's syndrome fibroblasts indicates cleavage sites after Phe", G1uZ2, and GlyZK and not after Lys". CV-1 cells expressing B-APP mutants lacking 4 and 6 amino acids adjacent to Lys" yielded -9-kDa fragments with two identical cleavage sites, neither of which occurred after the retained Lys" but were after Glu" and Hid3. These data suggest that secretion of &APP involves multiple proteinases and that the composition of these proteinases may vary within different cell backgrounds.

The deposition of @-amyloid in the brain parenchyma and cerebral vasculature is a histopathological characteristic of Alzheimer's disease. @-Amyloid is a 39-42-amino acid peptide that results from proteolytic processing of a larger precursor, the P-amyloid precursor protein (p-APP).' Three major isoforms of B-APP encoding proteins of 695,751, and 770 amino acids have been described (1-4). p-APP isoforms are highly modified membrane glycoproteins with a single transmem-

* This work was supported by Marion Merrell Dow, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduer- tisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Daiichi Pharmaceutical Co., 16-13 Kita-Kasai 1-Chome, Edogawa-ku, Tokyo Japan.

To whom all correspondence should be addressed Scios Nova Inc., 2450 Bayshore Parkway, Mountain View, CA 94043.

The abbreviations used are: p-APP, P-amyloid precursor protein; DS, Down's syndrome; VV, vaccinia virus; Tricine, N-[P-hyroxy-l,l- bis(hydroxymethyl)ethyl]glycine.

brane spanning domain followed by a short cytoplasmic tail (1,5-7). All /3-APP isoforms are proteolytically cleaved within the @-amyloid sequence to generate a large soluble amino- terminal derivative of p-APP which is secreted and a membrane-bound carboxyl-terminal fragment of -9 kDa (8-10). The secretion of soluble ,8-APP, extensively documented in vitro, appears also to occur in vivo based on the presence of carboxyl-terminal truncated ,6-APP in cerebrospinal fluid (6, 11) and serum? It has been determined that a constitutive secretory pathway produces soluble P-APP (12) and that proteolytic cleavage appears to occur only with fully glycosy- lated precursor protein (13), thereby localizing cleavage to the trans-Golgi, to a secretory vesicle, or to the cell membrane surface. Evidence for both intracellular cleavage and membrane surface cleavage has been reported (13-15). Esch et al. (8) and Sisodia et al. (9) were the first to demonstrate that the cleavage releasing soluble P-APP occurs within the p- amyloid peptide domain. In the report by Esch et al., the cleavage was found to occur after Lys" of the p-amyloid sequence. Since these original observations, the secretory cleavage site of @-APP has been studied in a variety of mammalian cultured cell systems (15-19), in cultured insect cells (20, 21), and in Alzheimer's disease brain (22).

Several different approaches have been employed to define the /3-APP secretory cleavage event. These approaches included characterization of the carboxyl terminus of secreted 8-APP (8, 18-20, 22), amino-terminal analysis of the -9-kDa fragment generated upon cleavage (8,17,21), and site-specific mutagenesis of the secretory cleavage site (15-17). In the direct biochemical studies with isolated protein, only the previously identified Lys" cleavage site was observed. In experiments in which p-APP was mutated at and around this cleavage site, @-APP secretion was seen with all mutants. Although the cleavage site in the mutants was not specifically defined (except for one mutant), it has been concluded that the cleaving proteinase, referred to as P-APP secretase, lacks residue or sequence specificity but instead cleaves at a fixed distance from the membrane (15-17). The primary goal of this study was to address the apparent lack of sequence specificity by P-APP secretase. In this report, we examine the cleavage event in greater biochemical detail and show that multiple cleavage sites are used for both native and recombinant P-APP751 secretion, indicating that a diverse set of proteinases may be responsible for the secretion of this mol- ecule.

EXPERIMENTAL PROCEDURES

Cell Culture, Drug Treatment, Radiolabeling, and Virus Infection- CV-1 cells, an African monkey kidney fibroblast line, were obtained from the American Type Culture Collection (Rockville, MD) as were

D. Quon and B. Cordell, unpublished observations.

627

628 P-APP Secretase Cleavage Sites the COS-7 cells. DS fibroblasts, a line derived by skin biopsy from an individual documented to be trisomic for chromosome 21, were obtained from the Human Genetic Mutant Cell Repository Coriell Institute for Medical Research (Camden, NJ) under the repository number GM02504G. Cells were propagated as suggested by the pro- viding agencies. Briefly, cell culture conditions were as follows: CV-I in Eagle's minimum essential medium with 10% fetal bovine serum, COS-7 cells in Dulbecco's modSed Eagle's medium-21 with 10% fetal bovine serum, and DS fibroblasts in Eagle's minimun essential medium with 20% fetal bovine serum. Infections with 8-APP recombinant vaccinia viruses were carried out as described previously (23) in which each titred virus stock was used to infect washed CV-1 cell monolayers at a multiplicity of infection of 0.1 in Eagle's minimum essential medium containing 0.2% fetal bovine serum. Following a 2- h adsorption period, monolayers were washed, and a 10-fold excess of serum-free medium was added for the remainder of the 16-h infection period. For experiments involving radiolabeling of 8-APP, cultures were washed then incubated for 30 min in serum-free medium lacking the amino acid(s) to be used in labeling, incubated with either 200 &i/ml [%]methionine/cysteine (ICN), 1 mCi/ml [3H]lysine, or [3H]isoleucine (Amersham Corp.) for 2 h. In experiments using chloroquine treatment, 100 pg/ml of drug was applied 30 min prior to the addition of radiolabel and was present during the entire labeling period.

Preparation of 8-APP Mutant Viruses-Mutants of P-APP695 were constructed using oligonucleotide-directed site-specific mutagenesis according to standard procedures. Briefly, an EcoRI-PuuII fragment encoding the carboxyl-terminal 103 amino acids of 8-APP was cloned into pBluescript I1 (KS-) vector (Strategene, La Jolla, CA). Single-stranded DNA was isolated after transfection of DNA into JMlOl bacteria with helper phage then used for mutagenesis. Oligonucleotides used to create Lys" replacement mutants were as follows.

LYS" to Leu: 5"AAAGAACACCAACAGTTGATGATGACC-3'. Lys" to Ala: 5'-AAAGAACACCAAGGCTl'GATGATGAAC-3'. Lys" to Gln: 5'-AAAGAACACCAACTGTl'GATGATGAAC-3'.

Lys" to Arg: 5'-AAAGAACACCAAGCGTTGATGATGAAC-3'. Oligonucleotides used to create deletion mutants were as follows. ALys'? 5'-CAAAGAACACCAATTGATGATGAACTT-3'. AGln16 Lys? 5'-CAAAGAACACCAAATGATGAACTTCATA-3'.

LYS" to Glu: 5'-AAAGAACACCAACTCTTGATGATGAAC-3'.

A H i ~ ' ~ - ~ s ' ' - G l n ~ ~ - L y s ~ ~ : 5'-TGCAAAGAACACCAAAACTTCA- TATCCTGA-3'. AGl~~~-Val'~-His'~-His"-Gln'~-Lys~~: 5"TGCAAAGAACACCA-

ALeu17: 5'-CTGCAAAGAACACTTTTTGATGATGAA-3'. A L ~ U ' ~ - V ~ ~ ' ~ : 5'-TCTTCTGCAAAGAATGATGATGAA-

ALeu'7-Val'S-Phe's-Phem (KM-3): 5'-CCACATCTTCTGCTTTT

ALeu"-Va11S-Phe'a-Phe20-Ala2'-Glu21 (KM-4): B'-TTTGAACCCA-

Double-stranded phage DNA, shown to be correct by DNA sequence analysis, was prepared for each mutant from which the EcoRI- PuuII plasmid was isolated then reinserted into the 8-APP695 cDNA carried on a pGEM-3 plasmid. An expression plasmid that harbors the human 8-actin promoter was engineered for each mutant and for wild-type B-APP695. Each mutant DNA was characterized for release of 8-APP into medium following transfection of COS-7 cells and Western blotting using an antiserum raised to the extracellular domain of B-APP695 (241, the predominating epitope of which maps near the amino terminus of 8-APP. Construction and isolation of recombinant vaccinia viruses harboring KM-3 and KM-4 deletion mutants were carried out as described for a vaccinia virus harboring wild type 8-APP751 (VV751).

Isolation of -9-kDa Carboxyl-terminal Secretase Fragment-Viral- infected CV-1 cells and uninfected DS fibroblasts were lysed and immunoprecipitated with an antiserum raised to the cytoplasmic domain of 8-APP (369Ab). Cell lysis was performed exactly according to the procedure of Gabuzda et al. (25). The resulting detergent layers containing the membrane fraction and -9-kDa fragment were used for immunoprecipitation with 369Ab. Protein A Sepharose (Phar- macia LKB Biotechnology Inc.) was used to recover the 369Ab--9- kDa protein complex. Immunoprecipitated protein was washed extensively then subjected to electrophoresis on a 16% SDS-Tris-Tricine- polyacrylamide gel. Protein in the gel was transferred by electro- blotting onto polyvinylene difluoride membrane, and autoradiography was used to locate the -9-kDa fragment. To localize samples labeled

AATATCCTGAGTCATG-3'.

3'.

TGATGATGAAC-3'.

CATCTTTTTGATGATGAAC-3'.

with tritium, a sample labeled with [36S]methionine/cysteine was electrophoresed in an adjacent lane on the gel. The region on the polyvinylene difluoride membrane containing the -9-kDa fragment was excised for amino-terminal amino acid sequence analysis.

Large Scale Preparation of -9-kDa fragment-For large scale production of -9-kDa fragment, -IO9 CV-1 cells grown in roller bottles (-5 X lo7 cells/bottle) were infected with VV:751, lysed, and the fragment partially purified by ion exchange chromatography. Cell lysates were diluted with an equal volume of 50 mM Tris-HC1, pH 7.5,150 mM NaCl, 1% sodium deoxycholate, 1% SDS) and 10 volumes of 10 mM Tris-HC1, pH 7.5. The mixture was loaded onto a 50-ml bed volume of DEAE Sepharose CL-GB (Pharmacia) equilibrated with 10 mM Tris-HC1, pH 7.5. After loading the sample, the column was washed with 10 mM Tris-HCI, pH 7.5, and then with 10 mM Tris- HCl, pH 7.5, 100 mM NaCl. The -9-kDa fragment was eluted with 10 mM Tris-HC1, pH 7.5, 200 mM NaCl. The column eluent was immunoprecipitated with 369Ab and protein A-Sepharose. Fragment isolation was carried out by SDS-polyacrylamide gel electrophoresis and polyvinylene difluoride membrane transfer as described above coupled with staining by Coomassie Blue dye.

Amino-terminal Sequence Analysk"olyviny1ene difluoride mem- branes containing -9-kDa protein were subjected to Edman degradation using an Applied Biosystems A77A protein sequenator (Foster City, CA) using the BLOTT-2 program according to the manufactur- er's recommendations. For unlabeled protein, amino acid analysis was achieved by an on-line Applied Biosystems model 120A phenyl- thiohydantoin derivative analyzer coupled to the sequenator. For radiolabeled protein, each degradation cycle was collected, mixed with scintillation fluid, and then counted in a Beckman LS 3801 scintillation counter.

RESULTS

To define the cleavage event allowing secretion of @-APP, we used an African monkey kidney fibroblastic cell line, CV- 1, and a fibroblast cell line established by skin biopsy from an individual with Down's syndrome (DS fibroblasts) as cellular systems for study and comparison. The DS fibroblast line was selected since all individuals with Down's syndrome develop Alzheimer's disease. The CV-1 cells were used to express the recombinant 751-amino acid isoform of P-APP (j3-APP751) using a vaccinia virus vector harboring the human cDNA (VV751). The vaccinia virus expression system produces large amounts of recombinant protein (-100-fold over endogenous levels) (23), thereby facilitating analysis of the /3-APP secretase cleavage site(s). The Down's fibroblasts predominantly express 8-APP751. P-APP from this cell line represents processed native protein.

Sequence of 8-APP Secretion Cleavage Site($-To charac- terize the secretion site of recombinant P-APP751 produced in CV-1 cells, the -9-kDa membrane-bound carboxyl-terminal product of secretase cleavage was studied. Metabolically radiolabeled -9-kDa fragment was obtained by infecting CV- 1 cells with VV751 in the presence of either [35S]methionine, [3H]lysine, or [3H]isoleucine. The fragment was purified from the infected cell lysates by immunoprecipitation with an antiserum directed to the cytoplasmic domain of P-APP (369Ab) followed by SDS-polyacrylamide gel electrophoresis. Fig. 1B illustrates the pattern of [Y3]methionine/cysteine- labeled P-APP carboxyl-terminal fragments present in the immunoprecipitate from the CV-1 cell lysate which is well above that from endogenous 8-APP (Fig. lA). Typically the -9-kDa fragment is the major carboxyl-terminal fragment of a consistent set of fragments reported by a number of investigators (10, 14, 17, 26). The carboxyl-terminal fragments larger than the -9-kDa fragment do not appear to be produced by secretase activity but by degradative processes (10, 14). Since the carboxyl-terminal half of (3-APP lacks cysteine residues, labeling of the -9-kDa fragment will be exclusively at methionine residues.

When the -9-kDa fragment was isolated and subjected to amino-terminal radiosequence analysis, 35S signals were found

p-APP Secretase Cleavage Sites 629 A B C D E M r

:--18 koa

L - 14

I "

' 6

FIG. 1. Immunoprecipitation of [86S]methionine-labeled -0-kDa and other B-APP carboxyl-terminal fragments separated by 16% SDS-Tris-Tricine-polyacrylamide gel electrophoresis. Lune A , pSCll control virus-infected CV-1 cell lysate; lane B, VV:751-infected CV-1 cell lysate; lane C, VV:751-infected CV-1 cell lysate treated with chloroquine; lane D, VV:KM-3-infected CV-1 cell lysate; lane E , VV:KM-4-infected CV-1 cell lysate; lane F, DS fibroblasts. The -9-kDa fragment characterized in each sample was the fastest migrating single protein band on the gel. The protein fragment migrating immediately above the -9-kDa carboxyl-terminal fragment in the KM-3 and KM-4 samples was not included in sequence analyses. For lanes A-E infection conditions and exposure periods were identical.

-1 16 Mtt

9

R 12

cycle

1 1 . - Y E V H H Q K " L V F " F A E D V C S N K C A I 1 CLM."

Sequencing cycle W: 1 I2 15 16 19 1 9 12 13 16

FIG. 2. Radiosequence analysis of -9-kDa carboxyl-terminal fragment isolated from VV:751-infected CV-1 cell lysates. Panel a, sequence of [35S]methionine-labeled fragment; panel b, sequence of [3H]lysine-labeled fragment; panel c, sequence of [3H] isoleucine-labeled fragment; panel d, partial sequence of 8-amyloid peptide illustrating secretase cleavage sites a t Lyd6 and Phelg (de- noted by arrows).

at cycles 16 and 19 (Fig. 2a). When the experiment was repeated with protein labeled with [3H]lysine or [3H]isoleucine, 3H radioactive signals were seen at cycles 9 and 12 from the lysine labeling (Fig. 2b) and at cycles 12, 13, 15, and 16 for isoleucine labeling (Fig. 2c). When these data are aligned with the known sequence of p-APP, a uniform fit for all radiolabeled amino acids indicates that the -9-kDa fragment

had two amino termini, one at Leu" of the @-amyloid peptide and the second at PheZ0 (Fig. 2 4 .

Direct amino-terminal sequence analysis was conducted on the /3-APP -9-kDa fragment to confirm the observed heterogeneity obtained by radiosequencing. Preparative amounts of -9-kDa fragment were generated from VV:751-infected CV-1 fibroblasts. The fragment was purified identically except for the inclusion of an ion exchange chromatographic step needed to remove bulk protein in the lysate prior to immunoprecipitation. For two independent preparations, direct amino-terminal microsequence of the -9-kDa fragment again revealed heterogeneity (Table I). Two sequences were obtained; one sequence initiates with Led7, the other with Phe2'. From the relative yields of amino acids at each cycle of degradation over four cycles, it can be deduced that the sequence resulting from cleavage after Lys" represents roughly 60% of the total -9-kDa fragment population. These direct amino-terminal results are in complete agreement with the cleavage sites determined by radiosequence analysis of the -9-kDa fragment. Together, the data confirm the presence of at least two secretase cleavage sites in 6-APP751 produced by recombinant means.

To address the possibility that the heterogeneity found with the -9-kDa fragment is a result of secretase cleavage and not a combination of secretase plus an intracellular degradation event, we determined the sequence of -9-kDa fragment produced in the presence of chloroquine, a drug that neutralizes acidic intracellular compartments such as lysosomes. It has been shown that secretion of @-APP is unaltered in the presence of chloroquine (26). CV-1 cells were infected with VV:751 during which protein was metabolically labeled with [35S]methionine/cysteine in the presence of 100 pg/ml chloroquine. As shown in Fig. lC, although chloroquine changed the abundance of some p-APP carboxyl-terminal fragments, the -9-kDa secretase product was present as the major spe- cies. When this -9-kDa fragment was sequenced, 35S radioactive signals were identical to that seen in the non-chloroquine-treated sample (Fig. 3).

Site-specific Mutugenesis of the Cleavage Domuin-To un- derstand better the apparent lack of sequence specificity of secretase, we constructed a set of mutants at and around Lyd6 of P-APP695 using site-direct mutagenesis (Table 11). Re- placement of Lys" in the @-amyloid domain by small hydrophobic residues (Ala or Leu), by acidic residues (Glu or Asp), or by an alternative basic reside (Arg) did not block secretion of @-APP into the medium after transfection and transient expression in COS-7 cells (data not shown). In addition, a

TABLE I NH2-terminal microsequencing of the -9-kDa fragment from CV-I

cells infected with VV:751 Sequence of fragment A Sequence of fragment B

Amino acid 1st run" 2nd run Amino acid 1st run' 2nd run Cycle

P m l P W l 1 Leu - 6.3 Phe - 4.5 2 Val - 5.5 Ala - 3.8 3 Phe - 4.8 Glu - 3.5 4 Phe 25 5.2 Asp 20 3.5 5 Ala 16 3.5 Val 17 3.2 6 Glu 16 3.8 Gly 18 2.9 7 Asp 18 2.9 Ser (2.44) (1.2) 8 Val 12 2.6 Asn 16 2.4 9 Gly 10 Lys 17

10 Ser (1.25) Gly 16 11 Asn 13 Ala 11 12 Lys 7 Ile 14

a Power failure in the first three cycles.

630 @-APP Secretase Cleavage Sites

series of mutants with deletions of up to 6 amino acid residues amino-terminal to and including Lys" as well as 6 residues immediately following Lyd6 were constructed and tested for production of soluble 8-APP695. All of these deletion mutations showed secretion of precursor protein into the culture medium (data not shown).

Two of the deletion mutants lacking 4 and 6 residues immediately adjacent to the carboxyl-terminal side of L y P (KM-3 and KM-4, respectively) were investigated with re- spect to the cleavage site(s) used for secretion. These mutants were selected because they each retain Lys" but also harbor significant deletions and might, therefore, reveal specific alterations in secretion site(s). To increase the expression effi- ciency and production of -9-kDa fragments, DNAs for these two mutants were individually engineered into vaccinia viruses. CV-1 cells were infected with the recombinant vaccinia viruses, metabolically labeled with [?'3]methionine/cysteine, and cell lysates prepared. Equivalent amounts of labeled lysate from each infection were immunoprecipitated with 369Ab and analyzed by electrophoresis on SDS-polyacrylamide gels (Fig. 1, D and E ) . The carboxyl-terminal fragments from the KM-3 and KM-4 cell lysates gave slightly higher molecular mass bands in the -9-10 kDa range than their wild type counterpart, suggesting that the secretory cleavages in these deletion mutants occurred amino-terminal to Lys". Sequencing analyses were next performed to identify the exact cleavage sites used to release soluble @-APP from the KM-3 and KM-4 mutants. The -9-kDa fragments present in [35S] methionine metabolically labeled KM-3 and KM-4 cell lysates were isolated and sequenced. 35S signals were found at cycles 18 and 20 for the KM-3 mutant, suggesting a two-site cleavage

0 5 10 15 20 25 30 cycle

FIG. 3. Radiosequence analysis of [s5S]methionine-labeled -B-kDa carboxyl-terminal fragment isolated from chloroquine-treated VV761-infected CV-1 cell lysates.

after Hid3 and Glu" within the @-amyloid domain (Fig. 4, a and c ) . Identical cleavage sites were found upon analysis of the KM-4 mutant protein harboring a larger 6-amino acid deletion. 35S radiosignals were observed at cycles 16 and 18 indicating a double cleavage, again at Hid3 and Glu" (Fig. 4, b and c) . The secretion sites for KM-4 are located 9 and 11 residues amino-terminal to the putative point of insertion into the membrane as surmised from the hydrophobic and hydrophilic nature of the sequence in this region. In contrast, the Lys" site is located 12 residues from the transmembrane domain.

Secretory Cleavage Sites in Down's Syndrome Fibroblusts- To compare the secretory cleavage sites identified in human P-APP synthesized by recombinant methods with the secretion of endogenous P-APP in a human cell line, we obtained a fibroblast cell line derived from the skin of an individual with Down's syndrome. The DS fibroblasts were metabolically labeled with [35S]methionine/cysteine, cell lysates prepared, immunoprecipitated with 369Ab, and analyzed by SDS-polyacrylamide gel electrophoresis. The pattern of carboxyl-terminal fragments obtained was very similar to that seen produced in CV-1 fibroblasts (Fig. 1F). When the -9-kDa fragment from DS fibroblasts was radiosequenced, a heterogeneous collection of cleavage sites was found. 35S radiosignals were observed at cycles 10, 13, and 16, indicative of multiple cleavages (Fig. 5). Based on these data, it can be concluded that one cleavage site occurred after Phel9 of the @-amyloid domain, with additional sites after G1u22 and Gly25. In contrast to the -9-kDa fragment generated by VV751 infected CV-1 cells, no radiosignal was found at cycle 19, which is expected if cleavage occurred at Lys". Hence, the proteolytic cleavage sites used for P-APP751 secretion in DS fibroblasts are heterogeneous and different from those found in the CV-1 cell system.

DISCUSSION

One potential mechanism to explain the excessive production of P-amyloid peptide in the brains of Alzheimer's disease victims is that the peptide is generated upon secretion of @- APP. Therefore, an understanding of the proteolytic cleavage process used to release soluble B-APP is important in eluci- dating the role of secretion in P-amyloidogenesis. A number of investigators have studied @-APP secretion. Esch et al. (8) first defined the major secretase cleavage site as Lys" of the &amyloid domain based on biochemical analyses of both recombinant 8-APP695 and P-APP751 proteins synthesized in human 293 cells. Subsequently, similar results have been

TABLE I1 Summary of /j"APPprocessing mutants

@-Amyloid domain Mutants

. . . .1 16 Membrane

. . . .DAEFRHDSGYEVHHQKLVFFAEDVGSNK§. . . WT L K16L A K16A R K16R E K16E Q Kl6Q AA K16A/L17A A AK16

AA AQ15, K16 AA AA AH13, H14, Q15, K16

AAAA AA AEll, V12, H13, H14 Q15, K16 A AL17 AA AL17, V18 AAAA AL17, V18, F19, F20 (KM-3) AAAAAA AL17, V18, F19, F20, A21, E22 (KM-4)

P-APP Secretase Cleavage Sites 631

18 KM3

16 KM4

0 s 10 1s 2 0 2 5 30 Cycle

C) + + l6 ... Y E V H H Q K { ( L V F F ) A E ) D V G S N K ... KM3 m 4

A A A A A A A A A A

FIG. 4. Radiosequence analysis of [s6S]methionine-labeled -9-kDa carboxyl-terminal fragments produced by deletion mutants of @-APP. Panel a, KM-3 -9-kDa fragment analysis; panel b, KM-4 -9-kDa fragment analysis; panel c, partial sequence of 8- amyloid peptide with secretase cleavage sites of KM mutants indicated by arrows. KM-3 and KM-4 deletions are also indicated on the sequence. The -9-kDa fragments were generated by infecting CV-1 cells with VVKM-3 or VVKM-4.

13

O l 0 5 10 1s 20 25 30

cycle

FIG. 5. Radiosequence analysis of [s6S]methionine-labeled -9-kDa fragment from DS fibroblasts.

obtained for human p-APP expressed in Chinese hamster ovary cells (16, 18), COS-1 cells (15-17), SF9 insect cells (20, 21), and native p-APP synthesized in PC-12 neuronal cells (19) and Alzheimer's disease brain (22). However, in all of the studies, methodology and/or yields of protein may have only permitted the detection of a major cleavage site by secretase. For example, in the study conducted by Wang et al. (18) in which the carboxyl terminus of soluble B-APP was characterized, only the CNBr product with an identical high perform-

ance liquid chromatography retention time as a synthetic peptide standard designed to correspond to cleavage at Lys" was analyzed. In the original study by Esch et al. (8), as well as those of Anderson et al. (19), and Ramabhadran et al. (21), molar yields of /i?-APP fragments characterized ranged from 2 to 12% depending on the study. Hence, sites other than the major Lys" cleavage site may not have been detected. In fact, this caveat was noted by Ramabhadran et al. (21).

Experiments in which the Lys" cleavage site has been mutated or extensively deleted revealed little negative effect on the secretion of p-APP (15-17). The secretase cleavage site used to secrete the various p-APP mutants was not identified except for one deletion mutant generated by Ma- ruyama et al. (17). Based on the position of the cleavage site found for this one large deletion mutant spanning the Lys" site, it was concluded that secretase is not a sequence-specific proteinase but is distance-specific, i.e. the site cleaved in p- APP is solely determined by its distance from the membrane surface. Since most proteinases demonstrate amino acid specificity, we investigated the possibility that a collection of proteinases, with different specificities, may be responsible for the secretion of p-APP. With this scenario, the primary cleavage at Lys" would be produced by a prevelant proteinase with trypsin-like activity in the collection of secretases. Dele- tion of Lysl' would then allow another proteinase in the collection with a different specificity to cleave and secrete p- APP. If a collection of proteinases comprises the secretase activity, then it would be predicted that other p-APP cleavage sites should exist. In addition, it is possible that the secretase cleavage area represents a structurally exposed region of p- APP which is vulnerable to proteolytic cleavage. The apparent preference for secretase to cleave at a set distance from the membrane surface could be accounted for by a susceptible region of amino acid sequence in p-APP.

To begin to refine our understanding of the p-APP secretase cleavage process, we have closely examined the cleavage site by conducting radiolabel and direct amino-terminal sequence analyses of the -9-kDa carboxyl-terminal product of p-APP secretion. Recombinant p-APP751 synthesized in CV-1 fibroblasts was one source of -9-kDa cleavage product characterized. Radiosequence data using three different amino acids to label the -9-kDa fragment metabolically were consistent with two sites of cleavage, one at Lys" and another at Phelg. Direct amino-terminal sequence of the fragment confirmed the radiosequence results. These same cleavage sites were obtained when the -9-kDa fragment was produced in the presence of chloroquine, a drug that suppresses protein degradation by acidifying intracellular proteolytic compartments. These results suggest that there is heterogeneity in secretase cleavage which is not caused by contributing degradative processes. Furthermore, the nature of the residues in the P1 positions would suggest that proteinases of different specificities are operating in the secretion event. This conclusion is further substantiated by analysis of p-APP mutants made at and around the primary cleavage site, Lys" of (3-amyloid.

Thirteen different mutations of p-APP, including five point mutations at Lys" and eight deletional mutations removing 1-6 residues at and adjacent to Lys", were constructed and tested for the ability to secrete p-APP. All mutants were found to produce soluble 6-APP. This result is in agreement with Sisodia (15), Sahasrabudhe et al. (16), and Maruyama et al. (17), who also found that mutations in this region of 6- APP would still permit secretion of p-APP. We defined the cleavage sites for two deletion mutants with preserved Lys". Metabolically labeled -9-kDa fragments were purified from cells expressing each mutant and radiosequenced. The results

632 B-APP Secretase Cleavage Sites

indicate that for both mutants, cleavage after Lys16 is ignored in favor of cleavage after other amino acids, namely, His13 and Glu" of the p-amyloid domain. These data again suggest heterogeneity in secretase cleavage. Exopeptidase activity does not appear to be responsible for generating this heterogeneity. The cleavage sites for wild type p-APP751 are separated by 3 amino acids, whereas the sites found for both p- APP751 mutants are separated by 2 amino acids; hence, it is difficult to envision selective activity by tripeptidyl and dipeptidyl aminopeptidases in the same cell background. The absence of cleavage at the retained Lys16 may reflect confor- mational alterations imposed by the deleted residues or may be a result of the loss of residues required for proteinase specificity resulting from deletion of the P1' and P2' positions adjacent to the P1 Lys". The new cleavage sites used to secrete the mutated p-APPs are located up-stream of L y P similar to that observed by Maruyama et al. (17), who studied a different deletion mutant of p-APP which also cleaved after a glutamate residue. Although the two mutants differed in the lengths of residues deleted (4 and 6 amino acids), the cleavage sites were identical. The use of the same cleavage site with both mutants suggests cleavage by an alternate proteinase(s) with a defined specificity versus cleavage by a distance-specific proteinase.

Further evidence for heterogeneity of secretase cleavage comes from our analysis of native p-APP synthesized by DS fibroblasts. Cleavage was not found at Lys16 but rather at Phe", GluZZ, and G1yz6 of the @-amyloid domain. Since it has been demonstrated that tripeptidyl and dipeptidyl aminopeptidase activity is unaltered in Alzheimer's disease (27), it seems unlikely that the multiple cleavage sites found in the DS fibroblasts result from tripeptidyl aminopeptidase action. The distances from these sites to the membrane surface are 9, 6, and 3 residues. These data deviate from the suggested specific distance of 9-11 residues. Whether this cleavage pattern is unique to Down's syndrome, Alzheimer's disease, or to cell type variation requires further investigation. Het- erogeneity of secretase action has also been recently reported by Seubert et al. (28) who employed specific antibodies to identify alternative cleavage sites used in p-APP release. In addition, our results describing secretase heterogeneity can explain the data of Busciglio et al. (29) who characterized the 3-kDa product generated by subsequent processing of the -9- kDa fragment. Busciglio et al. (29) reported a heterogeneous amino-terminal sequence for the 3-kDa peptide reflecting cleavages at Lys16, Phelg, and Valz4, which we believe is caused by the action of multiple secretory proteinases.

In summary, this study demonstrates that the secretion of p-APP is likely to involve a collection of proteinases that have been heretofore referred to as secretase. Cleavage of j3- APP also appears to be heterogeneous, occurring at several sites in a region of the precursor near the membrane surface. Although Lys" of the @-amyloid domain seems to be a pre-

ferred cleavage site for most cell systems studied, alternative sites may be used in different cellular backgrounds.

Acknowledgments-We thank Drs. Sam Gandy and Paul Green- gard for 369Ab, Daiichi Pharmaceutical Co. for support with mutant production, and Marion Merrell Dow Inc. for support of all other experiments.

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