Regulation of Amyloid Protein Precursor (APP) Binding to Collagen and Mapping of the Binding Sites on APP and Collagen Type I
1996; Elsevier BV; Volume: 271; Issue: 3 Linguagem: Inglês
10.1074/jbc.271.3.1613
ISSN1083-351X
AutoresDirk Beher, Lars Hesse, Colin L. Masters, Gerd Multhaup,
Tópico(s)Connective tissue disorders research
ResumoThe specific binding of the amyloid precursor protein (APP) to extracellular matrix molecules suggests that APP regulates cell interactions and has a function as a cell adhesion molecule and/or substrate adhesion molecule. On the molecular level APP has binding sites for collagen, laminin, and glycosaminoglycans which is a characteristic feature of cell adhesion molecules. We have examined the interactions between the APP and collagen types I and IV and identified the corresponding binding sites on APP and collagen type I.We show that APP bound most efficiently to collagen type I in a concentration-dependent and specific manner in the native and heat-denatured states, suggesting an involvement of a contiguous binding site on collagen. This binding site was identified on the cyanogen bromide fragment α1(I)CB6 of collagen type I, which also binds heparin. APP did not bind to collagen type I-heparin complexes, which suggests that there are overlapping binding sites for heparin and APP on collagen. We localized the site of APP that mediates collagen binding within residues 448-465 of APP695, which are encoded by the ubiquitously expressed APP exon 12, whereas the high affinity heparin binding site of APP is located in exon 9. Since a peptide encompassing this region binds to collagen type I and inhibits APP-collagen type I binding in nanomolar concentrations, this region may comprise the major part of the collagen type I binding site of APP. Moreover, our data also indicate that the collagen binding site is involved in APP-APP interaction that can be modulated by Zn(II) and heparin. Taken together, the data suggest that the regulation of APP binding to collagen type I by heparin occurs through the competitive binding of heparin and APP to collagen. The specific binding of the amyloid precursor protein (APP) to extracellular matrix molecules suggests that APP regulates cell interactions and has a function as a cell adhesion molecule and/or substrate adhesion molecule. On the molecular level APP has binding sites for collagen, laminin, and glycosaminoglycans which is a characteristic feature of cell adhesion molecules. We have examined the interactions between the APP and collagen types I and IV and identified the corresponding binding sites on APP and collagen type I. We show that APP bound most efficiently to collagen type I in a concentration-dependent and specific manner in the native and heat-denatured states, suggesting an involvement of a contiguous binding site on collagen. This binding site was identified on the cyanogen bromide fragment α1(I)CB6 of collagen type I, which also binds heparin. APP did not bind to collagen type I-heparin complexes, which suggests that there are overlapping binding sites for heparin and APP on collagen. We localized the site of APP that mediates collagen binding within residues 448-465 of APP695, which are encoded by the ubiquitously expressed APP exon 12, whereas the high affinity heparin binding site of APP is located in exon 9. Since a peptide encompassing this region binds to collagen type I and inhibits APP-collagen type I binding in nanomolar concentrations, this region may comprise the major part of the collagen type I binding site of APP. Moreover, our data also indicate that the collagen binding site is involved in APP-APP interaction that can be modulated by Zn(II) and heparin. Taken together, the data suggest that the regulation of APP binding to collagen type I by heparin occurs through the competitive binding of heparin and APP to collagen. INTRODUCTIONThe amyloid precursor protein (APP) 1The abbreviations used are: APPamyloid protein precursorAPPsα-secretase cleaved form of APPhs-APPAPP purified from human brainrn-APPAPP purified from rat brainβA4βA4 amyloid proteinAPLPamyloid precursor like proteinBSAbovine serum albuminPBSphosphate-buffered salinePAGEpolyacrylamide gel electrophoresisCBPcollagen-binding peptideNCAMneural cell adhesion molecule. belongs to a gene family in which three genes are known. In addition to the APP gene, the genes for the amyloid precursor-related proteins APLP1 and APLP2 map to the human chromosomes 19 (APLP1) (1.Wasco W. Bupp K. Magendantz M. Gusella J.F. Tanzi R.E. Solomon F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10758-10762Crossref PubMed Scopus (320) Google Scholar) and 11 (APLP2)(2.Wasco W. Gurubhagavatula S. Dparadis M. Romano D.M. Sisodia S.S. Hyman B.T. Neve R.L. Tanzi R.E. Nature Genet. 1993; 5: 95-100Crossref PubMed Scopus (320) Google Scholar, 3.Sprecher C.A. Grant F.J. Grimm G. O'Hara P.J. Norris F. Norris K. Foster D.C. Biochemistry. 1993; 32: 4481-4486Crossref PubMed Scopus (160) Google Scholar). APP is encoded by the APP gene on the long arm of human chromosome 21 and has attracted attention due to its involvement in the deposition of amyloid βA4 protein in the brain of patients with familial and sporadic Alzheimer's disease and individuals with trisomy 21(4.Müller-Hill B. Beyreuther K. Annu. Rev. Biochem. 1989; 58: 287-307Crossref PubMed Scopus (189) Google Scholar, 5.Selkoe D.J. Annu. Rev. Neurosci. 1994; 17: 489-517Crossref PubMed Scopus (825) Google Scholar).The role of APP in the pathogenesis of Alzheimer's disease has been underscored by the discovery of mutations within the βA4 or sequences flanking the βA4(6.Goate A. Chartier-Harlin M.-C. Mullan M. Brown J. Crawford F. Fidani L. Giuffra L. Haynes A. Irving N. James L. Mant R. Newton P. Rooke K. Roques P. Talbot C. Pericak-Vance M. Roses A. Rossor M. Owen M. Hardy J. 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Johnson-Wood K. Beattie E.C. Ward P.J. Blacher R.W. Dovey H.F. Sinha S. Nature. 1989; 341: 144-147Crossref PubMed Scopus (367) Google Scholar) and are endocytosed by the low density lipoprotein receptor-related protein(13.Kounnas M.Z. Moir R.D. Rebeck G.W. Bush A.I. Argraves W.S. Tanzi R.E. Hyman B.T. Strickland D.K. Cell. 1995; 82: 331-340Abstract Full Text PDF PubMed Scopus (442) Google Scholar). A possible in vivo function is provided by the discovery that APP is a very potent inhibitor of factor XIa, and APP-factor XIa complexes might be involved in the regulation of the coagulation cascade(14.Smith R.P. Higuchi D.A. Broze G.J. Science. 1990; 248: 1126-1128Crossref PubMed Scopus (253) Google Scholar, 15.Van Nostrand W.E. Schmaier A.H. Farrow J.S. Cunningham D.D. 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A. 1993; 90: 10150-10153Crossref PubMed Scopus (156) Google Scholar, 25.Narindrasorasak S. Lowery D.E. Altman R.A. Gonzalez-DeWhitt P. Greenberg B.D. Kisilevsky R. Lab. Invest. 1992; 67: 643-652PubMed Google Scholar, 26.Small D.H. Nurcombe V. Reed G. Clarris H. Moir R. Beyreuther K. Masters C.L. J. Neurosci. 1994; 14: 2117-2127Crossref PubMed Google Scholar).APP has been shown to bind Zn(II) and Cu(II) at two distinct sites (27.Bush A.I. Multhaup G. Moir R.D. Williamson T.G. Small D.H. Rumble B. Pollwein P. Beyreuther K. Masters C.L. J. Biol. Chem. 1993; 268: 16109-16112Abstract Full Text PDF PubMed Google Scholar, 28.Hesse L. Beher D. Masters C.L. Multhaup G. FEBS Lett. 1994; 349: 109-116Crossref PubMed Scopus (221) Google Scholar) and was recently found to belong to a family of zinc-modulated, heparin-binding proteins(29.Bush A.I. Pettingell Jr., W.H. DeParadis M. Tanzi R.E. Wasco W. J. Biol. Chem. 1994; 269: 26618-26621Abstract Full Text PDF PubMed Google Scholar). Zn(II) binding was shown to strengthen the binding of APP to heparin, thus demonstrating an interaction of residues involved in ligand binding which are located in different domains(21.Multhaup G. Biochimie (Paris). 1994; 76: 304-311Crossref PubMed Scopus (64) Google Scholar, 27.Bush A.I. Multhaup G. Moir R.D. Williamson T.G. Small D.H. Rumble B. Pollwein P. Beyreuther K. Masters C.L. J. Biol. Chem. 1993; 268: 16109-16112Abstract Full Text PDF PubMed Google Scholar). Binding of metallic cations like Zn(II) and Cu(II) may control APP conformation and stability (27.Bush A.I. Multhaup G. Moir R.D. Williamson T.G. Small D.H. Rumble B. Pollwein P. Beyreuther K. Masters C.L. J. Biol. Chem. 1993; 268: 16109-16112Abstract Full Text PDF PubMed Google Scholar, 28.Hesse L. Beher D. Masters C.L. Multhaup G. FEBS Lett. 1994; 349: 109-116Crossref PubMed Scopus (221) Google Scholar, 30.Multhaup G. Bush A.I. Pollwein P. Masters C.L. FEBS Lett. 1994; 355: 151-154Crossref PubMed Scopus (90) Google Scholar) and thus may promote the binding of APP to extracellular matrix elements like heparan sulfate proteoglycans(30.Multhaup G. Bush A.I. Pollwein P. Masters C.L. FEBS Lett. 1994; 355: 151-154Crossref PubMed Scopus (90) Google Scholar).We report here the regulation of APP binding to collagen type I by heparin and the mapping of the binding sites for APP to collagen type I and vice versa. Our binding studies reveal that APP binding to collagen type I is mediated by residues 448-465. Since synthetic peptides representing this region show self-aggregational properties, it is suggested that APP-APP binding may exist. APP was identified in the human platelet to be present in membrane associated and soluble forms (31.Bush A.I. Martins R.N. Rumble B. Moir R. Fuller S. Milward E. Currie J. Ames D. Weidemann A. Fischer P. Multhaup G. Beyreuther K. Masters C.L. J. Biol. Chem. 1990; 265: 15977-15983Abstract Full Text PDF PubMed Google Scholar), and collagen is particularly important for initiation of platelet activation leading to successful formation of a hemostatic plug(32.Santoro S.A. Cell. 1986; 46: 913-920Abstract Full Text PDF PubMed Scopus (289) Google Scholar). For this reason our findings suggest that APP as an adhesive glycoprotein may participate in collagen-induced platelet aggregation and as APPs may take part in the regulation of the coagulation cascade at sites of vascular injury.EXPERIMENTAL PROCEDURESMaterialsCollagen type I from bovine and human tissues (Sigma C-3511, C-7774) and collagen type IV (human placenta, Sigma C-7521) were used.Different collagen types were dissolved in 10 mM NaOAc, pH 5.5, and diluted with 10 mM Tris-HCl, pH 7.5, containing 100 mM NaCl, to appropriate concentrations just before use.Protein concentrations were determined by amino acid analysis.Collagen type I was dissolved at 10 mg/ml in 70% formic acid, and solid CNBr (Merck) was added to a final concentration of 20 mg/ml. Digestion was allowed to proceed for 18 h at 4°C.Peptides were synthesized according to published methods (33.Barany G. Merrifield B. Gross E. Meienhofer J. The Peptides. 2. Academic Press, Inc., New York1981Google Scholar) and purified on C-18 columns with a linear gradient of acetonitrile in 0.1% trifluoroacetic acid. Sequences were confirmed by amino acid sequence analysis (477A, Applied Biosystems).Construction of Expression VectorsThe plasmid pFd770sIgA is based on the procaryotic expression vector pFd770IgA(28.Hesse L. Beher D. Masters C.L. Multhaup G. FEBS Lett. 1994; 349: 109-116Crossref PubMed Scopus (221) Google Scholar). For in vitro mutagenesis according to Kunkel(34.Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4886) Google Scholar), M13mp18 replicative form DNA was cut with SalI-HindIII and religated in the presence of oligonucleotides TCGACCTGACTAGTTGCA and AGCTTGCAACTAGTCAGG to create a SpeI restriction site. A SacI-SpeI fragment of pFd770IgA was cloned into M13mp18-SpeI, and the oligonucleotide CCCACATCTTCTGCAAATCACTATTATTTTTGATGATGAAC was annealed to the single-stranded DNA. The mutated sequence was exchanged with the corresponding fragment of pFd770IgA (28.Hesse L. Beher D. Masters C.L. Multhaup G. FEBS Lett. 1994; 349: 109-116Crossref PubMed Scopus (221) Google Scholar) to construct the plasmid pFd770sIgA.Purification of APPAPP695 was isolated from rat brain as described previously(21.Multhaup G. Biochimie (Paris). 1994; 76: 304-311Crossref PubMed Scopus (64) Google Scholar).A full-length (Fd-APP770) and truncated recombinant forms of human APP (Fd-APPN262, TP-APP770s) were prepared and purified in the form of a prokaryotic expressed Fd fusion protein by methods essentially as described by Weidemann et al.(35.Weidemann A. König G. Bunke D. Fischer P. Salbaum J.M. Masters C.L. Beyreuther K. Cell. 1989; 57: 115-126Abstract Full Text PDF PubMed Scopus (1035) Google Scholar). TP-APP770s represents the secreted form of APP770 (α-secretase cleaved)(5.Selkoe D.J. Annu. Rev. Neurosci. 1994; 17: 489-517Crossref PubMed Scopus (825) Google Scholar).After preparative SDS-PAGE and electroelution, the soluble protein was separated from salts and SDS by Excellulose GF-5 columns (Pierce).Protein concentrations were determined by amino acid analysis according to the manufacturer's protocol after hydrolysis with 6 N HCl, 0.1% phenol for 24 h at 110°C (420A Amino Acid Analysis System, Applied Biosystems).Binding Assay of Type I CollagenThe binding of proteins to collagen was determined by either dot blot or a solid-phase assay. The dot blot assay was performed as described previously for APP-heparin binding(21.Multhaup G. Biochimie (Paris). 1994; 76: 304-311Crossref PubMed Scopus (64) Google Scholar). The inhibitory effect of heparin was studied in the dot blot assay after preincubation of 3.5 pmol of 125I-rn-APP with 125 μg/ml heparin in 1 × PBS for 4 h at room temperature and separating the protein from salts and free heparin by Excellulose GF-5 columns (Pierce) and preincubation of dots coated with collagen in the presence of 40 μg/ml heparin in 1 × PBS for 1 h.After adsorption of collagen to microtiter wells, nonspecific binding sites were blocked by incubation for longer than 1 h with 1% BSA in PBS. 125I-Labeled proteins were diluted with PBS and added to the wells (1 × 103 to 2 × 106 cpm/well), together with the reagents to be tested. After a 3-h incubation at room temperature, the wells were emptied. The plate was divided into separate wells, and the radioactivity of bound ligand was measured by liquid scintillation counting.The value obtained with wells coated with BSA was subtracted, as this represents nonspecific binding. Results were expressed as the mean of duplicate determinations, which usually did not differ by more than 10%.Determination of the Collagen Binding Domain of APPPurified rat brain APP695 and fusion proteins Fd-APP770 and Fd-APPN262 were incubated at 37°C overnight with endoproteinase Lys-C (5 μg) (Boehringer Mannheim). After inactivating the protease for 1 min at 110°C, fragments were incubated in PBS overnight at room temperature with collagen type I bound to microtiter wells. After being washed three times with cold PBS, bound peptides were extracted with formic acid. High performance liquid chromatography revealed one major fragment (16 kDa) in the eluate. The NH2-terminal sequence was AAQIRSQVMTHLRVIYERMNQSLSLLYNVPAVA, corresponding to residues 448-480 of APP695. Similar digestions of 125I-rn-APP generated the same sized fragment as visualized by SDS-PAGE and autoradiography.Synthetic peptides representing candidates for the collagen binding region on APP and control peptides were dissolved in 0.1 × PBS and tested as competing ligands for 125I-APP-collagen type I (human placenta) binding. Collagen type I was dot-blotted in duplicate onto nitrocellulose (0.05-10 μg) and nonspecific binding sites were blocked with 1% BSA in 1 × PBS for 1 h at room temperature. The dot blot was cut into strips and incubated with 125I-APP together with and without competing peptides for 3 h at room temperature in 0.1 × PBS. After incubation the dot blot was washed one time with blocking buffer, and the dots were excised, placed in scintillant, and assayed by counting. Variations of this binding assay were occasionally used and are indicated in the text and figure legends.Determination of the APP Binding Domain of CollagenIn order to separate chains α1(I) and α2(I) of collagen type I, 2-3 mg of protein was heat-denatured and loaded onto 6% SDS-polyacrylamide gels (α1(I)CB6 fragment: 15% polyacrylamide gel). Protein was electroeluted and extensively dialyzed against 1 × HEPES buffered saline (10 mM HEPES, 150 mM NaCl, 0.005% Nonidet P-40, pH 7.4) to remove the SDS, followed by dialysis against 0.1 M acetic acid. Purity of each preparation was checked by reverse phase-high performance liquid chromatography (Aquapore RP-300, buffer A: 0.1% trifluoroacetic acid, buffer B: 0.098% trifluoroacetic acid in 70% CH3CN, gradient from 0% buffer B to 100% buffer B in 60 min).CNBr cleavage of isolated chains was performed as described(36.Gross E. Witkop B. J. Am. Chem. Soc. 1961; 83: 1510-1511Crossref Scopus (190) Google Scholar). The identity of the α1(I)CB6 fragment has been confirmed by NH2-terminal sequencing.APP Binding to the Synthetic Peptide CBP (Residues 448-480)A synthetic peptide corresponding to residues 448-480 of APP695 was adsorbed to microtiter wells (0.5-5 μg/well) and incubated with 1% BSA in PBS for 1 h, 125I-labeled hs-APP was added (1 × 103 to 2 × 106 cpm/well) together with the competitors to be tested in PBS. After a 3-h incubation at room temperature the wells were emptied, dried, cut, and subjected to liquid scintillation counting. The mean of duplicate measurements is given which was corrected for nonspecific binding (BSA alone).Adsorption of Protein to Microtiter WellsMicrotiter wells (Falcon, microtest III, 96 flatbottom wells, flexible) were coated with either 0.05-10 μg of collagen/well or 1% BSA in 200 μl of PBS by incubation at room temperature overnight. After a brief rinse, nonspecific binding sites in the wells were blocked for 1 h with 200 μl of 1% BSA in PBS. The wells were then rinsed with PBS and prepared for the appropriate assay.To determine the amount of collagen adsorbed to microtiter wells, 5 μg of each collagen type were allowed to adsorb in microtiter wells overnight followed by a brief rinse. Then protein was extracted from microtiter wells with three successive rinses of 100 μl of formic acid and subjected to amino acid analysis after hydrolysis. The amount of protein adsorbed to the microtiter wells was quantified and calculated to be 50% of the amount of that was loaded.Ligand BlotProteins were denatured for 5 min in SDS-sample buffer, separated by SDS-PAGE, and transferred to nitrocellulose filters. Nonspecific binding sites were blocked with 1% BSA in 1 × PBS for 1 h at room temperature. Incubation was done either with 125I-APP (2 × 104 cpm/ml) or 35S-heparin (2.5 × 105 cpm/ml) in hypotonic binding buffer (14 mM NaCl, 0.27 mM KCl, 0.81 mM Na2HPO4× 2H2O, 0.15 mM KH2PO4) for 3 h at room temperature. Filters were then washed three times for 5 min with binding buffer and exposed to Kodak XAR-5 films at −70°C.Iodination of APP105-kDa rat brain full-length APP was iodinated by the chloramine-T method (IODO-BEADS, Pierce) and separated from free iodine by heparin-Sepharose chromatography.RESULTSCharacterization of Binding of APP to CollagenDuring our initial overlay studies using 125I-rn-APP to identify the cell surface receptor of APP that had earlier been proposed to exist(37.Kang J. Lemaire H.G. Unterbeck A. Salbaum J.M. Masters C.L. Grzeschik K.H. Multhaup G. Beyreuther K. Müller-Hill B. Nature. 1987; 325: 733-736Crossref PubMed Scopus (3916) Google Scholar), we observed an APP-collagen interaction and decided to study this in more detail.First, we tested which different collagen types interact with APP. A dot blot assay revealed that 125I-labeled rn-APP binding to fibrillar collagen type I and basement membrane collagen type IV (38.Vuorio E. deCrombrugghe B. Annu. Rev. Biochem. 1990; 59: 837-872Crossref PubMed Scopus (384) Google Scholar) was saturable and specific. APP bound most efficiently to collagen type I (data not shown).To investigate the kinetics of APP binding to collagen types I and IV and to gelatin in more detail, we used two distinct assays. Radiolabeled rn-APP was incubated in microtiter wells with immobilized collagen types I and IV at room temperature in microtiter wells or BSA as a control. APP-gelatin interaction was tested by incubating the protein with gelatin-Sepharose beads.The binding efficiency was observed to depend on salt concentration and showed substrate specificity (binding to collagens, but not to other extracellular matrix components, such as fibronectin or vitronectin). At the specific activity used, 10% of the 125I-labeled rn-APP added bound to collagen type I in a dot blot assay. Binding was saturable and analyzed by using the methodology of Scatchard(39.Scatchard G. Ann. N. Y. Acad. Sci. 1949; 51: 660-672Crossref Scopus (17759) Google Scholar). Assuming the presence of only one binding site on the collagen types I and IV, Kd values in the range of 0.5 × 10-8M for collagen type I (Fig. 1), 4.5 × 10-8M for collagen type IV, and 1.5 × 10-8 for gelatin (data not shown) were calculated for the binding of APP to native and heat-denatured collagen (gelatin).Determination of the Collagen Binding Domain of APPTo map the putative and single collagen binding site, we used proteolytic fragments of purified fusion protein APP (Fd-APP770) obtained by endoproteinase Lys-C digestion. A mixture of fragments was incubated with collagen type I that had been immobilized to microtiter plates. Proteolytic fragments of APP that specifically bound to collagen type I were extracted from microtiter wells with formic acid. Amino acid sequence analysis of the only resulting fragment revealed the sequence to commence at residue 448 of APP695 (Fig. 2). Other peaks were found to be caused by collagen (α1 and α2 chains of collagen type I elute between 20 and 25 min, Fig. 2) that was initially immobilized to the wells and was co-extracted with formic acid.Figure 2:HPLC purification of a putative collagen binding peptide obtained by endoproteinase Lys-C digestion of Fd-APP770 encoded by exons 1-18 of APP (A) and Fd-APPN262 encoded by exons 1-6 of APP (B). Only Fd-APP770 yielded the collagen binding peptide (black bar) determined by amino acid sequence analysis. Other fractions (hatched area) were revealed to contain collagen type I chains or fragments thereof.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To confirm that we identified the collagen binding site of APP, three peptides of residues 448-465, 448-480, and 471-493 (Table 1) were synthesized to perform inhibition studies. Two of these peptides were found to inhibit strongly the binding of 125I-hs-APP to collagen type I in a dot blot assay, with a calculated IC50 value of 150 nM (CBP1) and 75 nM (CBP) (Fig. 3), whereas control peptides did not have any influence at concentrations of up to 20 μg/ml. As an independent control we used iodinated NCAM140kDa purified from rat brain according to Probstmeier et al. (40.Probstmeier R. Kühn K. Schachner M. J. Neurochem. 1989; 53: 1794-1801Crossref PubMed Scopus (92) Google Scholar) and did not observe any inhibition of NCAM binding to collagen in the presence of CBP concentrations of up to 5 μg/ml (data not shown).TABLE 1 Open table in a new tab Figure 3:Dose-dependent inhibition of the binding of 125I-hs-APP to collagen by the synthetic peptides CBP (diamonds), CBP1 (open squares), and CBP2 (filled squares). The values represent residual binding determined in dependence of the concentration of the synthetic peptides as the competing ligands in a dot blot assay.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Since APP belongs to a superfamily, we further investigated synthetic peptides that represent homologous sequences of CBP in the APLP to inhibit APP binding to collagen type I. APP-collagen type I interaction could be less effectively inhibited by synthetic peptides of mouse APLP1 (residues 445-477) (1.Wasco W. Bupp K. Magendantz M. Gusella J.F. Tanzi R.E. Solomon F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10758-10762Crossref PubMed Scopus (320) Google Scholar) and human ALPL2 (2.Wasco W. Gurubhagavatula S. Dparadis M. Romano D.M. Sisodia S.S. Hyman B.T. Neve R.L. Tanzi R.E. Nature Genet. 1993; 5: 95-100Crossref PubMed Scopus (320) Google Scholar, 3.Sprecher C.A. Grant F.J. Grimm G. O'Hara P.J. Norris F. Norris K. Foster D.C. Biochemistry. 1993; 32: 4481-4486Crossref PubMed Scopus (160) Google Scholar) (residues 522-554). For both peptides, inhibition was found to be dose-dependent with an IC50 of about 1 μM (5 μg/ml) in a dot blot assay (data not shown). But mouse APLP1 and human APLP2 peptides (Table 2) were still able to inhibit binding of APP to collagen type I although with a reduced capacity by a factor of 10. A possible explanation for this is given by the secondary structure prediction for CBP/hs-APP, CBP/mm-APLP1, and CBP/hs-APLP2 according to Chou and Fasman (Table 3; (41.Chou P.Y. Fasman G.D. Adv. Enzymol. 1978; 47: 45-148PubMed Google Scholar)) that changes from β-structure for the NH2 terminus of CBP/APP to α-structure in the same site of APLPs (Table 3).TABLE 2 Open table in a new tab TABLE 3 Open table in a new tab Furthermore, direct binding of peptides CBP and CBP1 to collagen type I was proven by surface plasmon resonance (BIAcore, Pharmacia Biotech Inc.). Collagen type I was immobilized to the dextran surface of the sensorchip(21.Multhaup G. Biochimie (Paris). 1994; 76: 304-311Crossref PubMed Scopus (64) Google Scholar), but the kinetics of the binding reaction could not be determined, because two independent binding events, CBP to collagen and CBP to CBP, were observed to superimpose. This led us to the conclusion that both peptides showed a strong tendency for self-aggregation and suggested that CBP could represent a binding site for APP.If APP-APP binding could occur through the CBP sequence, APP should bind specifically to the synthetic peptide CBP. The binding was analyzed in
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