The Proteolytic Processing of the Amyloid Precursor Protein Gene Family Members APLP-1 and APLP-2 Involves α-, β-, γ-, and ϵ-Like Cleavages
2004; Elsevier BV; Volume: 279; Issue: 18 Linguagem: Inglês
10.1074/jbc.m311601200
ISSN1083-351X
AutoresSimone Eggert, Krzysztof Paliga, Peter Soba, Geneviève Evin, Colin L. Masters, Andreas Weidemann, Konrad Beyreuther,
Tópico(s)Amyloidosis: Diagnosis, Treatment, Outcomes
ResumoAmyloid precursor protein (APP) processing is of major interest in Alzheimer's disease research, since sequential cleavages by β- and γ-secretase lead to the formation of the 4-kDa amyloid Aβ protein peptide that accumulates in Alzheimer's disease brain. The processing of APP involves proteolytic conversion by different secretases leading to α-, β-, γ-, δ-, and ϵ-cleavages. Since modulation of these cleavages represents a rational therapeutic approach to control amyloid formation, its interference with the processing of the members of the APP gene family is of considerable importance. By using C-terminally tagged constructs of APLP-1 and APLP-2 and the untagged proteins, we have characterized their proteolytic C-terminal fragments produced in stably transfected SH-SY5Y cells. Pharmacological manipulation with specific protease inhibitors revealed that both homologues are processed by α- and γ-secretase-like cleavages, and that their intracellular domains can be released by cleavage at ϵ-sites. APLP-2 processing appears to be the most elaborate and to involve alternative cleavage sites. We show that APLP-1 is the only member of the APP gene family for which processing can be influenced by N-glycosylation. Additionally, we were able to detect p3-like fragments of APLP-1 and p3-like and Aβ-like fragments of APLP-2 in the media of stably transfected SH-SY5Y cells. Amyloid precursor protein (APP) processing is of major interest in Alzheimer's disease research, since sequential cleavages by β- and γ-secretase lead to the formation of the 4-kDa amyloid Aβ protein peptide that accumulates in Alzheimer's disease brain. The processing of APP involves proteolytic conversion by different secretases leading to α-, β-, γ-, δ-, and ϵ-cleavages. Since modulation of these cleavages represents a rational therapeutic approach to control amyloid formation, its interference with the processing of the members of the APP gene family is of considerable importance. By using C-terminally tagged constructs of APLP-1 and APLP-2 and the untagged proteins, we have characterized their proteolytic C-terminal fragments produced in stably transfected SH-SY5Y cells. Pharmacological manipulation with specific protease inhibitors revealed that both homologues are processed by α- and γ-secretase-like cleavages, and that their intracellular domains can be released by cleavage at ϵ-sites. APLP-2 processing appears to be the most elaborate and to involve alternative cleavage sites. We show that APLP-1 is the only member of the APP gene family for which processing can be influenced by N-glycosylation. Additionally, we were able to detect p3-like fragments of APLP-1 and p3-like and Aβ-like fragments of APLP-2 in the media of stably transfected SH-SY5Y cells. The amyloid precursor protein (APP) 1The abbreviations used are: APP, amyloid precursor protein; APLP, amyloid precursor like protein; Aβ, amyloid Aβ protein; CTF, C-terminal fragment; AICD, APP intracellular domain; PMA, phorbol 12-myristate-13-acetate; PS, presenilin; wt, wild type; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; DAPT, N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester; BACE, β-site APP-cleaving enzyme. 1The abbreviations used are: APP, amyloid precursor protein; APLP, amyloid precursor like protein; Aβ, amyloid Aβ protein; CTF, C-terminal fragment; AICD, APP intracellular domain; PMA, phorbol 12-myristate-13-acetate; PS, presenilin; wt, wild type; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; DAPT, N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester; BACE, β-site APP-cleaving enzyme. was first identified as the precursor to Alzheimer's Aβ amyloid peptides (1Kang J. Lemaire H.G. Unterbeck A. Salbaum J.M. Masters C.L. Grzeschik K.H. Multhaup G. Beyreuther K. Muller-Hill B. Nature. 1987; 325: 733-736Google Scholar). APP is a member of a multigene family comprising at least 16 orthologues (2Coulson E.J. Paliga K. Beyreuther K. Masters C.L. Neurochem. Int. 2000; 36: 175-184Google Scholar) that encode type I integral membrane proteins with similar multidomain structures (reviewed in Ref. 2Coulson E.J. Paliga K. Beyreuther K. Masters C.L. Neurochem. Int. 2000; 36: 175-184Google Scholar). There are two mammalian APP homologues, the APP-like proteins, termed APLP-1 (3Wasco W. Bupp K. Magendantz M. Gusella J.F. Tanzi R.E. Solomon F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10758-10762Google Scholar, 4Paliga K. Peraus G. Kreger S. Durrwang U. Hesse L. Multhaup G. Masters C.L. Beyreuther K. Weidemann A. Eur. J. Biochem. 1997; 250: 354-363Google Scholar) and APLP-2 (5Wasco W. Gurubhagavatula S. Paradis M.D. Romano D.M. Sisodia S.S. Hyman B.T. Neve R.L. Tanzi R.E. Nat. 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In the amyloidogenic pathway, APP is processed consecutively by β-secretase, which releases the Aβ N terminus, and by γ-secretase(s), which cleaves in the middle of the transmembrane domain to produce Aβ peptides ending at either Val40 or Ala42. The β-secretase BACE (β-site APP-cleaving enzyme) was identified by four independent groups (12Vassar R. Bennett B.D. Babu-Khan S. Kahn S. Mendiaz E.A. Denis P. Teplow D.B. Ross S. Amarante P. Loeloff R. Luo Y. Fisher S. Fuller J. Edenson S. Lile J. Jarosinski M.A. Biere A.L. Curran E. Burgess T. Louis J.C. Collins F. Treanor J. Rogers G. Citron M. Science. 1999; 286: 735-741Google Scholar, 13Yan R. Bienkowski M.J. Shuck M.E. Miao H. Tory M.C. Pauley A.M. Brashier J.R. Stratman N.C. Mathews W.R. Buhl A.E. Carter D.B. Tomasselli A.G. Parodi L.A. Heinrikson R.L. Gurney M.E. Nature. 1999; 402: 533-537Google Scholar, 14Sinha S. Anderson J.P. Barbour R. Basi G.S. Caccavello R. Davis D. Doan M. Dovey H.F. Frigon N. Hong J. Jacobson-Croak K. 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BACE cleavage releases the large ectodomain of APP (sAPPβ) and creates a membrane-anchored C-terminal fragment (CTF) of 99 amino acids. Whereas in neuronal cells APP is mostly processed by β-secretase, peripheral cells preferentially process APP in a non-amyloidogenic pathway by an alternative protease designated as α-secretase which cleaves within the Aβ domain, between Lys16 and Leu17, and therefore precludes generation of the Aβ peptide (16Esch F.S. Keim P.S. Beattie E.C. Blacher R.W. Culwell A.R. Oltersdorf T. McClure D. Ward P.J. Science. 1990; 248: 1122-1124Google Scholar, 17Wang R. Meschia J.F. Cotter R.J. Sisodia S.S. J. Biol. Chem. 1991; 266: 16960-16964Google Scholar). α-Secretase candidates include ADAM 10 (18Lammich S. Kojro E. Postina R. Gilbert S. Pfeiffer R. Jasionowski M. Haass C. Fahrenholz F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3922-3927Google Scholar), ADAM 17/TACE (19Buxbaum J.D. Liu K.N. Luo Y. Slack J.L. Stocking K.L. Peschon J.J. Johnson R.S. Castner B.J. 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Neurosci. 1996; 16: 899-908Google Scholar). Recently, we and others have identified a presenilin-dependent cleavage at the C-terminal end of the APP transmembrane domain between Leu49 and Val50, which we termed ϵ-cleavage (23Weidemann A. Eggert S. Reinhard F.B. Vogel M. Paliga K. Baier G. Masters C.L. Beyreuther K. Evin G. Biochemistry. 2002; 41: 2825-2835Google Scholar, 24Gu Y. Misonou H. Sato T. Dohmae N. Takio K. Ihara Y. J. Biol. Chem. 2001; 276: 35235-35238Google Scholar, 25Sastre M. Steiner H. Fuchs K. Capell A. Multhaup G. Condron M.M. Teplow D.B. Haass C. EMBO Rep. 2001; 2: 835-841Google Scholar, 26Yu C. Kim S.H. Ikeuchi T. Xu H. Gasparini L. Wang R. Sisodia S.S. J. Biol. Chem. 2001; 276: 43756-43760Google Scholar), that is homologous to the S3 cleavage of Notch (27Schroeter E.H. Kisslinger J.A. Kopan R. Nature. 1998; 393: 382-386Google Scholar, 28De Strooper B. Annaert W. Cupers P. Saftig P. Craessaerts K. Mumm J.S. Schroeter E.H. Schrijvers V. Wolfe M.S. Ray W.J. Goate A. 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Notch S3 and APP ϵ-cleavage liberate the Notch intracellular domain and APP intracellular domain (AICD), respectively, enabling their translocation to the nucleus where they can activate transcription (39Jarriault S. Brou C. Logeat F. Schroeter E.H. Kopan R. Israel A. Nature. 1995; 377: 355-358Google Scholar, 40Lu F.M. Lux S.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5663-5667Google Scholar, 41Cao X. Sudhof T.C. Science. 2001; 293: 115-120Google Scholar, 42Minopoli G. de Candia P. Bonetti A. Faraonio R. Zambrano N. Russo T. J. Biol. Chem. 2001; 276: 6545-6550Google Scholar). So far the processing of the APP-like proteins, APLP-1 and APLP-2, remains poorly understood. Large secretory fragments of APLP-1 (4Paliga K. Peraus G. Kreger S. Durrwang U. Hesse L. Multhaup G. Masters C.L. Beyreuther K. Weidemann A. Eur. J. Biochem. 1997; 250: 354-363Google Scholar) and APLP-2 (43Webster M.T. Groome N. Francis P.T. Pearce B.R. Sherriff F.E. Thinakaran G. Felsenstein K.M. Wasco W. Tanzi R.E. Bowen D.M. Biochem. J. 1995; 310: 95-99Google Scholar) corresponding to the size of α- and β-secretase-processed secretory fragments of APP have been detected in the media of transfected cell lines. In addition, the accumulation of an APLP-1 C-terminal fragment was observed in PS-1(–/–) neurons (44Naruse S. Thinakaran G. Luo J.J. Kusiak J.W. Tomita T. Iwatsubo T. Qian X. Ginty D.D. Price D.L. Borchelt D.R. Wong P.C. Sisodia S.S. Neuron. 1998; 21: 1213-1221Google Scholar). Presenilin-dependent processing of this fragment is consistent with our previous finding that PS-2 interacts with immature APLP-1 (45Weidemann A. Paliga K. Durrwang U. Czech C. Evin G. Masters C.L. Beyreuther K. Nat. Med. 1997; 3: 328-332Google Scholar). To elucidate further the processing of APLP-1 and APLP-2, we developed a method to detect C-terminal fragments derived from APP, APLP-1, and APLP-2 cleavage within their transmembrane domains by stabilizing the cytosolic domains as chimeric fusion proteins with two z-domains of protein A fused in tandem (2z tag). This method previously enabled us to detect the soluble APP C-terminal fragment (ϵ-CTF or AICD) generated by proteolysis at the ϵ-site, distal to the γ-cleavage site (23Weidemann A. Eggert S. Reinhard F.B. Vogel M. Paliga K. Baier G. Masters C.L. Beyreuther K. Evin G. Biochemistry. 2002; 41: 2825-2835Google Scholar). The analysis of APLP-1 and APLP-2 C-terminal fragments with the 2z tag system revealed a high similarity between the processing of APLPs and APP. Here we show that pharmacological studies with different inhibitors of α- and γ-secretases suggest for APLP-2 the production of several α- and ϵ-like C-terminal fragments, indicating therefore that the processing of this homologue is the most complex of all three APP gene family members. Furthermore, we could detect small secreted fragments in the conditioned media of APLP-2-transfected cell lines that very likely correspond to Aβ and p3-like peptides. For APLP-1, we have identified cellular α- and ϵ-like C-terminal fragments and secreted p3-like fragments using SH-SY5Y cells stably overexpressing APLP-1. In addition, we show that cleavage of APLP-1 can be regulated by N-glycosylation, leading to formation of alternative p3-like fragments. DNA Constructs—To generate the 2z-tagged APP derivatives, 2z-cDNA was amplified by PCR using pQE60–2z as template (23Weidemann A. Eggert S. Reinhard F.B. Vogel M. Paliga K. Baier G. Masters C.L. Beyreuther K. Evin G. Biochemistry. 2002; 41: 2825-2835Google Scholar), a sense primer encoding an XhoI restriction site, and a thrombin (LVPR GS) cleavage (5′-CCCCTCGAGCTGGTTCCGCGTGGATCGAAAGAGGAGAAATTAACC-3′) and an antisense primer encoding a stop codon and a ClaI restriction site. The resulting PCR fragment was subcloned into pBluescript SK+ (Stratagene) by using the XhoI and ClaI restriction sites. Generation of APP695–2z has been described previously (23Weidemann A. Eggert S. Reinhard F.B. Vogel M. Paliga K. Baier G. Masters C.L. Beyreuther K. Evin G. Biochemistry. 2002; 41: 2825-2835Google Scholar). To generate APLP-1-2z, the human APLP-1 cDNA cloned in pBluescript SK+ (4Paliga K. Peraus G. Kreger S. Durrwang U. Hesse L. Multhaup G. Masters C.L. Beyreuther K. Weidemann A. Eur. J. Biochem. 1997; 250: 354-363Google Scholar) was amplified by PCR with a T7 universal primer and an antisense primer that encoded a SalI restriction site just after the APLP-1 coding region (for the following 2z tag ligation). The corresponding 640-bp PCR product was digested with BamHI (coding region) and SalI (3′-end). The original cDNA (APLP-1 in pBluescript) was digested KpnI/BamHI and ligated with the PCR product in pBluescript SK+ (KpnI/SalI). The sequence of the 640-bp product was verified by sequencing. After digestion with KpnI/SalI, the full-length APLP-1 cDNA encoding the additional SalI-site at the 3′-end was ligated with the 2z-cDNA (XhoI/ClaI) into pBluescript SK+, digested with KpnI/ClaI. Subcloning of the APLP-1-2z construct in the expression vector pCEP4 cleaved with KpnI/NheI was performed by using the KpnI/XbaI sites. To generate the APLP-2-763-2z construct, the human APLP-2-763 cDNA cloned in pBluescript SK+ was amplified by PCR with T3 reverse primer and a 3′-antisense primer encoding a SalI restriction site (for the following 2z tag ligation). The PCR product was digested with AatII (in the coding region) and SalI (3′-coding region). The original APLP-2-763 pBluescript SK+ clone was digested with BamHI/AatII, and both fragments were cloned into pBluescript SK+ (cleaved with BamHI/SalI). The XbaI/SalI fragment encoding the full-length cDNA was cloned together with 2z-cDNA (XhoI/SalI cleaved) into pCEP4 (NheI/BamHI cleaved). APLP-1 Deletion Mutants—The human APLP-1 cDNA in pBluescript SK+ (4Paliga K. Peraus G. Kreger S. Durrwang U. Hesse L. Multhaup G. Masters C.L. Beyreuther K. Weidemann A. Eur. J. Biochem. 1997; 250: 354-363Google Scholar) was amplified by PCR using the following primer, APLP-1-2z sense primer (encoding a KpnI restriction site): D568-APLP-1-2z, 5′-GGGGGTACCCACCATGGATGAGCTGGCACCAGCTGGG-3′; R579-APLP-1-2z, 5′-GGGGGTACCCACCATGCGTGAGGCTGTATCGGCGTCTG-3′; M588-APLP-1-2z, 5′-GGGGGTACCCACCATGGGAGCGGGCGGAGGCTCCCTC-3′; M600-APLP-1-2z, 5′-GGGGGTACCCACCATGCTGCTCCTGCGCAGGAAGAAG-3′; and 3′-CGGACCTCCTTGCTGGGCAGCTGGGG-5′ for the antisense primer (encoding a SalI restriction site). The resulting PCR fragments were subcloned in pBluescript SK+, by using the KpnI and SalI restriction sites. The SalI site of the APLP-1 deletion constructs was ligated with the XhoI site of the 2z-DNA in pBluescript (XhoI/ClaI) and cloned into KpnI/ClaI-cleaved pBluescript SK+. The human APLP-2 cDNA in pBluescript SK+ was amplified by PCR using the following primer, APLP-2 deletion mutants, APLP-2 sense primer (encoding a KpnI restriction site): M664-APLP-2-2z, 5′-CCCGGTACCACCATGATTTTCAATGCCGAGAGAG-3′; S691-APLP-2-2z, 5′-CCCGGTACCACCATGAGTAGCAGTGCTCTCATTGG-3′; M715-APLP-2-2z, 5′-CCCGGTACCACCATGCTGAGGAAGAGGCAGTATGG-3′; and 3′-GGACCTCGTCTACGTCTAAACAGCTGGGG-5′ for the antisense primer (encoding a SalI restriction site). The resulting PCR fragments were subcloned into pBluescript SK+, using the KpnI and SalI restriction sites. The SalI site of the APLP-2 deletion constructs was ligated with the XhoI site of the 2z-DNA in pBluescript (XhoI/ClaI) and cloned in the vector pBluescript SK+ KpnI/ClaI. The template APLP-2-2z in pCEP4 was amplified by PCR using the following primer: R678-APLP-2-2z, 5′-CCCGGTACCACCATGCGGGAATCCGTGGGCCCAC-3′(encoding a KpnI restriction site); antisense primer, 3′-CCCTCCTAGGTCTAGAACTTAGCTACCG-5′. The resulting PCR fragments were subcloned in pBluescript SK+, by using the KpnI and SalI restriction sites. The SalI site of the APLP-2 deletion constructs was ligated with the XhoI site of the 2z-DNA in pBluescript (XhoI/ClaI) and cloned into the vector pBluescript SK+KpnI/ClaI. Mutant N551Q APLP-1 was generated by site-directed mutagenesis of the human APLP-1 cDNA in pBluescript SK+ and subcloned into the vector pCEP4. Mutant R567E APLP-1 c-Myc was generated by site-directed mutagenesis of the human APLP-1 cDNA with an C-terminal Myc tag in pBluescript SK+ and subcloned into the vector pCEP4. Antibodies—The human APLP-1-specific antiserum 150 was raised in rabbits using a synthetic peptide corresponding to residues 553–592 of human APLP-1 (46Walsh D.M. Fadeeva J.V. LaVoie M.J. Paliga K. Eggert S. Kimberly W.T. Wasco W. Selkoe D.J. Biochemistry. 2003; 42: 6664-6673Google Scholar). The human APLP-2-specific antiserum 158 was raised in rabbits using the synthetic peptide sequence 672–696 of APLP-2-763, coupled to keyhole limpet hemocyanin. The APLP-1-specific antiserum 57 was raised in rabbits using keyhole limpet hemocyanin conjugates of two peptides, corresponding to human APLP-1 residues 642–650 and residues 604–614 with an additional N-terminal cysteine. An antibody recognizing the APLP-2 C terminus was purchased from Calbiochem (residues 752–763). The polyclonal antibody CT13 against the APP C terminus was raised against a synthetic peptide comprising the C-terminal 13 residues of A4CT (Eurogenetech, Brussels, Belgium) (47Grziwa B. Grimm M.O. Masters C.L. Beyreuther K. Hartmann T. Lichtenthaler S.F. J. Biol. Chem. 2003; 278: 6803-6808Google Scholar). The human APLP-1-specific antiserum 42464 was raised in rabbits using a synthetic peptide raised to the ectodomain of APLP-1 (residues 499–557) (4Paliga K. Peraus G. Kreger S. Durrwang U. Hesse L. Multhaup G. Masters C.L. Beyreuther K. Weidemann A. Eur. J. Biochem. 1997; 250: 354-363Google Scholar). Cell Culture, Transfection, and Inhibitor Treatment—Human SH-SY5Y cells were cultivated and transfected with LipofectAMINE Plus (Invitrogen). For inhibitor experiments cells were treated for 7 h with Me2SO or Me2SO solutions of the following compounds: L-685,458, batimastat (provided by Merck, Sharp & Dohne), TAPI-2 (Peptides International), MDL28170/calpain inhibitor III (benzyloxycarbonyl-Val-Phe-CHO, carbobenzoxy-valinyl-phenylalanyl, Calbiochem), γ-secretase inhibitor IX (DAPT) (Calbiochem), MG132 (benzyloxycarbonyl-LLL-CHO, carbobenzoxy-l-leucyl-l-leucyl-l-leucinal, Calbiochem), ALLN (calpain inhibitor I, N-acetyl-leucyl-leucyl-norleucinal-CHO, Calbiochem), lactacystin (Calbiochem). Cells were pretreated for 1 h with tunicamycin (Sigma) or phorbol 12-myristate 13-acetate (PMA) (Calbiochem) and chased for 4 h. Control cells were treated with Me2SO. Immunoblotting—Lysis of cells and immunoprecipitation of APP, APLP-1, and APLP-2 was performed as described previously (48Weidemann A. Konig G. Bunke D. Fischer P. Salbaum J.M. Masters C.L. Beyreuther K. Cell. 1989; 57: 115-126Google Scholar). 2z-tagged proteins were immunoprecipitated using human IgG immobilized to Sepharose (Amersham Biosciences). For the analysis of the 2z-tagged CTFs, samples were denatured in sample buffer in the absence of reducing agents and electrophoresed on 15% Tris/glycine gels. To investigate the full-length 2z-tagged proteins, samples were denatured in sample buffer with β-mercaptoethanol and separated on 8% Tris/glycine gels. After Western blotting, samples were incubated with a nonspecific rabbit antiserum as the primary antibody followed by anti-rabbit serum coupled to horseradish peroxidase and ECL detection. Metabolic Labeling and Immunoprecipitation—Stably transfected SH-SY5Y cells cultivated in 6-cm dishes were incubated for 7 h with 200 μCi of [35S]methionine/dish (Amersham Biosciences) in 1.5 ml of minimum essential medium lacking methionine (Sigma), and 5% dialyzed fetal calf serum (Sigma). The conditioned media were harvested, centrifuged at 4 °C at 10,000 × g for 10 min to remove cellular debris, subsequently transferred to new microcentrifuge tubes, and finally subjected to immunoprecipitation for 3 h at room temperature. The cells were washed with phosphate-buffered saline, harvested, and lysed in ice-cold lysis buffer (50 mm Tris/HCl, pH 7.5, 150 mm NaCl, 1% Nonidet P-40 (Sigma), 5 mm EDTA, 1% Triton X-100), supplemented with protease inhibitors (Complete™ protease inhibitor mixture, Roche Applied Science). The 10,000 × g supernatants were diluted 1:5 in buffer A (10 mm Tris/HCl, pH 7.5, 150 mm NaCl, 0.2% Nonidet P-40, 2 mm EDTA, supplemented with 0.05% SDS) and subjected to immunoprecipitation for 3 h at room temperature. The Sepharose beads were washed three times with buffer A, twice with buffer B (10 mm Tris/HCl, pH 7.5, 500 mm NaCl, 0,2% Nonidet P-40, 2 mm EDTA), once with buffer A′ (10 mm Tris/HCl, pH 7.5, 150 mm NaCl, 0,2% Nonidet P-40, 2 mm EDTA, supplemented with 0,2% SDS), and once with buffer C (10 mm Tris/HCl, pH 7.5). The beads were resuspended in 40 μl 2× Laemmli SDS sample buffer plus β-mercaptoethanol and heated at 95 °C for 5 min. For analysis of the 2z-tagged CTFs, samples were denatured in sample buffer without β-mercaptoethanol and electrophoresed on 15% Tris/glycine gels. For analysis of the 2z-tagged full-length protein, samples were denatured in sample buffer with β-mercaptoethanol and electrophoresed on 8% Tris/glycine gels. To investigate the APLP-1 wt CTFs and the small secreted peptides, samples were denatured in sample buffer with β-mercaptoethanol and electrophoresed on 16.5% Tris/Tricine gels. The full-length protein was analyzed on 8% Tris/glycine gels. Detection of the C-terminal Fragments of APP695, APLP-1, and APLP-2-763—The investigation of the C-terminal proteolytic fragments of APLP-1 and APLP-2 was performed in analogy to our previous study on APP processing (23Weidemann A. Eggert S. Reinhard F.B. Vogel M. Paliga K. Baier G. Masters C.L. Beyreuther K. Evin G. Biochemistry. 2002; 41: 2825-2835Google Scholar), for which the APP cytosolic domain was stabilized as a chimeric fusion protein with a 2z tag. This tag corresponds to a tandem repeat of the z-domain of protein A that binds to the heavy chain of immunoglobulins. We have shown that the 2z tag does not alter the proteolytic processing of APP but stabilizes its C-terminal fragments and enhances their detection (23Weidemann A. Eggert S. Reinhard F.B. Vogel M. Paliga K. Baier G. Masters C.L. Beyreuther K. Evin G. Biochemistry. 2002; 41: 2825-2835Google Scholar). This method has enabled the detection of a novel ϵ-cleavage site of APP at the C-terminal end of the transmembrane domain, between Leu49 and Val50 (23Weidemann A. Eggert S. Reinhard F.B. Vogel M. Paliga K. Baier G. Masters C.L. Beyreuther K. Evin G. Biochemistry. 2002; 41: 2825-2835Google Scholar). Thus, the 2z tag was fused to the C termini of APLP-1 and APLP-2-763, and these constructs, which we termed APLP-1-2z and APLP-2-2z, respectively, were overexpressed in SH-SY5Y cells. Cell lysates from SH-SY5Y cells, stably transfected with either APP-2z, APLP-1-2z, or APLP-2-2z, were incubated with IgG-coupled Sepharose to precipitate 2z-encoding full-length proteins and C-terminal fragments (Fig. 1). Cells transfected with an empty vector were used as a control. Classification of the C-terminal fragments of APLP-1-2z and APLP-2-2z in Fig. 1 is based on pharmacological studies, and the size of these fragments is compared with deletion mutants as shown below. For APP-2z cells, two major fragments of 27 and 23 kDa were immunoprecipitated (Fig. 1A, lane 1) that represent the C-terminal cleavage products derived from APP processing at α- and ϵ-sites, respectively, as determined previously by N-terminal sequence analysis (23Weidemann A. Eggert S. Reinhard F.B. Vogel M. Paliga K. Baier G. Masters C.L. Beyreuther K. Evin G. Biochemistry. 2002; 41: 2825-2835Google Scholar). The two upper bands at 29 and 28 kDa correspond to C-terminal
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