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• W2015510614 abstract "α2-Macroglobulin (α2M) and its receptor, low density lipoprotein receptor-related protein (LRP), function together to facilitate the cellular uptake and degradation of β-amyloid peptide (Aβ). In this study, we demonstrate that Aβ binds selectively to α2M that has been induced to undergo conformational change by reaction with methylamine. Denatured α2M subunits, which were immobilized on polyvinylidene difluoride membranes, bound Aβ, suggesting that α2M tertiary and quaternary structure are not necessary. To determine whether a specific sequence in α2M is responsible for Aβ binding, we prepared and analyzed defined α2M fragments and glutathione S-transferase-α2M peptide fusion proteins. A single sequence, centered at amino acids (aa) 1314–1365, was identified as the only major Aβ-binding site. Importantly, Aβ did not bind to the previously characterized growth factor-binding site (aa 718–734). Although the Aβ binding sequence is adjacent to the binding site for LRP, the results of experiments with mutated fusion proteins indicate that the two sites are distinct. Furthermore, a saturating concentration of Aβ did not inhibit LRP-mediated clearance of α2M-MA in mice. Using various methods, we determined that the KD for the interaction of Aβ with its binding site in the individual α2M subunit is 0.7–2.4 μm. The capacity of α2M to bind Aβ and deliver it to LRP may be greater than that predicted by the KD, because each α2M subunit may bind Aβ and the bound Aβ may multimerize. These studies suggest a model in which α2M has three protein interaction sites with distinct specificities, mediating the interaction with Aβ, growth factors, and LRP. α2-Macroglobulin (α2M) and its receptor, low density lipoprotein receptor-related protein (LRP), function together to facilitate the cellular uptake and degradation of β-amyloid peptide (Aβ). In this study, we demonstrate that Aβ binds selectively to α2M that has been induced to undergo conformational change by reaction with methylamine. Denatured α2M subunits, which were immobilized on polyvinylidene difluoride membranes, bound Aβ, suggesting that α2M tertiary and quaternary structure are not necessary. To determine whether a specific sequence in α2M is responsible for Aβ binding, we prepared and analyzed defined α2M fragments and glutathione S-transferase-α2M peptide fusion proteins. A single sequence, centered at amino acids (aa) 1314–1365, was identified as the only major Aβ-binding site. Importantly, Aβ did not bind to the previously characterized growth factor-binding site (aa 718–734). Although the Aβ binding sequence is adjacent to the binding site for LRP, the results of experiments with mutated fusion proteins indicate that the two sites are distinct. Furthermore, a saturating concentration of Aβ did not inhibit LRP-mediated clearance of α2M-MA in mice. Using various methods, we determined that the KD for the interaction of Aβ with its binding site in the individual α2M subunit is 0.7–2.4 μm. The capacity of α2M to bind Aβ and deliver it to LRP may be greater than that predicted by the KD, because each α2M subunit may bind Aβ and the bound Aβ may multimerize. These studies suggest a model in which α2M has three protein interaction sites with distinct specificities, mediating the interaction with Aβ, growth factors, and LRP. β-amyloid peptide β-amyloid precursor protein Alzheimer's disease α2-macroglobulin transforming growth factor-β nerve growth factor-β low density lipoprotein receptor-related protein methylamine-activated α2M platelet-derived growth factor-BB bovine serum albumin dithiothreitol polyvinylidene fluoride bis(sulfosuccinimidyl) suberate receptor-associated protein glutathione S-transferase bovine serum albumin receptor binding fragment fusion protein amino acid(s) Accumulation of β-amyloid peptide (Aβ(1–40) and Aβ(1–42))1 in the brain plays a central role in the development and progression of Alzheimer's disease (AD) (1.Sisodia S.S. Price D.L. FASEB J. 1995; 9: 366-370Crossref PubMed Scopus (226) Google Scholar). Mutations in β-amyloid precursor protein (APP), which result in increased production of Aβ, are associated with autosomal dominant forms of familial AD in humans (2.Citron M. Oltersdorf T. Haass C. McConlogue L. Hung A.Y. Seubert P. Vigo-Pelfrey C. Lieberburg I. Selkoe D.J. Nature. 1992; 360: 672-674Crossref PubMed Scopus (1537) Google Scholar, 3.Cai X.D. Golde T.E. Younkin S.G. Science. 1993; 259: 514-516Crossref PubMed Scopus (835) Google Scholar, 4.Hardy J. Trends Neurosci. 1997; 20: 154-159Abstract Full Text Full Text PDF PubMed Scopus (1277) Google Scholar). Mutated forms of human APP may also induce changes consistent with AD when expressed as transgenes in mice (5.Games D. Adams D. Alessandrini R. Barbour R. Berthelette P. Blackwell C. Carr T. Clemens J. Donaldson T. Gillespie F. et al.Nature. 1995; 373: 523-527Crossref PubMed Scopus (2251) Google Scholar, 6.Chen G. Chen K.S. Knox J. Inglis J. Bernard A. Martin S.J. Justice A. McConlogue L. Games D. Freedman S.B. Morris R.G. Nature. 2000; 408: 975-979Crossref PubMed Scopus (9) Google Scholar, 7.Terai K. Iwai A. Kawabata S. Sasamata M. Miyata K. Yamaguchi T. Brain Res. 2001; 900: 48-56Crossref PubMed Scopus (30) Google Scholar, 8.Sturchler-Pierrat C. Abramowski D. Duke M. Wiederhold K.H. Mistl C. Rothacher S. Ledermann B. Burki K. Frey P. Paganetti P.A. Waridel C. Calhoun M.E. Jucker M. Probst A. Staufenbiel M. Sommer B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13287-13292Crossref PubMed Scopus (1255) Google Scholar). Furthermore, immunization with Aβ(1–42) prevents progression of AD in animal model systems and may reverse symptoms by promoting resorption of Aβ-containing plaques (9.Schenk D. Barbour R. Dunn W. Gordon G. Grajeda H. Guido T. Hu K. Huang J. Johnson-Wood K. Khan K. Kholodenko D. Lee M. Liao Z. Lieberburg I. Motter R. Mutter L. Soriano F. Shopp G. Vasquez N. Vandevert C. Walker S. Wogulis M. Yednock T. Games D. Seubert P. Nature. 1999; 400: 173-177Crossref PubMed Scopus (2965) Google Scholar, 10.Janus C. Pearson J. McLaurin J. Mathews P.M. Jiang Y. Schmidt S.D. Chishti M.A. Horne P. Heslin D. French J. Mount H.T. Nixon R.A. Mercken M. Bergeron C. Fraser P.E. St. George-Hyslop P. Westaway D. Nature. 2000; 408: 979-982Crossref PubMed Scopus (1379) Google Scholar). These results suggest that Aβ accumulation in the brain is a dynamic and reversible process. Proteins other than antibodies with the capacity to bind Aβ and promote its catabolism may influence disease progression. α2-Macroglobulin (α2M) is a 718-kDa homotetrameric glycoprotein, which is well characterized as an extracellular proteinase inhibitor (11.Sottrup-Jensen L. Putnam F.W. The Plasma Proteins. 5. Academic Press, Inc., Orlando, FL1987: 192-291Google Scholar) and as a carrier of specific growth factors, including transforming growth factor-β (TGF-β) and nerve growth factor-β (NGF-β) (12.Crookston K.P. Webb D.J. Wolf B.B. Gonias S.L. J. Biol. Chem. 1994; 269: 1533-1540Abstract Full Text PDF PubMed Google Scholar, 13.Gonias S.L. LaMarre J. Crookston K.P. Webb D.J. Wolf B.B. Lopes M.B. Moses H.L. Hayes M.A. Ann. N. Y. Acad. Sci. 1994; 737: 273-290Crossref PubMed Scopus (78) Google Scholar). At least two separate polymorphisms in the A2M gene may be associated with increased risk of late-onset AD. The first involves a region within intron 17, at the 5′ splice acceptor site for exon 18 (14.Blacker D. Wilcox M.A. Laird N.M. Rodes L. Horvath S.M. Go R.C. Perry R. Watson Jr., B. Bassett S.S. McInnis M.G. Albert M.S. Hyman B.T. Tanzi R.E. Nat. Genet. 1998; 19: 357-360Crossref PubMed Scopus (585) Google Scholar). This exon is important because it encodes part of the bait region, where proteinases initiate reaction with α2M by cleaving susceptible peptide bonds (15.Barrett A.J. Brown M.A. Sayers C.A. Biochem. J. 1979; 181: 401-418Crossref PubMed Scopus (413) Google Scholar, 16.Sottrup-Jensen L. Sand O. Kristensen L. Fey G.H. J. Biol. Chem. 1989; 264: 15781-15789Abstract Full Text PDF PubMed Google Scholar), and a segment of the growth factor binding sequence (17.Webb D.J. Wen J. Karns L.R. Kurilla M.G. Gonias S.L. J. Biol. Chem. 1998; 273: 13339-13346Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 18.Gonias S.L. Carmichael A. Mettenburg J.M. Roadcap D.W. Irvin W.P. Webb D.J. J. Biol. Chem. 2000; 275: 5826-5831Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 19.Webb D.J. Roadcap D.W. Dhakephalkar A. Gonias S.L. Protein Sci. 2000; 9: 1986-1992Crossref PubMed Scopus (28) Google Scholar). In the second A2M gene polymorphism, Val-1000 is replaced by Ile (20.Liao A. Nitsch R.M. Greenberg S.M. Finckh U. Blacker D. Albert M. Rebeck G.W. Gomez-Isla T. Clatworthy A. Binetti G. Hock C. Mueller-Thomsen T. Mann U. Zuchowski K. Beisiegel U. Staehelin H. Growdon J.H. Tanzi R.E. Hyman B.T. Hum. Mol. Genet. 1998; 7: 1953-1956Crossref PubMed Scopus (153) Google Scholar). The linkage of A2M gene polymorphisms to late-onset AD remains incompletely understood, because the original observations have been confirmed in only a limited number of populations (21.Dodel R.C. Du Y. Bales K.R. Gao F. Eastwood B. Glazier B. Zimmer R. Cordell B. Hake A. Evans R. Gallagher-Thompson D. Thompson L.W. Tinklenberg J.R. Pfefferbaum A. Sullivan E.V. Yesavage J. Alstiel L. Gasser T. Farlow M.R. Murphy Jr., G.M. Paul S.M. Neurology. 2000; 54: 438-442Crossref PubMed Google Scholar, 22.Myllykangas L. Polvikoski T. Sulkava R. Verkkoniemi A. Crook R. Tienari P.J. Pusa A.K. Niinisto L. O'Brien P. Kontula K. Hardy J. Haltia M. Perez-Tur J. Ann. Neurol. 1999; 46: 382-390Crossref PubMed Scopus (72) Google Scholar, 23.Koster M.N. Dermaut B. Cruts M. Houwing-Duistermaat J.J. Roks G. Tol J. Ott A. Hofman A. Munteanu G. Breteler M.M. van Duijn C.M. Van Broeckhoven C. Neurology. 2000; 55: 678-684Crossref PubMed Scopus (58) Google Scholar, 24.Jhoo J.H. Kim K.W. Lee D.Y. Lee K.U. Lee J.H. Kim S.Y. Youn J.Y. Youn J.C. Woo J.I. J. Neurol. Sci. 2001; 184: 21-25Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 25.Gibson A.M. Singleton A.B. Smith G. Woodward R. McKeith I.G. Perry R.H. Ince P.G. Ballard C.G. Edwardson J.A. Morris C.M. Neurology. 2000; 54: 433-438Crossref PubMed Google Scholar) and because there is no molecular explanation regarding how A2M gene mutations may affect α2M structure, function, and expression. α2M is expressed by microglia, which accumulate near amyloid plaques (26.Van Gool D. De Strooper B. Van Leuven F. Triau E. Dom R. Neurobiol. Aging. 1993; 14: 233-237Crossref PubMed Scopus (94) Google Scholar). Thus, locally synthesized α2M may affect AD progression by regulating the activity of various proteinases or by binding important growth factors. The previously demonstrated ability of α2M to bind and neutralize the activity of TGF-β (12.Crookston K.P. Webb D.J. Wolf B.B. Gonias S.L. J. Biol. Chem. 1994; 269: 1533-1540Abstract Full Text PDF PubMed Google Scholar, 13.Gonias S.L. LaMarre J. Crookston K.P. Webb D.J. Wolf B.B. Lopes M.B. Moses H.L. Hayes M.A. Ann. N. Y. Acad. Sci. 1994; 737: 273-290Crossref PubMed Scopus (78) Google Scholar, 27.Lysiak J.J. Hussaini I.M. Webb D.J. Glass 2nd, W.F. Allietta M. Gonias S.L. J. Biol. Chem. 1995; 270: 21919-21927Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 28.Weaver A.M. Owens G.K. Gonias S.L. J. Biol. Chem. 1995; 270: 30741-30748Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 29.Fabrizi C. Businaro R. Lauro G.M. Starace G. Fumagalli L. Exp. Neurol. 1999; 155: 252-259Crossref PubMed Scopus (18) Google Scholar) may be detrimental in AD, because TGF-β stimulates Aβ clearance by microglial cells and reduces Aβ accumulation in the brain parenchyma of mice that overexpress human APP (30.Wyss-Coray T. Lin C. Yan F. Yu G.Q. Rohde M. McConlogue L. Masliah E. Mucke L. Nat. Med. 2001; 7: 612-618Crossref PubMed Scopus (516) Google Scholar). Furthermore, TGF-β has been reported to antagonize the cytotoxic activity of Aβ (29.Fabrizi C. Businaro R. Lauro G.M. Starace G. Fumagalli L. Exp. Neurol. 1999; 155: 252-259Crossref PubMed Scopus (18) Google Scholar, 31.Kim E.S. Kim R.S. Ren R.F. Hawver D.B. Flanders K.C. Mol. Brain Res. 1998; 62: 122-130Crossref PubMed Scopus (45) Google Scholar, 32.Flanders K.C. Ren R.F. Lippa C.F. Progress Neurobiol. 1997; 54: 71-88Crossref Scopus (337) Google Scholar). Another mechanism whereby α2M may regulate AD progression involves its ability to bind Aβ, forming a complex that is internalized by the α2M receptor, low density lipoprotein receptor-related protein (LRP) and then degraded (33.Narita M. Holtzman D.M. Schwartz A.L. Bu G. J. Neurochem. 1997; 69: 1904-1911Crossref PubMed Scopus (233) Google Scholar, 34.Kang D.E. Pietrzik C.U. Baum L. Chevallier N. Merriam D.E. Kounnas M.Z. Wagner S.L. Troncoso J.C. Kawas C.H. Katzman R. Koo E.H. J. Clin. Invest. 2000; 106: 1159-1166Crossref PubMed Scopus (307) Google Scholar, 35.Hyman B.T. Strickland D. Rebeck G.W. Arch. Neurol. 2000; 57: 646-650Crossref PubMed Scopus (90) Google Scholar). Du et al. (36.Du Y. Ni B. Glinn M. Dodel R.C. Bales K.R. Zhang Z. Hyslop P.A. Paul S.M. J. Neurochem. 1997; 69: 299-305Crossref PubMed Scopus (126) Google Scholar) originally reported that Aβ(1–40) and Aβ(1–42) bind to native α2M and to α2M that has been transformed into the LRP-recognized or “activated” conformation by reaction with methylamine (α2M-MA). Narita et al. (33.Narita M. Holtzman D.M. Schwartz A.L. Bu G. J. Neurochem. 1997; 69: 1904-1911Crossref PubMed Scopus (233) Google Scholar) subsequently reported selective binding of Aβ(1–40) and Aβ(1–42) to the activated conformation of α2M. α2M-MA apparently binds Aβ(1–40) and Aβ(1–42) with equivalent affinity (33.Narita M. Holtzman D.M. Schwartz A.L. Bu G. J. Neurochem. 1997; 69: 1904-1911Crossref PubMed Scopus (233) Google Scholar). Hughes et al. (37.Hughes S.R. Khorkova O. Goyal S. Knaeblein J. Heroux J. Riedel N.G. Sahasrabudhe S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3275-3280Crossref PubMed Scopus (200) Google Scholar) executed a yeast two-hybrid screen using Aβ(1–42) as bait and identified a 250-amino acid peptide from the C terminus of α2M as a strong and specific interactor. The same group also reported experiments confirming the interaction of Aβ with intact α2M; however, they did not demonstrate that the sequence identified by yeast-two hybrid screen is responsible for the binding of Aβ to intact α2M. The growth factor binding site in α2M is contained within a 16-amino acid peptide located ∼500 amino acids N-terminal to the Aβ-binding site identified by yeast-two hybrid screen (19.Webb D.J. Roadcap D.W. Dhakephalkar A. Gonias S.L. Protein Sci. 2000; 9: 1986-1992Crossref PubMed Scopus (28) Google Scholar). The growth factor binding sequence is composed mainly of hydrophobic amino acids with two potentially important acidic residues. TGF-β, platelet-derived growth factor-BB (PDGF-BB), and NGF-β all interact with the growth factor-binding site in α2M (18.Gonias S.L. Carmichael A. Mettenburg J.M. Roadcap D.W. Irvin W.P. Webb D.J. J. Biol. Chem. 2000; 275: 5826-5831Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 19.Webb D.J. Roadcap D.W. Dhakephalkar A. Gonias S.L. Protein Sci. 2000; 9: 1986-1992Crossref PubMed Scopus (28) Google Scholar), despite the fact that these proteins demonstrate limited sequence identity. Based on this promiscuous behavior, we hypothesized that the growth factor-binding site in α2M may also function as an Aβ-binding site. To test our hypothesis, we undertook a comprehensive molecular analysis to identify sequences in α2M with Aβ binding activity. Our results demonstrate that a single sequence, located near the C terminus of the α2M subunit, constitutes the only significant Aβ-binding site. Importantly, this sequence is entirely distinct from the growth factor-binding site. The LRP recognition sequence is also located near the C terminus of the α2M subunit (38.Sottrup-Jensen L. Gliemann J. Van Leuven F. FEBS Lett. 1986; 205: 20-24Crossref PubMed Scopus (87) Google Scholar, 39.Nielsen K.L. Holtet T.L. Etzerodt M. Moestrup S.K. Gliemann J. Sottrup-Jensen L. Thogersen H.C. J. Biol. Chem. 1996; 271: 12909-12912Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 40.Howard G.C. Yamaguchi Y. Misra U.K. Gawdi G. Nelsen A. DeCamp D.L. Pizzo S.V. J. Biol. Chem. 1996; 271: 14105-14111Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 41.Jenner L. Husted L. Thirup S. Sottrup-Jensen L. Nyborg J. Structure. 1998; 6: 595-604Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 42.Huang W. Dolmer K. Liao X. Gettins P.G.W. J. Biol. Chem. 2000; 275: 1089-1094Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar); however, our evidence indicates that the LRP recognition site and the Aβ binding sequence are distinct. Thus, in addition to the bait region, the α2M subunit has at least three distinct “protein interaction sites” with distinct binding specificities. These sites mediate interactions with growth factors, Aβ and LRP. α2M was purified from human plasma by the method of Imber and Pizzo (43.Imber M.J. Pizzo S.V. J. Biol. Chem. 1981; 256: 8134-8139Abstract Full Text PDF PubMed Google Scholar). α2M-MA was prepared by dialyzing α2M against 200 mm methylamine-HCl in 50 mmTris-HCl, pH 8.2, for 12 h at 22 °C and then exhaustively against 20 mm sodium phosphate, 150 mm NaCl, pH 7.4. Modification of α2M by methylamine was confirmed by demonstrating the characteristic increase in α2M electrophoretic mobility by non-denaturing PAGE (15.Barrett A.J. Brown M.A. Sayers C.A. Biochem. J. 1979; 181: 401-418Crossref PubMed Scopus (413) Google Scholar). α2M-MA was radioiodinated using IODO-BEADs (Pierce) and stored at 4 °C for no more than 2 weeks. The specific activity was 0.5–1.0 μCi/μg. Receptor-associated protein (RAP), which blocks binding of α2M-MA to LRP (65.Herz J. Goldstein J.L. Strickland D.K. Ho Y.K. Brown M.S. J. Biol. Chem. 1991; 266: 21232-21238Abstract Full Text PDF PubMed Google Scholar), was expressed as a glutathione S-transferase (GST) fusion protein in bacteria and purified by chromatography on glutathione-Sepharose. Aβ(1–40) was purchased from Bachem and radioiodinated using 125I-labeled Bolton-Hunter reagent (di-iodinated, PerkinElmer Life Sciences). Biotinylated Aβ(1–40) was prepared by reacting Aβ(1–40) with 4 μmsulfo-N-hydroxysuccinimide biotin (Pierce) for 2 h at 4 °C in siliconized tubes. The reaction mixture was dialyzed extensively against water. Biotinylated Aβ was stored for up to 1 month at 4 °C or frozen at −80 °C and thawed once without affecting its ability to bind to α2M. GST-specific IgG, bovine serum albumin (BSA, greater than 99% pure), dithiothreitol (DTT), and iodoacetamide were from Sigma Chemical Co. Bis(sulfosuccinimidyl) suberate (BS3) and horseradish peroxidase-conjugated avidin were from Pierce. Polyclonal Aβ-specific rabbit antibody was from Zymed Laboratories Inc. When α2M is treated with papain under mildly acidic conditions, an 18-kDa fragment is released from the C terminus of each α2M subunit (aa 1314–1451) (38.Sottrup-Jensen L. Gliemann J. Van Leuven F. FEBS Lett. 1986; 205: 20-24Crossref PubMed Scopus (87) Google Scholar). The 18-kDa fragment includes the intact receptor-binding site and is thus referred to as the receptor binding fragment (RBF). The residual 600-kDa α2M remnant retains the major structural features of the parent molecule (45.Hussaini I.M. Figler N.L. Gonias S.L. Biochem. J. 1990; 270: 291-295Crossref PubMed Scopus (17) Google Scholar). To obtain the 18- and 600-kDa α2M fragments, 4.0 μm α2M-MA was treated with 2.4 μm papain in 50 mm sodium acetate, 1 mm cysteine, pH 5.0, for 20 h at 22 °C. The pH of the reaction mixture was increased to 7.4, and the products were purified by molecular exclusion chromatography on Ultrogel AcA-22. Each α2M subunit has a single thiol ester bond formed by the side chains of Cys-949 and Gln-952 (11.Sottrup-Jensen L. Putnam F.W. The Plasma Proteins. 5. Academic Press, Inc., Orlando, FL1987: 192-291Google Scholar, 46.Sottrup-Jensen L. Petersen T.E. Magnusson S. FEBS Lett. 1980; 121: 275-279Crossref PubMed Scopus (247) Google Scholar, 47.Swenson R.P. Howard J.B. J. Biol. Chem. 1980; 255: 8087-8091Abstract Full Text PDF PubMed Google Scholar). When α2M is heated in the presence of SDS, the thiol esters react internally, and, as a result, the α2M peptide backbone is cleaved (47.Swenson R.P. Howard J.B. J. Biol. Chem. 1980; 255: 8087-8091Abstract Full Text PDF PubMed Google Scholar, 48.Harpel P.C. Hayes M.B. Hugli T.E. J. Biol. Chem. 1979; 254: 8669-8678Abstract Full Text PDF PubMed Google Scholar). The products include a 120-kDa N-terminal heat fragment and a 60-kDa C-terminal heat fragment. To produce α2M heat fragments, native α2M was incubated at 100 °C in 2% SDS (w/v) and 14 mm DTT for the indicated periods of time. The products were treated with iodoacetamide (70 mm) and subjected to SDS-PAGE. Six previously described fusion proteins, which collectively encode amino acids 99–1451 of the human α2M sequence, were expressed in BL-21 cells (17.Webb D.J. Wen J. Karns L.R. Kurilla M.G. Gonias S.L. J. Biol. Chem. 1998; 273: 13339-13346Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 18.Gonias S.L. Carmichael A. Mettenburg J.M. Roadcap D.W. Irvin W.P. Webb D.J. J. Biol. Chem. 2000; 275: 5826-5831Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). These fusion proteins include: FP1 (aa 99–392), FP2 (aa 341–590), FP3 (aa 591–774), FP4 (aa 775–1059), FP5 (aa 1030–1279), and FP6 (aa 1242–1451). Constructs encoding new GST fusion proteins, including FP6a (aa 1242–1365), FP6b (aa 1242–1400), and FP6c (aa 1365–1451), were generated using PCR and the intact A2M cDNA in pBluescript as a template. The oligonucleotides included recognition sequences for BamHI and EcoRI, to allow direct cloning into the vector, pGEX-2T. Final constructs were subjected to sequence analysis to verify proper orientation and reading frame. Two constructs, labeled FP6d, correspond in sequence exactly to the 18-kDa RBF (aa 1314–1451). In FP6d-AA, Lys residues at aa 1370 and 1374, which are critical for LRP binding (39.Nielsen K.L. Holtet T.L. Etzerodt M. Moestrup S.K. Gliemann J. Sottrup-Jensen L. Thogersen H.C. J. Biol. Chem. 1996; 271: 12909-12912Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 40.Howard G.C. Yamaguchi Y. Misra U.K. Gawdi G. Nelsen A. DeCamp D.L. Pizzo S.V. J. Biol. Chem. 1996; 271: 14105-14111Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), were mutated to Ala, using the QuikChange system (Stratagene). In FP6d-AR, Lys-1370 was mutated to Ala, and Lys-1374 was mutated to Arg. All of the fusion proteins were partially purified from induced bacterial suspensions by selective detergent extraction, as previously described (17.Webb D.J. Wen J. Karns L.R. Kurilla M.G. Gonias S.L. J. Biol. Chem. 1998; 273: 13339-13346Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The resulting preparations yielded clearly defined bands, with the correct molecular masses when assessed by Coomassie Blue staining of SDS gels or immunoblot analysis with GST-specific antibody. FP3, FP6, and FP6d-AA were purified to homogeneity by chromatography on glutathione-Sepharose. Fig. 1 shows the sequences of the GST fusion proteins used in this investigation. 125I-Aβ (2.5 nm) was incubated with native α2M, α2M-MA, or the purified 600-kDa fragment (0.3–1.0 μm) in 20 mm sodium phosphate, 150 mm NaCl, pH 7.4, for 2 h at 37 °C. In some experiments, increasing concentrations of the 18-kDa RBF (0.2–2.8 μm) were co-incubated with125I-Aβ and α2M-MA. Reaction mixtures were subjected to non-denaturing PAGE, using the buffer system described by Van Leuven et al. (49.Van Leuven F. Cassiman J.J. Van den Berghe H. J. Biol. Chem. 1981; 256: 9016-9022Abstract Full Text PDF PubMed Google Scholar). 125I-Aβ binding to α2M was detected as radioactivity co-migrating with the α2M band. In control experiments, free125I-Aβ did not migrate near α2M. To quantitate 125I-Aβ binding to α2M, gels were subjected to PhosphorImager analysis using ImageQuant software. Non-denaturing PAGE preserves non-covalent interactions; however, the amount of binding detected may be influenced by dissociation of protein complexes during electrophoresis (13.Gonias S.L. LaMarre J. Crookston K.P. Webb D.J. Wolf B.B. Lopes M.B. Moses H.L. Hayes M.A. Ann. N. Y. Acad. Sci. 1994; 737: 273-290Crossref PubMed Scopus (78) Google Scholar). Because Aβ binding to α2M is reversible and probably subject to rapid dissociation when methods such as non-denaturing PAGE or chromatography are used, we utilized the BS3 rapid cross-linking method to determine the apparent KD for the binding of Aβ to α2M-MA. This method has been used previously to determine KDvalues for the interaction of α2M with multiple growth factors and cytokines (12.Crookston K.P. Webb D.J. Wolf B.B. Gonias S.L. J. Biol. Chem. 1994; 269: 1533-1540Abstract Full Text PDF PubMed Google Scholar, 13.Gonias S.L. LaMarre J. Crookston K.P. Webb D.J. Wolf B.B. Lopes M.B. Moses H.L. Hayes M.A. Ann. N. Y. Acad. Sci. 1994; 737: 273-290Crossref PubMed Scopus (78) Google Scholar, 50.Wolf B.B. Gonias S.L. Biochemistry. 1994; 33: 11270-11277Crossref PubMed Scopus (44) Google Scholar). Increasing concentrations of α2M-MA were incubated with 25 nm125I-Aβ for 2 h at 37 °C. Freshly dissolved BS3 (5 mm) or vehicle (H2O) was then added for 5 min. Cross-linking reactions were quickly terminated by rapid acidification, followed by transfer to buffered SDS. Under pseudo-first order conditions, a constant fraction of the non-covalent 125I-Aβ·α2M-MA complex is covalently stabilized by the BS3 (13.Gonias S.L. LaMarre J. Crookston K.P. Webb D.J. Wolf B.B. Lopes M.B. Moses H.L. Hayes M.A. Ann. N. Y. Acad. Sci. 1994; 737: 273-290Crossref PubMed Scopus (78) Google Scholar). To quantitate the amount of covalently stabilized complex, BS3-treated and vehicle-treated samples were subjected to SDS-PAGE. 125I-Aβ that was covalently cross-linked to α2M-MA (bound) and free 125I-Aβ (free), which includes free Aβ and Aβ that was bound to α2M-MA but not cross-linked, were quantitated by PhosphorImager analysis. Results were analyzed according to the following equation (12.Crookston K.P. Webb D.J. Wolf B.B. Gonias S.L. J. Biol. Chem. 1994; 269: 1533-1540Abstract Full Text PDF PubMed Google Scholar),free/bound=(KD/z)(1/α2M­MA)+1/z−1Equation 1 The cross-linking efficiency, z, is a constant, derived from the y intercept, for each set of proteins and conditions (12.Crookston K.P. Webb D.J. Wolf B.B. Gonias S.L. J. Biol. Chem. 1994; 269: 1533-1540Abstract Full Text PDF PubMed Google Scholar). z is referred to as the BS3-cross-linking efficiency but may also be affected if a fraction of the radioiodinated protein is incapable of binding to the α2M. The apparent KD was determined from the slope when free/bound was plotted against 1/[α2M-MA]. This value is based on the assumption that there is a single binding site for Aβ in α2M. Assuming one Aβ-binding site/α2M subunit, as suggested by our data, then the KD must be corrected by multiplying the apparent KD by a factor of four. This method has been previously used to demonstrate specific and saturable binding of growth factors to denatured α2M subunits, α2M fragments, and GST-α2M-peptide fusion proteins (17.Webb D.J. Wen J. Karns L.R. Kurilla M.G. Gonias S.L. J. Biol. Chem. 1998; 273: 13339-13346Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 18.Gonias S.L. Carmichael A. Mettenburg J.M. Roadcap D.W. Irvin W.P. Webb D.J. J. Biol. Chem. 2000; 275: 5826-5831Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 19.Webb D.J. Roadcap D.W. Dhakephalkar A. Gonias S.L. Protein Sci. 2000; 9: 1986-1992Crossref PubMed Scopus (28) Google Scholar). Protein preparations were denatured in 2% SDS or treated with 1 mmDTT in 2% SDS and then with 5 mm iodoacetamide for 2 h, as previously described (17.Webb D.J. Wen J. Karns L.R. Kurilla M.G. Gonias S.L. J. Biol. Chem. 1998; 273: 13339-13346Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Samples were then subjected to SDS-PAGE and electrotransferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% milk in 20 mm sodium phosphate, 150 mm NaCl, 0.1% Tween 20, pH 7.4, and probed for 2 h with 125I-Aβ or biotinylated-Aβ. 125I-Aβ ligand blots were washed and subjected to PhosphorImager analysis. Biotinylated Aβ ligand blots were probed with horseradish peroxidase-conjugated avidin (1:5000 dilution). The membranes were then subjected to enhanced chemiluminescence (ECL) and densitometry. Equivalent loading and transfer of proteins were demonstrated by Coomassie Blue staining or, when applicable, by immunoblot analysis with GST-specific antibody (17.Webb D.J. Wen J. Karns L.R. Kurilla M.G. Gonias S.L. J. Biol. Chem. 1998; 273: 13339-13346Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Denatured α2M subunits and BSA were treated with 1 mm DTT and then with 5 mm iodoacetamide to block free sulfhydryl groups, subjected to SDS-PAGE, and electrotransferred to PVDF. The membranes were blocked with 5% milk. Unlabeled Aβ(1–40) was incubated with the immobilized α2M and BSA in PBS-T at 37 °C for 2 h. The membranes were then washed extensively and probed with rabbit Aβ-specific IgG (1:4000) in PBS-T and 0.1% milk (v/v), followed by anti-rabbit IgG-horseradish peroxidase conjugate (1:10,000). Membranes were analyzed by ECL and densitometry. 125I-α2M-MA (20 nm) was incubated with 20 μm Aβ or with vehicle for 2 h at 37 °C. The 125I-α2M-MA (0.3 μCi) was then injected, in the presence and absence of GST-RAP (40 or 80 μg), into the lateral tail veins of CD-1 female mice (30 g). Blood samples" @default.
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• W2015510614 title "Distinct Binding Sites in the Structure of α2-Macroglobulin Mediate the Interaction with β-Amyloid Peptide and Growth Factors" @default.
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