Proc. Natl. Acad. Sci. USA Vol. 89, pp. 10758-10762, November 1992 Medical Sciences

Identification of a mouse brain cDNA that encodes a protein related to the Alzheimer disease-associated amyloid IB protein precursor WILMA WASCO*t4, KEITH BUPP*§, MARGARET MAGENDANTZ*, JAMES F. GUSELLAt, RUDOLPH E. TANZIt, AND FRANK SOLOMON* *Department of Biology and Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139; and tMolecular Neurogenetics Laboratory, Massachusetts General Hospital East, Charlestown, MA 02129

Communicated by Phillips W. Robbins, June 26, 1992

We have isolated a cDNA from a mouse brain ABSTRACT library that encodes a protein whose predicted amino acid sequence is 42% identical and 64% similar to that of the amylold (3 protein precursor (APP). This 653-amino acid protein, which we have termed the amyloid precursor-like protein (APLP), appears to be similar to APP in overall structure as well as amino acid sequence. The amino acid homologies are concentrated within three distinct regions of the two proteins where the identities are 47%, 54%, and 56%. The APLP cDNA hybridizes to two messages of approximately 2.4 and 1.6 kilobases that are present in mouse brain and neuroblastoma cells. Polyclonal antibodies raised against a peptide derived from the C terminus of APLP stain the cytoplasm in a pattern reminiscent of Golgi sai . In addition to APP, APLP also displays significant homology to the Drosophila APP-like protein APPL and a rat testes APP-like protein. These data indicate that the APP gene is a member of a strongly conserved gene family. Studies aimed at determining the functions of the proteins encoded by this gene family should provide valuable clues to their potential role in Alzheimer disease neuropathology.

The 39- to 43-amino acid peptide (BA4 is the major component of the senile plaques and cerebrovascular amyloid deposits that are characteristic of Alzheimer disease, Down syndrome, and to a lesser extent, normal aging. (3A4 is derived from the larger amyloid (3 protein precursor (APP), which resembles an integral membrane-associated protein with a small cytoplasmic C-terminal domain and a larger extracellular N-terminal domain (1-4). The APP gene produces at least four major transcripts predicted to encode proteins of 563, 695, 751, and 770 amino acids (5-8). With the exception of APP 695, these transcripts contain an alternatively spliced exon encoding a Kunitz type protease inhibitor domain (6-8). Secreted forms of APP containing the protease inhibitor domain are identical to protease nexin-Il (9, 10), an inhibitor of various serine proteases. It has been shown that normally APP is secreted by cleavage at a site near the membraneextracellular junction within the f3A4 domain (11-14). Therefore, the normal constitutive processing of APP precludes the formation of fA4. In the course of cloning the human APP gene, other cross-hybridizing cDNAs were also obtained, leading to the suggestion that APP is a member of a larger gene family (4). We have now identified a mouse brain cDNA that encodes a protein whose predicted amino acid sequence is 42% identical and 64% similar to APP.L This amyloid precursor-like protein (APLP) is clearly not the mouse homologue of human APP, which has previously been cloned and found to be 96.8% identical to human APP (15). APLP contains three particularly conserved regions of homology with APP and is en-

coded by a 2.4-kilobase (kb) message that is present in mouse brain and neuroblastoma cells. Antibodies raised against a synthetic peptide derived from the C terminus of APLP stain the cytoplasm of neuroblastoma cells in a pattern similar to that obtained with an antibody to a known Golgi protein. The cDNA described here, together with the gene encoding APP and two other genes-one from Drosophila (16) and one from rat (17)-form a gene family producing proteins that are highly conserved and may therefore share common functions and, perhaps, undergo similar processing.

MATERIALS AND METHODS Cells. Neuroblastoma NB2A cells were maintained as previously described (18). Screening of Agtll Libraries. Three mouse brain Agtl1 cDNA libraries (Stratagene, Clontech) were screened with cDNA probes (19). Positive clones were sized by PCR amplification and subcloned in pBluescript (Stratagene) or M13 (New England Biolabs) vectors, and both strands were sequenced with Sequenase (United States Biochemical). Rapid Ampfication of cDNA Ends (RACE) Procedure for Obtining 5' cDNA Extensions. The RACE procedure (20,21) was carried out with primers corresponding to nucleotides 699-719 and 672-692 of the sequence presented in Fig. 2. Products were subcloned in pBluescript, screened by hybridization to the 5' 120-base-pair (bp) EcoPJ-Pst I fragment of 69A, and sequenced. RNA Analysis. Poly(A)+ RNA (22) was analyzed by Northern blotting according to standard methods (23, 24). Production of Antisera to an APLP Peptide. A 20-mg sample of the peptide CQQLRELQRH (Biopolymers Laboratory, Howard Hughes Medical Institute and Center for Cancer Research, Massachusetts Institute of Technology) was conjugated to keyhole limpet hemocyanin (25) and used to immunize four New Zealand White rabbits (26). Protein Preparation. Protein from neuroblastoma cells was isolated by rinsing the cells with phosphate-buffered saline (PBS) followed by lysing the cells in SDS sample buffer (gel loading) and boiling. Protein from mouse brain was isolated by homogenizing in a modified RIPA buffer (50 mM Tris-HCl, pH 7.4/150 mM NaCl/5 mM EDTA/1% Triton X-100/1% sodium deoxycholate/0.1% SDS/protease inhibitors) and centrifuging at 10,000 x g for 30 min at 40C. Abbreviations: APP, amyloid ,( protein precursor; APLP, amyloid precursor-like protein; APPL, amyloid precursor protein-like protein; RACE, rapid amplification of cDNA ends; ORF, open reading frame. *To whom reprint requests should be addressed at the present address: Department of Neurology, Molecular Neurogenetics Laboratory, Massachusetts General Hospital East, 149 13th Street, Charlestown, MA 02129. §Present address: Department of Molecular and Cellular Biochemistry, University of Paris, 91405, Orsay, France. IThe sequence reported in this paper has been deposited in the GenBank data base (accession no. L04538).

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 10758

Proc. Natl. Acad. Sci. USA 89 (1992)

Medical Sciences: Wasco et al.

protein (MAP), a clone was isolated from a mouse brain cDNA library which was found to have an open reading frame (ORF) homologous to that of APP. The epitope recognized by the antibody was translated from the noncoding strand of the APLP cDNA, which does not display homology to any known MAP gene. The cDNA clone in which the APP homology was originally identified contained a portion of the C-terminal coding sequence as well as aportion of the 3' untranslated region. To extend the APLP ORF in the 5' direction, we screened two Clontech Agtll libraries with probes from the 5'-most regions of our available cDNA clones. Repetitive screens using progressively more upstream probes resulted in the isolation of a 1.8-kb cDNA clone, 69A (Fig. 1), whose 5' terminus has an EcoRI site that is present in the coding sequence of APLP. A variation of the RACE procedure (20, 21) allowed the isolation of several independent overlapping cDNA clones that extended the APP homology past the 5' EcoRI site. A PCR product amplified with primers to the 5'-most 100 bp of clone J was then used to screen a Stratagene mouse brain cDNA library. Full-length APLP clones were obtained and sequenced (Fig. 2). The predicted initiator methionine is in agreement with the eukaryotic consensus initiation sequence

E

r

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APLP IA 69A J

-100 bse pairs = open reaing frame

FIG. 1. Schematic representation of the mouse APLP ORF and the relationships of various cDNA clones. The 2361-bp ORF and noncoding region of the APLP cDNA are shown. Also shown are the relative locations of two representative cDNA clones isolated from Agtll libraries, 69A and lA, and a clone obtained through the RACE procedure, J. Restriction enzyme sites: E, EcoRl; P, Pst I.

t3-Galactosidase fusion protein was generated from an EcoRI-EcoRI fragment of a Agtll clone containing 666 nucleotides of the 3' coding portion and 260 nucleotides ofthe untranslated region of the APLP ligated into the pUEX5 vector (23). For Western blot analysis, protein samples were subjected to polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with rabbit antibodies and 125I-labeled staphylococcal protein A (27). Antibody R37 was provided by T. Ishii (Tokyo Psychiatric University). Immunofluorescence. Neuroblastoma cells were plated onto glass coverslips 48 hr before fixation; 24 hr before fixation, the concentration of fetal calf serum was changed from 10% to 0.1% to induce neurite extension. Twenty minutes before fixation, concanavalin A was added to 20 mg/ml to promote cell adhesion to the coverslips. Cells were fixed in 3.7% (vol/vol) formaldehyde/PBS, permeabilized in acetone, and blocked for 30 nmn at 37°C in PBS containing 1% calf serum. Primary antibody (1:10,000) was added and visualized with fluorescein isothiocyanate-conjugated goat anti-rabbit antibody (Cappel Laboratories). A rabbit antibody against Golgi mannosidase II was provided by K. Moremen (28, 29). RESULTS Identification and Cloning of APLP. In an antibody-based screen for cDNA clones encoding a microtubule-associated 1

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APLP Is a Member of a Family of APP-Like Proteins. The alignment of the APP and APLP sequences (Fig. 3) reveals that the two proteins are 42% identical and 64% similar at the amino acid level. The identities are particularly strong in three distinct regions (Fig. 4), where the proteins are 47%, 54%, and 56% identical and 67%, 73%, and 74% similar. These same three regions have been shown previously to be shared between APP and a APPL, and they have been termed the extracellular I (El), extracellular II (EII), and cytoplasmic (C) domains by these investigators (16). The cytoplasmic domain homology is also present in a partial cDNA clone that has been isolated from a rat testis library (17). Only APP contains the PA4 sequence that is found in amyloid plaques. In APLP the extracellular portion of ,BA4 is absent; however, a small degree of homology is observed in the transmembrane portion of the domain. All four proteins contain a 3- or 4-amino acid span ofcharged residues (arginine/lysine) at the cytoplasmic face of the membrane (Fig. 4). This motif has been hypothesized to allow interaction with membrane phosQ

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FIG. 2. Composite nucleotide sequence of APLP cDNA and predicted amino acid sequence of APLP. The predicted membrane-spanning region is underlined. The location of the peptide sequence used for the production of antisera is double underlined. Predicted N-glycosylation sites and a region surrounding a potential tyrosine phosphorylation site are underlined by dots. The polyadenylylation signal is indicated by boldface type and the stop codon is shown by an asterisk.

10760

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Medical Sciences: Wasco et al.

APLP 21 LLLPLSLLLLRAQLAVGNIAVG3SPSAAEAPGSAQVAGLCGRLTLHRDLRT

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FIG. 3. Comparison ofthe APLP and APP amino acid sequences. The GCG BESTFIT analysis of the mouse APLP and human APP 695 (2) is shown. Identities are indicated by a vertical line between the symbols for the two amino acids. Similarities are indicated by a single or double dot. Gaps produced by the BESTFIT alignment are shown by dots in the sequence. The /3A4 peptide is underlined in the APP sequence. The identities are concentrated in three regions: APLP amino acids 21-211, 316-488, and 608-653.

pholipids or to provide a stop-transfer signal for membranebound proteins (31). Northern Blot Analysis. Northern blots (Fig. 5) reveal that in mouse brain and neuroblastoma cells there are two messages of approximately 2.4 and 1.6 kb, with the larger message present in greater abundance. The cDNA that we have cloned corresponds to the 2.4-kb message, although both messages are consistently seen in Northern blots that are probed and washed under stringent conditions. The predominant message in brain is 2.4 kb, while in peripheral organs (lung, heart, kidney, spleen, and liver; data not shown) only the 1.6-kb message was observed. The mouse APLP cDNA does not hybridize to the 3.2- and 3.4-kb APP messages under the conditions used. Generation of Antibodies Against an APLP Peptide. Antibodies were raised against a synthetic peptide which corresponds to a unique sequence in the C terminus of mouse APLP. Two of four injected rabbits (301 and 302) produced sera that strongly recognize a 65-kDa mouse brain protein that is not recognized by preimmune sera (Fig. 6A). A smaller protein of approximately 33 kDa that is recognized by antiserum 301 may be a proteolytic degradation product of the larger protein; however, this remains to be shown. In Fig. 6A, the specificity of the interaction of the antibody with these proteins is demonstrated by the ability to block the binding of antibody 301 to the proteins by preabsorbing with the original peptide (lanes 2-5); an irrelevant peptide has no effect on the

Proc. Natl. Acad. Sci. USA 89 (1992)

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APLP 172 RrHQeAqEACSsqG1lLHgsGKLLPCasDrIROyWVCCP APP 148 hWHtvAkEtCSEkstnLHdyGNLLPCOIDkPRQVUFVCCP APPL 159 RWnQtgaaACqgrGmgmrtfaULIovm= 5Y XRACNLLULAR DOMAIN 1I 1 II*... II.. .. . .I APLP 316 AKmdLErrrmrqineVIRW amAdsQsKNL P&A DrQAlneH]lQsilQtL APP 318 AKeRL~akHRErMsqNVRE eEAerQaKNL ; PKA DlkAviqHlQekVesL APPL 413 sqkRLBEsHRgkvtrVxkdWsdlEekyQdmrLadaqsafIfKQrmtarPQtsVQaL I **0 I** 11*0**1 *le 11 I .10001 1*0 * 110 11 * 01 APLP 365 UEqvsg3RQrLVETlatRViAl IXDqRhaALEgflaALQgdPPqAerVlmaLrrYL APP 368 UqEaanNRQQLVETmaRVeOAm1NDRRNlALEnYitALQavPPrprHVfrunLkKYv APPL 470 ZEEgnalkhQLaamuqqaVlAhllqRkReAmtcYtqALteqPPnAhHVekcLqKl L 0 1 * * I I* I*.*, 1 I . . 1. 1 *j . . LI * *.. ** *.. * I I .I APLP 421 RAEQleqrNTLrHYqN VaaVDP EKAqQmRfQVqTHLqVleERMIQ8LgLL APP 424 NAEQKDRqNTLk~fe. VrmVDP kKAAQiRsQVmTHLrVtyERMNQSLsLL APPL 523 RAlhEDRahaLaNYrNl1nsggPgglEaAAseRprt 1erLididravnfptmL

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FIG. 4. Domains of homology. Regions of the amino acid sequences of the mouse APLP, the human APP, the Drosophila amyloid precursor protein-like protein (APPL), and the rat testis cDNA product (test) are compared. Amino acids that are identical in all of the sequences in the domain are shown as uppercase letters in boldface type and are identified by the presence of a vertical line above the sequences. Amino acids that are the same in more than one sequence are shown as uppercase letters and have a dot over the sequences. Amino acids that are not identical to any others are shown as lowercase letters. The conserved cysteines (see text) are identified by the presence of a caret (A) underneath the sequence. Spans of particularly conserved amino acids are underlined. An N-glycosylation signal is identified by a double underline. Stop codons are indicated by an asterisk and the amino acid numbers of the sequences are shown at the beginning of each line.

interaction of the antibody with either the 65-kDa or the 33-kDa protein (lane 6). Antiserum 301 also recognizes these two proteins in neuroblastoma cell extracts (Fig. 6B). To further confirm the specificity of the 301 antiserum, we determined whether the antiserum would recognize a ,B-galactosidase fusion protein containing the 222 C-terminal amino acids encoded by the APLP cDNA. Fig. 6C shows a Western blot of bacterially produced proteins that were probed with antiserum 301. As can be seen in lane 6, antiserum 301 does specifically interact with the ,B-galactosidase-APLP fusion protein. There are a number of antibodies that have been generated against the C terminus of APP. Because the identity between APP and the mouse APLP in this region is particularly strong,

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FIG. 5. Northern blots of mouse brain and neuroblastoma RNA. Poly(A)+ RNA (10 ,ug) from neuroblastoma (lane 1) and mouse brain (lane 2) was probed with DNA corresponding to nucleotides 1791-2305 of the nucleotide sequence shown in Fig. 2. Sizes of hybridizing messages are indicated in kb.

Medical Sciences: Wasco et al.

Proc. Nati. Acad. Sci. USA 89 (1992)

are stained with 301 (or antiserum 302; data not shown), the pattern that is observed is a reticular staining near the nucleus (Fig. 7 a and b). When these cells are stained with an antibody to a known Golgi enzyme, mannosidase II, a similar pattern is observed (Fig. 7c). It remains unclear whether the cytoplasmic protein stained corresponds to the 65-kDa or the 33-kDa species observed on Western blots. Because these cells were permeabilized with acetone it was not possible to determine whether APLP resides in the plasma membrane.

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1 2

FIG. 6. Western blots using antiserum 301. Mouse brain and neuroblastoma proteins were separated on a 7.5% polyacrylamide gel. (A) Mouse brain protein was probed with antiserum 301 or preimmune serum at a dilution of 1:100. The binding of antiserum 301 to the 65- and 33-kDa proteins is inhibited by the presence of increasing amounts of the peptide used to immunize the rabbit. Lane 1, preimmune serum with no peptide; lane 2, immune serum with no peptide; lane 3, immune serum preabsorbed with peptide at 5 ng/ml; lane 4, immune serum preabsorbed with peptide at S0 ng/ml; lane 5, immune serum preabsorbed with peptide at 500 ng/ml. Preabsorption with an irrelevant yeast P-tubulin peptide at 500 ng/ml had no effect on the binding (lane 6). (B) Neuroblastoma cell extracts probed with preimmune serum (lane 1) and 301 antiserum (lane 2). Both sera were used at a dilution of 1:100. (C) Anti-peptide antiserum recognizes a P-gaIactosidase-APLP fusion protein. Western blots on bacterially produced proteins. Lanes 1-3 were stained with preimmune serum from rabbit 301. Lanes 4-6 were stained with immune serum. Lanes 1 and 4, induced cells containing a plasmid with its P-galactosidase gene fused to an APLP cDNA fragment inappropriately oriented for production of an APLP epitope. Lanes 2 and 5, uninduced cells containing a plasmid with its .-galactosidase gene fused in frame to the APLP ORF. Lanes 3 and 6, same cells as in lanes 2 and 5 except induced. Induced cells were grown at 42°C. Uninduced cells were grown at 30°C. The arrowhead indicates a P-galactosidase-APLP fusion protein recognized by immune serum but not by preimmune serum. That protein is approximately 24 kDa larger than P-galactosidase alone-as predicted due to the insertion of 222 additional residues of APLP ORE. we predicted that some of these antisera would also interact with the mouse APLP. One of these antisera (R37; refs. 2 and 32) is directed against the C-terminal 15 amino acids of APP, a region where the two proteins are particularly similar (see Fig. 4). R37 recognizes the ,-galactosidase-APLP fusion protein and the 65-kDa protein in mouse brain (data not shown). The 15-amino acid sequence used to raise the anti-APP antibody does not overlap the 9 amino acids used to generate antiserum 301. These data suggest that the 65-kDa protein contains at least two epitopes in common with the APLP fusion protein. AaU-APLP Immunocytodemstry. The subcellular localization of the protein recognized by antiserum 301 was assayed by immunofluorescence. When neuroblastoma cells

DISCUSSION The APLP cDNA, whose predicted product displays 42% identity with APP, encodes a murine member ofthe APP-like gene family. Homology among APP, APLP, and APPL is strongest in three distinct domains (Figs. 3 and 4) and includes 11 cysteine residues, an acidic residue-rich region, a potential N-glycosylation site, a hydrophobic membranespanning region, a highly conserved cytoplasmic domain, and several specific amino acid sequence motifs with exact identity. These findings indicate that the mouse APLP, the Drosophila APPL, the rat testis protein, and APP constitute a family of related proteins. The extensive conservation of amino acid identity as well as both the overall and specific domain structure within this family of proteins suggests that these proteins may share common functions and, perhaps, be processed similarly. Immunocytochemical analysis of APLP in neuroblastoma cells reveals a pattern suggestive of Golgi staining. Antisera to APPL and APP have also been shown to recognize a protein in the endoplasmic reticulum or Golgi complex (33, 34). The N-terminal extracellular portion of both APPL and APP can be secreted by means of cleavage near the membrane (13, 34, 35). Our results suggest that APLP, like APP and APPL, may be processed in the Golgi complex; however, it is not yet clear whether APLP is secreted. The cytoplasmic region is the most strongly conserved domain in the APP-like proteins. A peptide containing a portion of the APP cytoplasmic domain can be phosphorylated on serine and threonine residues in vitro (36). In APLP, a threonine residue resides in a position analogous to the potentially phosphorylated serine in APP. Agents that are known to regulate protein phosphorylation at these sites in APP appear to affect the rate of proteolytic processing of mature forms (37), raising the possibility that protein phosphorylation may also affect processing of APLP. A seven-amino acid sequence in APLP contains a potential tyrosine phosphorylation site residing eight or nine amino acids from the C terminus of all four proteins (Fig. 4; ref. 38). The seven-amino acid sequence also contains the tetrameric motif NPXY required for the ligand-independent, coated pit-mediated internalization of the low density lipoprotein receptor (39). The NPXY sequence is present in the cytoplasmic tails of at least 16 other cell surface receptor molecules, including the ,-integrin receptor and members of the epidermal growth factor receptor family (39), and appears to play a role in binding clathrin. The existence of a family of APP-like proteins implies that they may share common functions. The conservation of 11 specifically spaced cysteines in the extracellular portion of these proteins is indicative of conserved higher-order structure and suggests that these molecules may interact with a common extracellular molecule(s). Moreover, the strong amino acid conservation within the intracellular C termini suggests that the proteins in this family may be similarly phosphorylated and undergo clathrin-mediated internalization. A distinct physiological role for the ubiquitously distributed APP has yet to be determined. Clues to the function of any of the members of this family of proteins should help to elucidate the role of APP and APP-like genes in the neuropathogenesis of Alzheimer disease.

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Proc. Natl. Acad. Sci. USA 89 (1992)

FIG. 7. Immunofluorescence staining of mouse neuroblastoma cells with antiserum 301. Cells were stained with antiserum 301 at a dilution of 1:10,000. (a and b) Neuroblastoma cells stained with antiserum 301. This staining pattern is similar to that seen with an antibody to a known Golgi enzyme (mannosidase IL1 c). The perinuclear staining is blocked by the addition of the peptide that was used as the antigen (d) and is not seen in the presence of preimmune serum (e). (a. c, d, and e. x720; b.

x950.) Note. We have now isolated human APLP cDNAs and mapped APLP to the proximal long arm of human chromosome 19 (40).

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